International Journal of
Reproductive Biomedicine
Fri, Jan 3, 2025
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Volume 13, Issue 4 (Supl.1- Poster Presentations 2015)
IJRM 2015, 13(4): 31-93 |
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Abstracts of the 6th Yazd International Congress and Student Award in Reproductive Medicine with 1st Reproductive Genetic Congress. IJRM 2015; 13 (4) :31-93
URL: http://ijrm.ir/article-1-1678-en.html
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Poster Presentations
P-1
Fertility preservation in cancer patients
Abtahi NS, Ebrahimi B, Rezazadeh Valojerdi M, Fathi R.
Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: b.ebrahimi@royaninstitute.org
Introduction: Fertility preservation before gonadotoxic treatments is necessary in cancer patients. Ovarian tissue cryopreservation is the only available option for prepubertal girls and women who cannot delay cancer treatment or when ovarian stimulation is contraindicated.
Materials and Methods: Human ovarian tissue biopsies were obtained from six women (29-40 years old) who were candidates for oophorectomy for benign gynecologic conditions. Their medulla were removed, cortical parts were thinned and then cut into 10×5×1 mm strips. Cortical strips were vitrified in two steps by using vitrification solutions including; V1 [HTCM as base medium (BM)+ 7.5% DMSO+ 7.5% EG+ 20% HAS] and V2 (BM+ 15% DMSO+ 15% EG+ 0.25M sucrose+ 20% HSA) for 15 and 10 min respectively. Morphology of ovarian tissue and enclosed follicles were compared in control and vitrification groups by histological assessment.
Results: Ovarian stromal integrity, granulosa cells distribution, follicular population and morphology were well preserved in vitrified-warmed strips as fresh one. Oocyte degeneration, vacuolization and granulation were rarely observed in vitrified-warmed ovarian tissues. Primordial follicles were more distributed in cortical region in both groups. The mean percentage of intact follicles (primordial, primary and pre antral) are higher in control groups compared to vitrification one but the difference was not significant.
Conclusion: Accordingly, used vitrification method could be suitable for human ovarian tissue preservation in cancer patients.
Key words: Human, Fertility preservation, Vitrification, Ovarian tissue.
P-2
Effects of sesame-supplemented diet on the histology of adult rat reproductive system
Amini Mahabadi J1, Hassani Bafrani H1, Nikzad H1, Taherian AA2, Seyed Hoseini ES1.
Introduction: Studies show that antioxidants are important in improving male infertility. There are several antioxidant compounds in sesame seeds.
Materials and Methods: This experimental study was carried out on 30 adults Wistar rat (200 gr). Rats were randomly divided into experimental and control groups. The control group received standard diet and experimental group received a diet containing 70% standard diet and 30% sesame seed for 12 weeks. At the end of the study, body weight, testis weight and volume were measured and histology of testis, epididymis and prostate were evaluated. Serum FSH, LH and Testosterone levels were measured as well. SPSS software was used to calculate t-test and p<0.05 was considered as significant.
Results: The measured sperm count and motility, the number of epithelial cells and lumen diameter were increased significantly in the experimental group (p<0.0001). The number of spermatogonial cells, primary spermatocytes, spermatid and spermatozoa were increased significantly in the experimental group compared to the control group (p<0.0001). The epididymal diameter, lumen diameter and epithelial thickness did not change significantly. Fibromascular and epithelium diameter of seminal vesicle in treatment group were very significant differences and volume density epithelium significantly increased in treatment group compared to control group but volume density, Fibromascular and lumen significantly decreased in treatment group compared to control group. LH concentration increased significantly in the experimental group (p<0.03).
Conclusion: This is the first study, which evaluated the histology effect of sesame seed on reproductive system of adult Wistar rat. These results highly suggest that sesame seed can improve male reproductive parameters.
Key words: Rat, Male Sex hormones, Sesame seed, Testis, Prostate, Epididymis.
P-3
The effects of sperm preparation media on motility, viability and DNA integrity of human spermatozoa
Anbari F1, Halvaei I1, Nabi A1, Ghazali Sh1, Khalili MA1, Johansson L2.
Introduction: The main goal was to compare the effects of three different sperm preparation media on sperm motility, viability and DNA integrity of semen samples from normozoospermic men.
Materials and Methods: A total of 15 normozoo spermic males were included in the study. The semen analysis was performed in accordance with the WHO guidelines (2010). After semen analysis, each sample was divided into three aliquots and swim-up performed with three different sperm preparation media (Sperm Preparation Media, Origio, Denmark, Ham’s F10, Biochrome, Berlin, Germany, and VitaSpermTM, Innovative Biotech, Iran). Sperm motility, viability and DNA fragmentation,were evaluated at 0, 1, 2, and 24 hr after swim-up.
Results: There were no significant differences, at any time intervals, in the totalsperm motility between the different sperm preparation media. However, the rate of progressive motility was significantly higher in spermatozoa prepared by the media from Origio in comparison to VitaSperm TM (68.3±11.5 vs. 58±9.8, p=0.03), whereas no significant difference was found against Ham’s F10. No significant differences in sperm viability were seen between the media products .One hour after swim-up therate of sperm DNA fragmentation was also significantly lower inthe medium from Origio versus VitaSpermTM (p=0.02).
Conclusion: The type of medium for preparation of semen samples from normozoospermic men significantly affects the performance of spermatozoain assisted conception.
Key words: Sperm preparation media, DNA fragmentation (SCD), Viability, Motility.
P-4
Investigating the underlying factors of preterm delivery prevalence preceded by preeclampsia among pregnant visitors of Shariati Hospital, Bandar-Abbas, Iran: 2011-2012
Aslavi N1, Fallahi S2, Nowroozi S1, Najafian A3, Ghanbarnejad A1.
Introduction: Along with blood loss and infection, disorders pertaining to blood pressure are the three deadly factors in preterm childbirth morbidities and disabilities. In other countries the prevalence rate of this crisis is maximally 10%, while in Iran, it is 7%.
Materials and Methods: The present descriptive/ analytic research is a cross-sectional retrospective study. It was carried out on 863 pregnant women (167 had a preterm delivery due to pre-eclampsia and 696 were diagnosed with preterm delivery due to other reasons). The data were collected via a checklist about mother’s age, blood group, pre-eclampsia history, systemic disease, preterm delivery pain, etc. SPSS 16 analyzed the data. T-test and Chi-squared tests were used too. Significance level was set at p<0.05.
Results: Participants with a preterm delivery after pre-eclampsia were found to have a higher average age than peers afflicted with preterm delivery for other reasons. Most of the women afflicted with pre-eclampsia were of the B+ blood type. Significant differences were observed in these variables: pre-eclampsia history, preterm delivery pain, preterm rupture in fetal membrane, vaginal bleeding, intrauterine growth restriction, mother’s systemic diseases, blood group, history of intrauterine death and amniotic fluid disorders.
Conclusion: There is an increase in the probability of preterm childbirth due to pre-eclampsia with intrauterine growth restriction, history of intrauterine death and mother’s background diseases. Timely medical and preventive attempts and instructions for pregnant mothers on pre-natal healthcare can help to decrease irrevocable a consequence that endanger mothers and baby’s health.
Key words: Pre-eclampsia, Premature childbirth, Disease prevalence, Bandar-Abbas.
P-5
Infertility and quality of life in women with polycystic ovary syndrome
Bahri Khomami M1, Ramezani Tehrani F1, Azizi F2.
Introduction: Polycystic ovary syndrome (PCOS), the most common endocrine disorder, may cause infertility among affected women. Infertility is a life crisis and blamed for psychosocial distress and poor quality of life (QoL). Ethnicity and socio-cultural factors may have an impact on this negative influence on QoL.
Materials and Methods: The study procedure was completed by 796 women with polycystic ovary syndrome, aged 15-49 years. A reliable validated Persian version of the health related quality of life questionnaire (HRQoL) for polycystic ovary syndrome patients (PCOSQ) was filled for each participant. They were subdivided into 2 groups according to their fertility status: fertile and infertile. Using linear regression the association between infertility and health related quality of life in women with PCOS was assessed.
Results: Out of all participants 120 (15.1%) and 482 (60.6%) women were infertile and fertile, respectively. Infertility was associated with lower HRQoL score before and after adjustment for age, body mass index and other perceived PCOS symptoms (CI 95%: -16.76, -3.81; p=0.002 and CI 95%: -15.02, -3.67; p=0.001). Women who had longer duration of infertility, had got better scores.
Conclusion: We found that infertility causes poor quality of life in PCOS women. Care providers should pay attention to the effect of each PCOS symptoms on patients emotional health along with physical health and plan to treat them accordingly.
Key words: Quality of life, Health-related quality of life, Infertility, Polycystic ovary syndrome.
P-6
The effect of hydrostatic pressure on parthenogenetic activation of mouse oocytes
Bashiri Z, Azadbakht M.
Departeman of Biology, Faculty of Sciences, Razi University, Kermanshah, Iran.
Email: zbashiri88@gmail.com
Background: Parthenogenetic activation of mammalian oocytes using artificial stimuli is commonly used in various reproductive biotechniques. Hydrostatic pressure can act as a mechanical stimulator that rearranges egg contents. In this study, we investigated the effect of hydrostatic pressure on parthenogenetic activation of MП oocytes derived from superovulation and matured oocytes in vitro.
Materials and Methods: In experiment 1, immature oocytes were dissected from ovary of female NMRI mouse (8-week-old) and transferred to α-MEM medium for in vitro maturation. After 24 hr, MП oocytes were transferred to T6 medium. In experiment 2, mice were superovulated by injections of 10 IU of PMSG and 10 IU of HCG 48 hr apart. MП oocytes were collected 12 hr after HCG injection and transferred to T6 medium. Then oocytes from two groups (experiment 1, 2) were divided into experiment and control groups. Oocytes of experiment group were subjected to 20 mmHg pressure for 10, 20, 30 min (treatments I, П, III). Oocytes without exposure to pressure were considered as control. Oocytes were cultured for 72 hr and embryo development was assessed.
Results: In experiment 1, cleavage rate in treatments I, П, III and control was 21.42%, 15.71%, 8.52%, 5.71% respectively. The best cleavage rate were associated with treatment I which were significantly different with treatment III and control group (p<0.05). In experiment 2, cleavage rate in treatments I, П, III and control was 22.91%, 53.12%, 29.15% and 9.34% respectively. Oocyte activation rate in experiment group was higher than control group. The highest cleavage rate associated with treatment П which were significantly different with treatments I, III and control (p<0.05).
Conclusion: Exposure of MП oocytes derived from superovulation to hydrostatic pressure, on account of cumulus cells presence around oocyte, could improve embryonic development and by affecting on calcium channels, probably leading to increase rate of cleavage in the mouse oocyte.
Key words: Parthenogenetic activation, Hydrostatic pressure, Oocyte, Mouse.
P-7
Evaluation the knowledge and awareness of nulliparous women from adverse outcome of delayed childbearing
Behboudi-Gandevani S.
Reproductive Endocrinology Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Email: s.behboudi@hotmail.com
Introduction: Recently the numbers of women who delay childbearing in older age were rapidly increased. The aim of this study was to evaluate the nulliparous women’s knowledge and awareness from adverse outcome of delayed childbearing.
Materials and Methods: In this cross-sectional survey study, a total of 700 healthy nulliparous women 35 years and older that had self-choice delayed pregnancy were selected by cluster sampling in Tehran. To evaluate the knowledge and awareness of these women, a standard questionnaire about adverse pregnancy outcomes in older age were used. The questionnaire consists of three parts: demographic characteristics, assessment of maternal adverse outcomes (10 questions) and to assessment of fetal-neonatal adverse outcomes in maternal older age (6 questions). Data were analysis by SPSS software and the significance level was set at p˂0.05.
Results: The psychometric properties of questionnaire including forward and backward translation, face and content validity and reliability with cronbach's-alpha: 0.078 were done. The result of the study showed that women’s awareness about the risks of delayed childbearing including gestational diabetes, congenital anomaly, Down syndrome, caesarean section, low birth weight, stillbirth and long-term health problems like learning difficulties among neonate varies between 40%-72%. In this respect, most of the women were aware about the increased risk of infertility in older age (82.9%), but a little were aware about the possibility of a twin or multiple pregnancy and gestational hypertension in older ages of mother (35.2%). Awareness of delayed childbearing risks were independently related to the women’s educational levels (OR: 1.87; 95% CI: 1.25-2.78) and family income (OR: 3.12, 95% CI: 0.01-5.12).
Conclusion: It seems that women are largely unaware about the adverse consequences of pregnancy in older age among mother and child. Women’s should be more inform about these risks by health care professional.
Key words: Awareness, Delayed childbearing, Adverse outcome.
P-8
Study the protective role of jujube extract on teratogenic effects of Carbamazepine in Balb/c mice embryos
Doostabadi MR, Hasanzadeh Taheri MM, Afshar M, Hami J, Hosseiny M.
Birjand University of Medical Sciences, Birjand, Iran.
Email: doostabadim@yahoo.com
Introduction: Carbamazepine (CBZ) is an anticonvulsant medication that is consumed during pregnancy and can produce congenital anomalies including; neural tube defects, cardiac, skeletal and craniofacial abnormalities.
Materials and Methods: In this experimental study, 100 pregnant mice of Balb/c 25±3 gr body weight randomly divided into 8 experimental groups (E) and 2 control groups (C) (n=10). Experimental groups (E1, E5 and E6) and (E2, E7 and E8) received daily intraperitoneal injection (IP) of 50 and 100 mg/kg/ of CBZ respectively from gestational day zero (GD 0) to GD 15 and also groups (E5, E7) and (E6, E8) in addition to medicine, received jujube extract (AJE) at doses of 200 or 400 mg/kg/ from ten days prior to gestation, to GD 15. E3 and E4 groups gavaged only by 200 and 400 mg/kg of AJE respectively. Two control groups (C1, C2) received normal saline or tween -20. Dams underwent cesarean section on GD 18 and fetuses harvested from uterine. First, absorbed embryos were counted. Thereafter, morphological studies were done on the offspring by stereomicroscope. All malformed fetuses were stained with Alizarin red S and alcian blue for detection of skeletal anomalies. Data were analyzed by ANOVA, Tukey and c2 tests and using SPSS software version 18 and p<0.05 were considered significant.
Results: The findings of this study showed that administration of CBZ purely in pregnant mice induced various anomalies in their fetuses such as; limb defects, deformities of vertebral column, craniofacial malformations and etc in experimental groups so that their differences were significant when compared with control groups. But these anomalies decreased significantly in those experimental groups which received CBZ and AJE synchronously when compared with experimental groups that received CBZ only.
Conclusion: According to our findings it can be concluded that although administration of CBZ can induce several malformations in fetuses of pregnant mice, but consumption of AJE synchronously with CBZ, can prevent teratogenicity of CBZ. Therefore, probably AJE can play a protective role against CBZ induced anomalies.
Key words: Teratogenic, Jujube, Carbamazepine.
P-9
Depression and clinical markers in polycystic ovary syndrome
Enjezab B1, Ghadiri anari A2, Eftekhar M3, Mohajeri M1.
Introduction: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among reproductive-age women. Previous studies have raised conflicting results of depressive disorders in PCOS compared with healthy people.
Materials and Methods: In this cross-sectional analytic study, 62 patients with PCOS diagnosed by Rotterdam criteria and 61 women without PCOS were selected after an initial survey conducted to determine demographic characteristic, systolic and diastolic blood pressure and reproductive status, the Beck depression questionnaire short form was used to assess depression in both groups. All women were introduced to determine fasting insulin and blood sugar and testosterone.
Results: The mean age, BMI, systolic and diastolic blood pressure of PCOS and Non PCOS was respectively (29.96±6.85, 29.49±7.44 years), (29.15±6.56, 25.65±5.84), (117.42±10.17, 117.18±66.57) and (75.41±9.27, 76.42±6.56) respectively that only mean of BMI was different (p˂0.05). The average Beck score in patients was 7.47±5.54 and in the control group was 7.57±5.79, (p˂0.05), which was not statistically significant. 37.1% of the patient group and 36.1% of the control group have degree of moderate to severe depressed mood (p˃0.05).
Conclusion: Despite non significant difference in prevalence of depression in both groups, due to the high prevalence of depression in both groups may be there is other reasons for depression in this population that overcomes on the psychological effects of this disease.
Key words: Depression, Polycystic ovary syndrome, BMI.
P-10
Comparison of metabolic syndrome in postmenopausal women with natural or surgical menopause: Community based cohort study
Farahmand M, Ramezani Tehrani F, Azizi F.
Introduction: The ovaries produce female sex hormones that after natural menopause gradually and following surgical menopause abruptly end.
Materials and Methods: The study subjects were selected from 5019 women, 59-35 years old, which were participated in the Tehran Lipid and Glucose Study. They include 357 natural menopauses and 63 surgical menopauses during the follow-up that was 10 years. Data were collected by questionnaires in this project.
Results: Changes in metabolic and biochemical profiles of these two groups of women during follow-up were compared with each other. The findings of our study showed that the incidences of metabolic syndrome in surgical menopause and natural menopause subjects were 14.5 and 8.9% respectively. Mean serum low density lipoprotein in surgically menopausal women was significantly lower than natural menopause subjects. Also, mean systolic blood pressure in naturally menopausal women was significantly higher than surgical menopause women.
Conclusion: It seems the metabolic disorders associated with menopause differ in surgical and natural menopause women. The metabolic complications should be considered following menopause.
Key words: Surgical menopause, Natural menopause, Metabolic syndrome.
P-11
Medical staffs’ viewpoints on “ART nursing” working at Reproductive Institutes in Iran
Faramarzi A, Khalili MA, Borzouei Z, Hosseini A, Rahiminia T.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: faramarzi_90@yahoo.com
Introduction: As, assisted reproductive techniques (ART) advances, multifaceted changes are noted in the roles, responsibilities, and commitments of each members of the infertility team. The aim was to determine the level of knowledge and attitude of medical staff working at reproductive institutes about role of nursing in ART program.
Materials and Methods: In this descriptive cross-sectional study, knowledge and attitudes of 199 Bio-medical staffs working at reproductive institutes were investigated through a questionnaire including 20 questions. All participants based on work experience, education and communication with patients were classified. Chi-square test was used for data analysis and p<0.05 was considered as statistically significant.
Results: There was no significant relationship between educational levels and viewpoints on ART nursing. But, significant relationship between work experience, kind of relation to the patients and viewpoints on ART nursing were found. 52.5% of participants were familiar with ART nursing. By increasing experience, knowledge and attitudes of Bio- medical staffs enhanced. Laboratory technician, embryologist, ART Laboratory technician and nurses were more familiar with ART nursing; but, Urologists were less familiar with ART nursing. Over 97% of participants requested offering a master degree of ART nursing in Iran (p<0.05).
Conclusion: There is a need for nursing professionals to assume an ongoing, visionary, scientific, and academic approach to advancement. It seems that appropriate continuing education and opportunities are essential to support this group of nursing professionals.
Key words: Viewpoint, Nurse, ART, Medical staffs.
P-12
The effect of microtubule stabilizer pretreatment in IVM program
Fesahat F1, Khalili MA1, Khoradmehr A1, Anbari F1, Bazrgar M2, Taheri F1.
Introduction: It has been reported that in vitro maturation (IVM) increases the risk of abnormal spindles and chromosome configurations of oocytes compared with oocytes matured in vivo. This is one possible explanation for the reduced developmental potential of IVM oocytes compared with those matured in vivo. Paclitaxel is known to stabilize the microtubules that constitute the spindle. The aim was to investigate whether pretreatment by a microtubule stabilizer, paclitaxel, would improve IVF outcomes in IVM program.
Materials and Methods: In this ongoing experimental study, 75 GV oocytes were retrieved from 5 mice primed with rec-FSH for 48 hr. The immature oocytes were cultured 24-48 hr for IVM. After identifying mature oocytes by the presence of a first polar body extrusion under a stereomicroscope, Normal morphologically MII oocytes were divided in control and 5 experiment groups. Experiment groups were incubated in presence of 1μM paclitaxel for different times (30 min/group 1,1 hr/group 2,2 hr/group 3,3 hr/group 4,4 hr/ group 5 and group 6 as control) prior to IVF. Standard IVF program was performed for the 60 MII oocytes after pretreatment with paclitaxel and embryo development was followed until blastocyst formation.
Results: Total maturation rate of GV oocytes was 80% and fertilization rates of MII oocytes were 85% in groups 6, 83% in groups 1, 2 and 3 and 74% and 65% in groups 4, 5 respectively. Blastocyst formation rates of embryos were 95% in group 1, 2 and 6, 60% in group 3 and 40% in groups 4 and 5. The final results, which include probable relationship between paclitaxel pretreatment and the aforementioned variables, will be presented.
Conclusion: The preliminary results showed by performing critical concentration with optimized timing of microtubule stabilization procedure IVM, MII oocytes could efficiently develop to the blastocyst stage. Paclitaxel may cause cell toxicity with irreversible harm on normal fertilization and embryo development in higher concentrations.
Key words: Paclitaxel, IVM, IVF outcomes.
P-13
The effect of ovarian drilling on poly cystic ovary syndrome (PCOS) women
Ghasemi-Esmailabad S, Eftekhar M, Pourmasumi S, Rajabi Firoozabadi Sh, Sabeti P, Heidarian Meimandi H.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: Qasemisaeed@yahoo.com
Introduction: One of the most common causes of female infertility factors is ovulation disorder. Poly cystic ovary syndrome (PCOS) has the highest prevalence rate about 5-10% among women. There are different diagnostic methods and treatments for PCOS syndrome, including medical and surgical treatments. Laparoscopic surgical treatment is for the patients resistant to medical treatment, and could be done with ovarian drilling at some points.
Materials and Methods: In this retrospective study we evaluated 289 patients. All patients had proven clomiphene resistant PCOS with ultrasound and hormonal tests. Laparoscopic ovarian cautery was performed for all these women. Age, duration of infertility and assisted reproductive techniques in each patient were asked.
Results: Of the 289 patients, we access to the information of 135 patients. The mean age and the duration of infertility were 26.90 and 4.83 years respectively. Among these, 63 patients (46.6%) had not pregnancy and 72 patients (53.33) were pregnant, 52 patients (72.22%) were naturally pregnant and 20 patients (27.77%) used assisted reproductive techniques.
Conclusion: PCOS patients who resistant to clomiphene could be treated with ovarian drilling.
Key words: Polycystic ovary, Clomiphene, Ovarian drilling.
P-14
Comparison of two different embryo loading techniques for embryo transfer in IVF/ET cycles
Halvaei I, Mangoli E, Omidi M, Khalili MA.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: ihalvaei@gmail.com
Introduction: Embryo loading (EL) is one of main steps in embryo transfer (ET) technique that plays an important role in IVF success. This study was aimed to compare the effect of two different techniques for EL on rates of pregnancy and delivery in IVF/ET cycles.
Materials and Methods: In total 195 fresh ET and 171 frozen-thawed ET were included in this retrospective study in which 76 and 119 cycles in fresh ET and 76 and 95 cycles in frozen-thawed ET were placed in two groups of A and B, respectively. Embryo catheter loading techniques were divided into two groups of A and B. In group A, the whole catheter was flushed with a 1-ml air-tight syringe with Ham’s F10 medium. Then the embryos were drawn into ET catheter between two air brackets. In group B, 70 µl airs was held in the syringe and the catheter was flushed by Ham’s F10 medium. Then the media, air, embryos, and air were respectively drawn into catheter. The main outcome measure of the study was delivery rate.
Results: The groups were matched for number of fertilized oocytes, etiology of infertility, source of sperm, type of stimulation protocol, percent of conventional IVF or ICSI, high quality embryos, type of embryo loading catheter, fresh ET and ease of transfer. In fresh ET cycles, delivery rate showed an increasing trend in group B compared to group A (21% vs. 11.8%, respectively, p=0.1). In frozen-thawed ET cycles, the rate of delivery rate was also higher in group B compared to group A (16.8% vs. 13.1%, respectively), but the difference was not significant (p=0.5).
Conclusion: It seems different embryo catheter loading has no effect on delivery rate.
Key words: Embryo loading technique, Embryo transfer, Pregnancy.
P-15
Application of sonography in infertility treatment cycles: instructions for midwives and nurses
Irani Sh, Ahmadi F, Javam M.
Department of Reproductive Imaging at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: Irani_shohreh@yahoo.com
Introduction: Ultrasound has a pivotal role in imaging modality in the study of the female pelvis, and provides fundamental information in detecting and characterizing pelvic masses of uterine, ovarian, or adnexal origin.
Materials and Methods: A narrative review was performed within articles published at PubMed, Elsevier, SID and original text books to reach the aim.
Results: Every patient in the process of infertility treatment needs to go through four steps of sonography to roll out disorders and malformations in ovaries, uterus and tubes then to opt for the best treatment method and to follow up the results. Infertility Workup: 1) Base sonography is applied in examination of the following organs: -For overall evaluation of the pelvis and determining any pathologic condition in uterus such as polyps, fibroms, uterus malformations. -Ovarians are examined for ovarian cysts and Poly Cystic Ovary Syndrome (PCOS). 2) Pre-treatment sonography is applied in checking the result of operative surgeries- like Hysteroscopy, Laparoscopy, Myomectomy. 3) Monitoring sonography is done at different stages of treatment cycle, especially at ovulation stimulation period, in order to investigate ovarians and in picking up the eggs as well as endometrial thickness in response to drugs. 4) Post-Assisted reproductive techniques (ART) cycles is done for confirmation of pregnancy (gestational sac, fetal heart), and the side effects of treatment cycles such as ovarian hyper stimulation syndrome (OHSS), pregnancy complications such as ectopic pregnancy (EP) and mole.
Conclusion: Ultrasound has a key role in diagnosis and proposing of different options of treatment regimes, and the post-treatment follow-ups. It is important to know the application of the aforementioned four steps to improve the accuracy and efficiency of infertility treatment.
Key words: Ultrasonography, ART cycle, Infertility.
P-16
Evaluation of anxiety, depression and risk factor associated with them in women after tubal ligation
Jahanian Sadatmahalleh Sh1, Ziaei S1, Kazemnejad A2, Mohamadi E3.
Introduction: Tubal ligation is recommendable for women completed their family. The existence of anxiety and depression following this procedure has been the subject of debate for decades.
Materials and Methods: A historical cohort study was carried out on 200 subjects with tubal ligation and on 200 subjects using condom as contraceptive method. The two groups were matched in demographic and personal characteristics. Data collection tool was a questionnaire including questions regarding demographic and obstetrical characteristics. Anxiety and depressive symptoms were evaluated by the Hospital Anxiety and Depression Scale (HADS). All statistical analyses were carried out using software package used for statistical analysis (SPSS) version 20 (SPCC Inc., Chicago, IL, USA). Student’s t-test and chi-square test were carried out to reveal the statistical differences between the groups. Multiple linear regression was done to build a prediction model in anxiety and depression.
Results: The mean (SD) duration of tubal ligation was 4.1 (1.6) years. The overall prevalence of anxiety and depression in the two groups was 81.5% and 48.5% respectively (p<0.0001). Multiple linear regression analysis revealed a significant association between low education (p<0.0001), post-sterilization regret (p=0.03) and no consultation prior tubal ligation (p=0.03) with risk for anxiety and depression in women who have undergone tubal ligation.
Conclusion: Termination of fertility with tubal ligation may be a risk factor for anxiety and depression. We found significant differences in anxiety and depression between women with and without tubal ligation. Therefore, women should be informed by the health providers regarding the advantages and disadvantages of tubal ligation before the procedures.
Key words: Historical cohort study, Tubal Ligation, Anxiety, Depression.
P-17
Polymorphism in CGA affects the function of miR-1302 and increases the risk of men infertility
Jamalvandi M, Motevali bashi M.
Department of Biology, Faculty of Sciences, Esfahan University, Esfahan, Iran.
Email: mina.jamalvandi@yahoo.com
Introduction: Infertility occurs in 10-15% of couples worldwide and close to half of it is caused by male factors. Despite decades of efforts to clarify mechanism of male infertility, most of cases are still idiopathic. Many factors such as genetics and sexual problems can affect infertility. Among these problems, genetic disorders are the most common factors. A study has shown that one of genes that can affect male infertility is CGA. This gene is involved in miotic. CGA, α subunit of glycoprotein hormones, is the main part of thyrotropin glycoprotein hormone (pituitary TSH), lutropin (LH), follitropin (FSH), and Chorionic gonadotropin (human placental gonadotropin, hCG) that has essential role in development and function of tyroid and gonads. CGA gene is located on 6q14-q21. Rs6631 in CGA has strong association with men infertility. Studies have shown that miR-1302 can negatively regulate CGA and substitution of T with A may interfere this process. This miRNA can band with rs6631-A more strongly than rs6631-T.
Materials and Methods: Tetraprimer technique is an appropriate way to study this polymorphism; because it is faster and cheaper than ordinary PCR. Also laboratories with low equipment can use this method.
Results: Primers designed by the use of Primer1 and then checked by Oligo7 software. By the use of these primers and tetraprimer technique, this polymorphism can be studied for the first time in Iran.
Conclusion: SNP in genes specially the ones that are target of miRNAs, can have important role in complex disease such as infertility. In this study, miR-1302 negatively regulates CGA, and the substitution of T by A at rs6631 within the binding site disrupts its regulation. It is not exactly clear that what is the real role of this polymorphism in idiopathic male infertility; but it has been suggested that the variant allele of rs6631 may elevate the risk of idiopathic male infertility through up-regulation of the expression of CGA. It has been showed that α-subunit, that is produced by CGA, can has growth factor activity and can induce the differentiation of lactotrope and secretion of RPL. Because CGA and PRL play an important role in the development of gonads, the abnormal hormone level may elevate the risk of male infertility.
Key words: Men infertility, miRNA, SNP, CGA.
P-18
Evaluation of testosterone levels after GnRH agonist administration in the adult male rat
Khadivi B1, Peirouvi T1, Zirak Javanmard M2, Rasmi Y3, Mohammadbeigi H1.
Introduction: Gonadotropin-releasing hormone (GnRH) is secreted from the hypothalamus in a pulsatile manner and stimulates the biosynthesis and release of gonadotropins, LH and FSH, via GnRH receptor located in gonadotropin cells. These gonadotropins regulate various gonadal functions such as gametogenesis and steroidogenesis.
Materials and Methods: 24 male adult Wistar rats were divided into three groups. In the first study group, 300 µg/kg buserelin, in the second study group, 600 µg/kg buserelin and in the control group, salin was injected subcutaneous for 5 days. Thirty day after the first injection, blood samples were collected from the heart, centrifuged and plasma was isolated from blood. Plasma testosterone level was measured by ELISA.
Results: The findings reveal no significant differences in plasma level of testosterone in the first study group (2.71±0.71) and second study group (2.12±1.74) compared with control group (3.14±1.20) (p>0.05).
Conclusion: Short-term administration of buserelin has no effect on the plasma testosterone level.
Key words: GnRH, Buserelin, Testosterone, Adult rat, ELISA.
P-19
Apoptotic cells and loss of follicle development were resulted after administration of Nano dioxide titanium on immature mouse ovary
Khoradmehr A1, Danafar AH1, 2, Hosseini M3, Halvaei I1, Golzadeh J1, Akyash F1, Anvari M1.
Introduction: Titanium dioxide (TiO2) is used as an antimicrobial and whitening agent in food and products such as chewing gums, candies, toothpastes, lip balms, shampoos, deodorants and sunscreens. Previous studies showed that ovary might be one of the target organs that TiO2 nanoparticles induced genotoxicity and cytotoxicity in ovary cells and exposure to TiO2 can effect on follicle development and fertility with changes the levels of sex hormones. The aim of the present study was to evaluate ovarian dysfunction by detect of follicle development.
Materials and Methods: 40 immature Balb/C female mice (4week ages) randomly divided into five groups. First, second and third groups were administrated by oral gavage 2.5, 5 and 10 mg/kg TiO2 for thirty days respectively. Forth group was gavaged normal saline as placebo and in control group we didn’t gavage anything. Finally female mice were dislocated and ovary was fixed in bouin's fluid. Ovaries were dehydrated with ethanol series, embedded in paraffin and then serially sectioned at 5μm thickness. Hematoxylin and Eosin used to evaluate primordial, primary, preantral and antral follicles by light microscope. TUNEL assay was used to detect cell apoptosis. Statistical significance was analyzed by Kruskal-Wallis test and the level of significance was determined to be at p≤0.05.
Results: According to data, primordial, primary, preantral and antral follicles were significantly decreased in third group in comparison with the controls and shams (p≤0.05) and also, there was increasing trend in primary and preantral follicles in first and second groups compared to control group during TiO2 exposure. Although, significant reduction of apoptotic cells was observed in third group compared to control and sham groups (p≤0.05).
Conclusion: Furthermore, there is little knowledge about physicochemical characteristics of TiO2 nano materials; our findings suggested that TiO2 exposure can defect follicle development and perhaps fertility potential in young mice.
Key words: Titanium dioxide, Ovary, Follicle development, TUNEL.
P-20
Correlation between 25-OH vitamin D in follicular fluid and implantation rate in infertile women undergo IVF/ICSI
Latifi Z1, Farzadi L2, Khayatzadeh Bidgoli H2, Fattahi A1, Nouri M2.
Introduction: Vitamin D in combination with its receptors can be involved in implantation by regulating gene expression, endometrium immune response and stimulation of endometrium decidualization. So, it seems that the amount of vitamin D in follicular fluids (FF) may has an association with ART success.
Materials and Methods: In a prospective study, 80 infertile female candidates for IVF/ ICSI were enrolled. Blood samples (on the day of human chorionic gonadotropin) and follicular fluids were taken, and then levels of serum estradiol and follicular fluids 25-OH vitamin D were measured. Also clinical characteristics of patients (duration of infertility, causes of infertility, and menstrual status), number and quality of oocytes, number of fertilized oocytes, estradiol levels, and clinical pregnancy were evaluated.
Results: Concentration of FF 25-OH vitamin D in pregnant women was significantly higher than non-pregnant women (p=0.007) but there were no significant differences in age, body mass index (BMI), duration of infertility, menstrual status, number of oocytes, oocytes quality, number of fertilized oocytes and serum estradiol levels between the two groups (p>0.05). Statistically positive correlation was found between 25-OH vitamin D levels with patient age and implantation rate (r=0.264, p=0.018 and r=0.301, p=0.007 respectively).
Conclusion: The obtained results suggest that vitamin D without affecting the number and quality of oocytes can independently improve implantation rate and IVF outcome.
Key words: Embryo implantation, Vitamin D, IVF outcome.
P-21
The protective effect of N-acetyl-L-cysteine on spermatogenesis and sperm characteristics in mice following exposure to Para-Nonylphenol
Malmir M, Soleimani Mehranjani M, Naderi Noreini S.
Faculty of Science, Arak Univercity of Medical Scinecs, Arak, Iran.
Email: mehhdi@yahoo.com
Introduction: Para-Nonylphenol (p-NP) is an environmental contaminant with wide industrial applications which causes oxidative stress in different organs such as the reproductive system. The purpose of this study was to investigate the protective effect of N-acetyl-L-cysteine (NAC), as a powerful antioxidant, on spermatogenesis indices, sperm parameters, chromatin quality and tail length following treatment with para-Nonylphenol in adult mice.
Materials and Methods: 24 adult male NMRI mice (32±4 gr) were divided randomly into 4 groups (n=6), control, NAC (150 mg/kg/day), p-NP (250 mg/kg/day) and p-NP+NAC, and they were treated orally for 35 days. By the end of the treatment, mice were weighed and sacrificed, Their right testis was also weighed then the left caudal epididymis was cut in Ham's F10. Released spermatozoa were used to analyze the motility, viability and the abnormalities of the sperm. The sperm tail length was estimated by stereological methods. Sperm chromatin quality was also assessed by nuclear staining using acridine orange and aniline blue dyes.In continue, the right testis were taken out, fixed, sectioned, processed and stained using heidenhain azan method. Spermatogenesis indices including the tubular differentiation index (TDI), Sertoli cell index (SCI) spermatogenesis index (SI), meiotic index (MI) and repopulation index (RI) were studied. Data were statistically analyzed using one way ANOVA and means were considered significantly different at p<0.05.
Results: A significant decrease in the spermatogenesis indices, sperm motility, viability, sperm tail length and the number of sperms with normal morphology was observed in the p-NP group compared with the control (p<0.002), while no significant change was found in the sperm chromatin quality. The above parameters significantly increased in the p-NP+NAC group compared to the p-NP treated ones (p<0.01).
Conclusion: N-acetylc-L-ysteine, as an antioxidant, can prevent the adverse effects of para-Nonylphenol exposure on spermatogenesis indexes and sperm parameters in mice.
Key words: Para-Nonylphenol, N-acetyl-L-cysteine, Spermatogenesis indices, Sperm parameters.
P-22
The effect of vitamin C on human vitrified sperm parameters in normozoospermic men: raw semen and washed semen
Mangoli E1, Anvari M2. Talebi AR3, Taheri F1, Rahiminia T1, Vatanparast M1.
Introduction: Vitamin C is a common component in seminal fluid. It can improve sperm parameters as an antioxidant. The objective was to evaluate the effect of Vitamin C on human vitrified sperm parameters in normozospermia men.
Materials and Methods: Semen samples were collected from 40 normozoospermic samples and divided into 4 groups. Group 1, raw semen was vitrified. Group 2, semen processed by swim up method and then vitrified. Group 3, vitamin C (600 µm) was added to raw semen and then vitrified. Group 4, vitamin C (600 µm) was added to prepared spermatozoa and then vitrified. The semen analysis was performed according to WHO criteria before and after vitrification.
Results: In this study data showed that progressive motility and immotile spermatozoa after vitrification were significantly different between groups (p=0.014, 0.005 respectively). All groups were compared, and the results revealed that progressive motility and immotile spermatozoa after vitrification were statistically significant in washed semen+vitamin C group compared with wash semen and raw semen groups (p=0.05, 0.01 for progressive motility and p=0.04, 0.007 for immotile spermatozoa respectively).
Conclusion: Adding vitamin C, as an antioxidant, to washed semen has shown beneficial effect on sperm motility after vitrification.
Key words: Sperm parameters, Vitamin C, Vitrification.
P-23
Review of fertility diet and its effect on infertility among women from the viewpoint of Iranian traditional medicine in comparison with modern medicine
Tork Zahrani Sh1, Mirahi A1, Tansaz M2, Alaeddini F3, Hekmatdoost A4.
Introduction: In the recent years the infertility diagnosis and treatment methods have undergone a radical change and development but still, the success rate of modern treatments like in vitro fertilization (IVF) even in the best centers is between 30-40%.
Materials and Methods: The available texts on the Iranian traditional medicine were reviewed. Besides, the relationship between fertility diet factors and infertility among women was investigated through reviewing databases on the world-wide web.
Results: Iranian traditional medicine investigates the relationship between nutrition and infertility from a perspective postulating that complying with fertility diet principles helps maintain healthiness and improve fertility. From this view, body members have their special temperaments; if balanced, body members function normally. Any kind of abnormal temperament happened to the semen or uterus results in infertility or hardship in fertility. These are seen as disorders in sanguine, choleric, melancholic, and phlegmatic temperaments. It should be noted that the bad temperaments are mainly caused by not following the health and fertility diet.
Conclusion: The different viewpoint of the Iranian traditional medicine towards the relationship between infertility and nutrition and its focus on the nature of food and nature of a person could be of interest to researchers in the field and if approved, help to prevent infertility or treat infertile couples.
Key words: Infertility, Iranian traditional medicine, Infertility diet.
P-24
The in vitro fertilization outcome and luteal phase GnRH antagonist administration
Eftekhar M1, Miraj S1, 2.
Introduction: Genital tissues (ovary, endometrium and placenta) express GnRH receptors. GnRH plays essential roles in embryo implantation, invasion of trophoblastic tissue and steroid synthesis in the placenta. In IVF-ICSI cycles, the use of GnRH antagonists is limited to the last days of ovulation. The aim of this study was to evaluate the effects of GnRH antagonist at pharmacological doses given in the early implantation period on pregnancy.
Materials and Methods: This retrospective study was performed in Yazd Research and Clinical Center for Infertility, 2014-2015. Women under 40 years old, with >20 follicles (>11mm) and risk of OHSS were included. Participants with history of endometriosis, hysteroscopy and history RIF were excluded. The treatment for all of participants was antagonist protocol. Twenty seven patients did not receive Cetrotide in luteal phase, and 67 patients received Cetrotide. Pregnancy outcomes were assessed based on chemical and clinical pregnancy.
Results: Totally 94 patients were included. The mean age of participants was 28.40±4.25 years old. There were no differences in ART and demographic characteristics (p>0.05). The most frequent causes of infertility were: male factor (45.2%) and polycystic ovary syndrome (19.4%). The pregnancy outcome was not significantly different between Cetrotide and non Cetrotide group (p=0.224).
Conclusion: The present study proposed that luteal phase GnRH antagonist administration does not influence the chance of successful pregnancy outcome. The incidence of chemical and clinical pregnancy in two groups was not significantly different.
Key words: GnRH antagonist, Pregnancy outcome, IVF.
P-25
Hazardous effect of acrylamide on development landmarks in rat offspring and the role of Glycyrrhiza Glabra
Mirgalili A1, Yadegari M1, Talebi AR2, Dortaj H1.
Introduction: Acrylamide (AA) is a chemical substance used mainly in certain industrial process. Monomer of Acrylamide formed in food stuffs containing carbohydrates during high temperature cooking.
Materials and Methods: 24 pregnant rats were selected for this study. The pups of these mothers divided into four groups: group A:(control group); group B: (ACR administration 10 mg/kg/day orally); group C: (GG administration 150 mg/kg/day orally); group D: (ACR+GG). At day 21 the pups were evaluated for developmental study. The results were analyzed by SPSS software (15) and p<0.05 was significant.
Results: The result demonstrate that eye opening and fur development appeared occurred slightly later in AA administration group.
Conclusion: These results showed that AA treatments induce delay in developing offspring and GG as an antioxidant reduce these changes.
Key words: Acrylamide, Glycyrrhiza glabra, Eye opening, Fur appearance, Offspring.
P-26
The effect of long-term exercise via TNF-α level on the type-1 diabetic rats in testis tissues
Moghaddami Z1, Sheikhzadeh Hesari F1, Banan khojasteh SM1, Hemmati AR2, Dastranj A3.
Introduction: Approximately 90% of diabetes patients often show sexual abnormalities and impotence infertility. Damages caused by hyperglycemia and oxidative conditions result in inflammatory mediators increasing such as cytokines in tissues of sex organs. The TNF-α is the first locally produced cytokines in chronic inflammation pathway. TNF-α level in diabetics blood is higher than normal people that probably histological studies of TNF-α could determine source of this increasing. Also exercise can reduce complications of diabetes especially in tissue by reducing of oxidative elements and establishing of Hypoglycemia.
Materials and Methods: In this study, 40 male rats weighing 200-250 gr were used and randomly were classified into four groups; each group consist 10 rats (control, diabetic, healthy with period of 60-day exercise, diabetic with period of 60-day exercise). Treadmill exercise daily was 1 hr with 22 m/min speed. Diabetes was induced by injection intraperitoneal of streptozotocin with the amount of 60 mg/kg. After expelling of the testis under general anesthesia, samples were homogenized and TNF-α protein levels were measured according to ELISA kit (special rat TNF-α).
Results: The level of TNF-α showed no significant changes among diabetic groups in testicular tissue compare to control groups. Also, exercise didn’t have any negative effects on the level of TNF-α in exercise control group compare with control group. Therefore, we didn’t show any significant change of TNF-α level between our groups.
Conclusion: In this study, the period of disease time may be is not enough to determine the level of TNF-α via exercise treatment in testis tissues. It seems that duration of diabetic developing, severity and type of exercise could be many important reasons to change inflammatory mediators.
Key words: Cytokine, Hyperglycemia, ELISA Kit, Sexual dysfunction.
P-27
Evaluation of progesterone and estradiol levels after GnRH agonist administration in the adult female rat
Mohammadbeigi H1, Peirouvi T1, Rasmi Y2.
Introduction: Gonadotropin-releasing hormone (GnRH) is secreted from the hypothalamus in a pulsatile manner and stimulates the synthesis and release of gonadotropins, LH and FSH, via GnRH receptor located in adenohypophysis. These gonadotropins, regulate various gonadal functions such as folliculogenesis and steroidogenesis. Various data are reported of GnRH agonists effects on ovary steroidogenesis.
Materials and Methods: 24 female adult Wistar rats were divided into three groups. In the first study group, 300 µg/kg buserelin, in the second study group, 600 µg/kg buserelin and in the control group, the same amount of salin was injected subcutaneous for 5 days. 30 days after the first injection, blood samples were collected from the heart, centrifuged and plasma was isolated from blood. Plasma esteradiol and progesterone levels were measured by ELISA.
Results: The findings reveal a significant incereas in plasma level of estradiol in the second study group (165±104.89 pg/ml) in comparison with control group (55.47±50.32 pg/ml) and in the second study group compared with the first study group (47.28±32.95 pg/ml) (p<0.05). But no significant differences were observed in plasma level of progesterone in the three groups (p>0.05).
Conclusion: Short-term administration of high doses buserelin increase plasma estradiol level, while has no effect on the plasma progesterone level.
Key words: Buserelin, Progesterone, Estradiol, Adult rat.
P-28
A comparative study on the effect of face-to-face or group education during the pregnancy period on sexual function of the couples under coverage of selected clinics in Isfahan in 2013
Mohammadi M1, Khani MB1, Bahadoran P2.
Introduction: Pregnancy can conflict with sexual function that can be affected by physical and psychological changes during Pregnancy.
Materials and Methods: In this quasi-experimental pre-post-test study, 64 couples with pregnant women were selected and randomized in two groups in Isfahan. The data were collected via the triangulation of FSFI, BFSI and demographic characteristics questionnaires. SPSS -18 was used to analyze the data by descriptive and inferential statistics.
Results: No significant difference was found in the demographic characteristics between two groups. Education was effective on sexual function in two groups of women (p<0.001), but no significant difference was found between two groups (p=0.61). Also education was effective on sexual function of the men in both groups (p<0.001), and there was a significant difference between the two groups (p=0.003). Meanwhile, there was no significant difference between couples regarding the education (p=0.104).
Conclusion: The results of the study showed that type of education plays a role in improvement of sexual function in pregnancy. In addition, sex education is effective on prevention of sexual disorders in pregnancy. Therefore, having a special approach toward sex education classes during pregnancy is inevitable for the health providers, particularly midwifery professionals.
Key words: Group education, Face to face education, Sexual function, Pregnancy, Iran.
P-29
Assisted reproductive technology outcomes in couples with hepatitis virus infection
Mohsenzadeh M, Omidi M, Nabi A, Khalili MA.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, iran.
Email: mohsenzade@sums.ac.ir
Introduction: Currently, many hepatitis positive infertile couples attend assisted reproductive technology (ART) program, so there is concern as this viral infection may be deleterious to human fertility. Some have suggested that hepatitis virus have an adverse effect on pregnancy outcomes.
Materials and Methods: The study was conducted in two groups of patients: A) seropositive for hepatitis B virus (HBV) and hepatitis C virus (HCV) (n=46); and B) patients lacking these viruses as a control group (n=32). Patients included couples seeking ART from January 2010 to October 2014. ART cycle characteristics and clinical outcomes were assessed between two groups.
Results: Demographic characteristics and age were similar between two groups. The mean±SD age was 30.19±5.54 and 34.31±6.86 years in female and male, respectively. Regarding to oocyte number and embryo score, there was no significant differences between groups A and B. The rate of pregnancy was, however, significantly higher in group B in comparison with A (p=0.03).
Conclusion: Although, the data on embryo formation was similar in both groups of infertiles, but the pregnancy outcomes were noticeably reduced in infertile patients with hepatitis virus infections.
Key words: Hepatitis virus infection, ART, Pregnancy outcome.
P-30
The influence of the Corpus Luteum (CL) on hormonal and biochemical metabolites composition of follicular fluid from different sized follicles and their relationship to serum concentrations in dairy cows
Moradi kor N.
Research Center of Physiology, Faculty of Medicine, Semnan University of Medical Sciences, Semnan, Iran.
Email: moradikor@semums.ac.ir
Introduction: Metabolic changes in blood serum may be rejected in the biochemical composition of follicular fuid (FF) and could indirectly influence oocyte quality. In addition, the levels of hormonal and biochemical metabolites in the FF were related to follicular size and interestingly to the presence or absence of a corpus luteum. The purpose of this study were to examine the influence of the corpus luteum on hormonal and biochemical metabolites composition of follicular fluid (FF) harvested from different sized follicles and their relationship to blood serum in dairy cows.
Materials and Methods: Ovaries were recovered from 30 female adult cows (Holstein Friesian) 4-7 years of age with clinically normal reproductive tracts after slaughtering. Blood samples were collected from the jugular vein before slaughter from each cow. The stage of the cycle in the cows slaughtered was diestrus determined post mortem. The ovaries collected per cow were classified with corpus luteum (CL+) and without corpus luteum (CL-). Visible follicles on the surface of the ovaries were classified, based on their diameter, into (I) small (3-5 mm), (II) medium (6-9 mm) and (III) large (10-20 mm) categories. Follicular fluid was aspirated from different sized follicles in CL+ and CL- ovaries. Serum and FF samples were analyzed for hormones (estradiol-17β, progesterone, testosterone, T3 and T4) and biochemical metabolites (glucose, cholesterol, triglyceride, total protein, albumin and globulin).
Results: Results showed that the FF concentration of estradiol-17β, progesterone and testosterone in different size follicles categories (small, medium and large follicles in CL+ and CL- ovaries) were significantly higher (p˃0.05) when compared with the serum. The FF concentration of estradiol-17β, testosterone, glucose and cholesterol in same follicle size categories of CL- ovaries were significantly higher (p˃0.05) when compared with CL+ ovaries. In the present study, the serum concentration of glucose, cholesterol and triglyceride were significantly higher (p˃0.05) when compared with the fuid from different sized follicles categories in CL+ and CL- ovaries. The differences between follicle size categories in CL+ ovaries were only significant for concentration of estradiol-17β and in CL- ovaries were significant for concentrations of estradiol-17β, glucose and triglyceride.
Conclusion: According to the results of the present study, the levels of the biochemical metabolites in the FF were related to follicular size and interestingly to the presence or absence of corpus luteum.
Key words: Dairy cow, Follicular fluid, Corpus luteum, Hormone, Metabolite.
P-31
Health related quality of life in women with polycystic ovary syndrome
Moridi M1, Rahebi SM1, Seyf Zadeh Z2.
Introduction: Polycystic ovary syndrome (PCOS) is associated with symptoms that affect psychological wellbeing and health-related quality of life (HRQoL).
Materials and Methods: This was a retrospective case-control study which was conducted on 116 women from April-August 2014 in Rasht, Iran. Cases were 60 newly diagnosed women with PCOS, according to the Rotterdam criteria while the controls were 60 healthy women and groups were matched on sociodemographics characteristics. Data needed to determine health-related quality of life were collected via convenience sampling using modified PCOS quality-of-life questionnaire (MPCOSQ) that contains 6 subscales: emotional disturbances, hirsutism, infertility, weight, menstrual and acne. Data collected were analyzed using statistical T-test, chi square and Kolmogorov-Smirnov.
Results: Finding demonstrated that groups were matched in age, body mass index, marital status, occupation, educational status and socioeconomic status. The mean HRQoL score was significantly lower among women with PCOS (20.96±7.4) than among controls (29.8±4.5) (p<0.001). The mean scores of all subscales in cases (32.01±13.56 for emotional disturbances, 23.34±9.35 for hirsutism, 12.02±6.6 for infertility, 21.25±10.16 for weight, 16.89±6.71 for menstrual and 20.27±7.25 for acne) were significantly lower than controls (47.53±8.28 for emotional disturbances, 31.81±6.68 for hirsutism, 25.83±4.19 for infertility, 20.23±7.70 for weight, 21.15±7.27 for menstrual and 24.26±6.17 for acne) (p<0.001).
Conclusion: According to the results of this study, the average of HRQoL and all the six subscales of MPCOSQ in the case group was significantly lower than in the control group. The psychological implications of PCOS are easily underestimated and have been largely ignored. Clinicians has a pivotal role in recognizing these concerns and implementing therapy to improve quality of life in women with PCOS.
Key words: Polycystic ovarian syndrom, Quality of life, Infertility.
P-32
Counseling during pregnancy and women with precedent infertility
Naeimi N.
Department of Biology, Faculty of Science, University of Sistan and Baluchestan, Zahedan, Iran.
Email: nnaeimi@rocketmail.com
Introduction: Delivery is physiological phenomenon and among the beautiful events in the life of every woman and yet stressful reality. In the present era considering the increasing of caesarean sections, it require using consulting services for informed, correct decisions.
Materials and Methods: In this descriptive-analytical study, 271 pregnant women, completed a researcher-made questionnaire including, demographic characteristics (age and precedent of infertility) and kind and rates of counseling. To analyze the obtained data, chi-square test was used through applying SPSS19 software.
Results: Our results showed that 7 pregnant women (2.6%) had a precedent of infertility while 259 pregnant women had not (95.6%). In total 53 (19.2%) subject did consulting during pregnancy and 213 (80.8%) did not. From these, 6 women with precedent infertility (85.7%) and 47 (14.3%) without, perform counseling during pregnancy. There was a significant relationship between counseling during pregnancy and fertility (p<0.05).
Conclusion: The results showed that women with precedent infertility because of the importance of pregnancy and because of the history of information which were obtained during the course of treatment were more acquainted with the role of consultant, and therefor used consultant services mostly during pregnancy. Ferthermore, considering to the high prevalence of elective cesarean, counseling during pregnancy can informe the pregnant women about the advantage of normal delivery, causing the reduction in cesarean section prevalance.
Key words: Counseling, Precedent infertility, Pregnancy termination.
P-33
Overweight and obesity of patients: Blind spot in assisted reproductive technology treatment
Naghibzadeh M.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: naghib330@yahoo.com
Introduction: The effects of lifestyle on health were investigated via several studies. But, the impacts of overweight and obesity on fertility and reproductive system are poorly understood. Overweight and obesity may reduce fertility rate via reducing the IVF success rate in both women and men undergoing treatment via assisted reproductive technology (ART). Improve lifestyle, including physical exercise and weight loss program, may improve fertility and ART outcome.
Materials and Methods: The results of the related epidemiological and randomised control trials (RTCs) sited on Pubmed and ISI database were selected as references.
Results: The results indicated that the patients with a BMI above 30 have about 68% less chance to have a live birth following their first ART cycle compared with women with a BMI less than 30. Also, overweight and obesity of the patients were associated with both lower implantation and more risk for cycle cancellation. A correlation between obesity and the risk of spontaneous abortion (22% increased risk) was observed in obese women compared with normal weight women. The body composition, specific fat mass, was adversely associated with the success of the IVF procedure. Finally, some studies indicate that weight loss is associated with a higher spontaneous pregnancy rate.
Conclusion: Lifestyle behaviours may have a significant impact on pregnancy rates in women with infertility problems. The results support the clinical recommendation of advising overweight and/ or obese women to lose weight prior to ART. Nevertheless, it seems that the prospective randomized controlled trials are required to prove efficient evidence-based guidelines for weight loss interventions in overweight and/ or obese women before the ART procedure.
Key words: Lifestyle, Obesity, Fertility, ART.
P-34
Role of nitric oxide in the developmental process of ovarian follicles in pregnant rats
Naseri Sh, Khanehzad M, Noori Moogehi SMH.
Department of Histology, Faculty of Medicine, Tehran University of Medical Sciences (TUMS), Tehran, Iran.
Email: naserishafighe@yahoo.com
Introduction: Nitric Oxide (NO)- as one of the smallest active products, involved in control of a number of physiological processes including cell growth, apoptosis, regulation of blood pressure, defense mechanism and especially in the reproductive process.
Materials and Methods: We used 40 Wistar pregnant rats between 200 and 250 grams in weight and aged eight weeks. Based on observation of vaginal plug, pregnant mice were divided into five groups. The first group was received 2 mg/kg normal saline and the others were received respectively 200 mg/kg L-Arginine, 20 mg/kg L-NAME and a mixture of the same doses of L-Arginine and L-NAME on 3, 4 and 5th gestational days via intraperitoneal. The control group did not received any injection. Ovaries were removed on 18th gestational days, and after fixation and tissue preparation via staining by the routine H&E method, studied by Light microscopy.
Results: Comparing the groups using ANOVA, there was a significant difference in reducing the number of primary follicles and increasing atretic follicles in the L-Arginine group in comparision with the other groups. The histological changes were also observed in the L-Arginine group.
Conclusion: The study results showed that nitric oxide during pregnancy has damaging effects on ovaries and is recommended to be used with caution during pregnancy.
Key words: Ovarian follicles, Nitric oxide, L-Arginine, L-NAME.
P-35
Comparison of ovarian stimulation protocols base on AMH level in patients undergoing intracytoplasmic sperm injection
Nikmard F1, Hosseini E1, Bakhtiyari M1, Ashrafi M2, Aflatoonian A3, Aflatoonian B4, Aflatoonian R2.
Introduction: Controlled ovarian hyperstimulation (COH) plays important role in reproductive medicine because selection of appropriate ovarian stimulation strategy can improve assisted reproductive technology outcomes. Antimullerian hormone (AMH) is a predictor of ovarian response, which can help to select the best treatment strategies in women undergoing agonist and antagonist protocols to optimize safety and clinical pregnancy rates.
Materials and Methods: This study is a retrospective study conducted in a private assisted reproductive unite and a total of 243 patients with tubal factor infertility are selected.
Results: In both of GnRH agonist and antagonist protocol, with increasing of AMH level, oocyte and good embryo number is increased. On the other hand in AMH˂1.1, ˃ 2.8 ng/ml levels, GnRH agonist leads to higher oocyte and embryo number that is significant. On the contrary, pregnancy rate with increasing of AMH level is not increased and the highest rate of pregnancy is observed in AMH 1.1-2.8 ng/ml levels that it observed with antagonist protocol.
Conclusion: Based on AMH levels we can predict assisted reproductive outcomes. In three range of AMH levels, GnRH agonist protocol can lead to better results. In the women with poor prognosis and low and high levels of AMH, it should focus on improving results with increasing of endometrial receptivity or embryo quality.
Key words: Antimullerian hormone, Ovarian response, ovarian stimulation protocols.
P-36
Prenatal exposure to a single dose of testosterone leads to appearance of polycystic ovary syndrome in female rat’s offspring in adulthood
Noroozzadeh M1, Ramezani Tehrani F1, Zahediasl S2, Piryaei A3, Azizi F2.
Introduction: Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women, affecting 8-12% of reproductive-aged women. PCOS is associated with reproductive and metabolic disorders, including infertility, hyperandrogenism, luteinizing hormone hypersecretion, polycystic ovaries, insulin resistance and dyslipidaemia, with an increased risk of cardiovascular disease and type 2 diabetes in those affected. PCOS is known as a genetic disease. Beside genetic factors, environmental factors may contribute to appearance of this syndrome.
Materials and Methods: Pregnant rats were divided into two groups, experimental and control groups (n=10 in each group). Experimental group were subcutaneously injected with 5 mg free testosterone on gestational day 20 and control rats received only solvent at the same time. The female offspring of these mothers were examined for the functioning of their reproductive system in adulthood.
Results: Levels of testosterone and luteinizing hormone and the luteinizing hormone/follicle-stimulating hormone ratio were increased in prenatally androgenized offspring compared with control rats (p<0.05). The numbers of preantral and antral follicles in the ovaries of prenatally androgenized offspring were also increased compared with control rats (p=0.07 and p<0.01, respectively). The number of corpora lutea was decreased in prenatally androgenized offspring compared with control rats. Cystic follicles were observed in the ovaries of prenatally androgenized offspring.
Conclusion: Prenatal exposure to a single dose of testosterone during the critical period of fetal development leads to appearance of PCOS phenotype in female rats with minimal morphological disorders in their reproductive system in adulthood. Production of a functional rat model that resembles many features of PCOS may contribute to a better understanding of this syndrome.
Key words: Fetal life, Testosterone, Polycystic ovary syndrome, Rat.
P-37
Advanced paternal age does not influence the outcomes of ART cycles
Omidi M, Halvaei I, Khalili MA, Ghazali Sh, Tahajjodi S, Sabeti P.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: omidi.marjan@ymail.com
Introduction: The reports in assisted reproductive technology (ART) cycles noted that the fertilization rates were significantly decreased in men over 50 years of age. However, data have shown that there was no adequate substantiation to prove the effects of paternal age on fertility outcomes.
Materials and Methods: In a retrospective study, the outcome of intracytoplasmic sperm injection (ICSI) in ART cycles in two groups of patients was studied. Patients regarding etiology of infertility were divided into two groups: A) male factor infertility (n=47), and B) female factor infertility (n=16). Sperm parameters, ART cycle characteristics and clinical outcomes were assessed between two groups.
Results: There were no significant differences for rates of sperm count and morphology between two groups, but rate of spermatozoa with progressive motility were higher in group B in comparison with A (p=0.002). The rates of high quality embryos, pregnancy and live birth showed no significant differences between the groups (p=0.4, p=0.4, p=0.2, respectively).
Conclusion: It appears that the paternal age has no detrimental effect on rates of pregnancy and live birth in ART program.
Key words: Paternal age, ART, Live birth.
P-38
Effect of non-polar extract of Phaleria macrocarpa (Mahkota dewa) on serum testosterone level and libido behavior in male rats
Aziz Dollah M1, Parhizkar S2, Ferdaus Binti N1.
Introduction: Phaleria macrocarpa (PM) has been claimed to overcome male fertility problems related to reduction of testosterone level.
Materials and Methods: Non-polar hexane extract of PM was prepared using soxhlet extraction technique, dried to powder using rotary evaporator and been kept for later use. In total 30 male adult Spraque Dawley rats weight about 250 gr were divided into five groups. For libido study, 30 female rats weight about 250 gr each were used for the purpose of mounting latency and mounting frequency study. The male rats were kept in individual cage while for female rats, there were five rats for one cage. Three groups of rats were given three different concentration of PM non-polar extract [High dose (60 mg/kg), Medium dose (12 mg/kg) and Low dose (6 mg/kg)]. One group was given tween 20 solution (negative control) and another group was given commercial testosterone hormone (positive control) orally for seven weeks. At the end of experiment period each male rat was introduced to one female rat to determine the libido behavior. Blood sample was then collected using cardiac puncture and the testosterone level was determined by radioimmunoassay kit (TESTO-CTK P3093).
Results: Administration of non-polar hexane extract of PM showed no significant changed for the serum testosterone level and mounting latency of the rats. While, there was a significant change (p<0.05) in the mounting frequency in different groups; 6, 14, 14, 4 and 7 times for the negative control, low dose, medium dose, high dose, and positive control respectively.
Conclusion: PM non-polar hexane extract did not show a potential value as an alternative way to improve the sexual strength. However it showed a potential for the improvement of the mounting frequency.
Key words: Phaleria macrocarpa (PM), Testosterone level, Libido behavior.
P-39
Effect of vitamin C on sperm parameters and serum malondialdehyde levels in mice fallowing treatment with sodium arsenite
Pirozmanesh H, Soleimani Mehranjani M, Naderi Noreini S.
Faculty of Science, Arak University of Medical ASciences, Arak, Iran.
Email: hp457@yahoo.com
Introduction: Arsenic as an environmental toxicant is able to exert malformation in male reproductive system by inducing oxidative stress. Ascorbic acid with potent antioxidant property is able to restrict oxidative stress.
Materials and Methods: In this experimental study, 24 NMRI mice were divided into four groups (n=6): control, sodium arsenite (7 mg/kg), ascorbic acid (150 mg/kg) and ascorbic acid + sodium arsenite. Oral treatment was performed for five weeks. At the end, left cauda epididymis was removed and used to analyze sperm viability, morphology, motility and sperm tail length. Serum MDA levels, as lipid peroxidation index, was measured by spectrophotometeric method. Data were analyzed using one way ANOVA and means difference was considered significant at p<0.05.
Results: The mean of sperm viability, morphology, motility and sperm tail length significantly decreased in sodium arsenite group compared to control (p<0.04). A significant increase in the serum MDA levels was found in sodium arsenite group compared to control (p<0.05). In ascorbic acid+ sodium arsenite group, a significantly reversed in adverse effect of sodium arsenite on these parameters was observed when compared with sodium arsenite group (p<0.02). In addition sperm viability, motility and sperm tail length was significantly increased in the mice treated with ascorbic acid alone in comparing to the control ones (p<0.001). Also, a significant reduction in MDA levels was found in ascorbic acid group compared with control group (p<0.001).
Conclusion: Ascorbic acid could compensate the adverse effects of sodium arsenite on viability, morphology, motility, sperm tail length and serum MDA levels in adult mice. Hence, this study suggests that ascorbic acid may improve the male fertility by improving sperm parameters.
Key words: Sodium arsenite, Ascorbic acid, Viability, Motility, Sperm tail length.
P-40
The predictors of spiritual growth and interpersonal relations in infertile couples referring to the Infertility Center, Al-zahra Hospital, Tabriz, Iran.
Rahimi M, Sehhatie Shafa'I F, Mirghaforvand M.
Tabriz University of Medical Sciences, Tabriz, Iran.
Email: marieh.rahimi@yahoo.com
Introduction: In all cultures, involuntary childlessness is recognized as a crisis that has the potential to threaten the stability of individuals, relationships, and communities. Every society has culturally approved solutions to infertility involving, either alone or together with alterations of social relationships (e.g., divorce or adoption), spiritual intercession (e.g., prayer or pilgrimage to spiritually powerful sites), or medical interventions.
Materials and Methods: This study is a descriptive cross-sectional study on 322 infertile couples referring to the Infertility Center at Al-zahra Hospital in Tabriz. Samples were selected through simple random sampling method. Questionnaires used in the study were demographic data questionnaire and Lifestyle Profile II (HPLP-II) questionnaire with 52 questions. The multivariate linear regression analysis method was used for defining the social-individual predictors of Spiritual Growth and Interpersonal relations. Data were analyzed by SPSS Win/13.
Results: The findings showed that the mean (SD) score of Spiritual Growth and Interpersonal relations in couples was 2.6 (0.5) out of 1-3 grade range. The variable of job, family member, history of contraception, reason of infertility, and education level were the predictors of Spiritual Growth and Interpersonal relations.
Conclusion: According to the results of the study, the mean score of Spiritual Growth and Interpersonal relations in infertile couples is average, therefore we should focus on predictive variables for recovering the infertile couples’ Health-promoting behaviors status.
Key words: Health-promoting behaviors, Spiritual Growth, Interpersonal relations.
P-41
Comparing in-vitro maturation of oocytes in presence of mature or immature cumulus cells in mice
Rahiminia T, Faramarzi A, Khoradmehr A, Khalili MA.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: tahere_1980@yahoo.com
Introduction: Cumulus cells (CCs) are somatic cells which are coupled with oocyte during maturation. During process of maturation, the secretions by CCs influence oocyte to maintain the functional competence.
Materials and Methods: Superovulated mice were killed and ampulla was ruptured to release cumulus oocyte complex (COCs) in IVM culture media. COCs were incubated for 30 min before removing oocytes. Then, denuded mature oocytes were removed and remaining mature CCs were collected for culturing germinal vesicles (GVs) for exp II. After dissecting ovaries, denuded GVs were considered as control group and put in IVM medium. In exp I, collected GVs with intact immature CCs were put in IVM medium and observed for maturation after 24 and 48 hr. In exp II, GVs were cultured with mature CCs and maturation was checked as mentioned for exp I group.
Results: The rate of maturation was (77.36±14.4) in control group. In exp I and exp II, the rate of maturation was (91.32±22.5) and (63.33±7.4) respectively. Difference in maturation rate was significant between two groups of exp I and exp II (p=0.04) at 24 hr. However, maturation rate did not increase after 24 hr. The average of maturation rate in metaphase I (MI) oocytes was as low as about 15% in different groups at 24 hr and did not increase by 48 hr. Degeneration rate was increased from time 0-48 hr in exp II more than this rate in the other two groups.
Conclusion: Presence of mature CC did not improve maturation of GVs comparing to exp I with immature CCs, or control group as conventional IVM.
Key words: In vitro maturation, Cumulus cells, Mice.
P-42
Investigating the effect of culture pH, on Chinese hamster ovary (CHO) cells growth and recombinant human chorionic gonadotropin (hCG), expression
Rahnama M, Sanati MH, Amiri Yekta A, Gourabi H.
Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: mhsanati@yahoo.com
Introduction: The demand for production of recombinant therapeutic proteins by mammalian cell lines, such as Chinese hamster ovary (CHO) cells, continues to grow. Significant achievements in process optimization including development of cell culture strategies for large-scale, cost effective production have been made. One of the key parameters, that has effect on mammalian cell growth, metabolism and productivity, is pH. Human chorionic gonadotropin (hCG), is a member of pituitary glycoprotein hormone family, which has an important role in regulating human reproductive functions and is widely used in assisted reproductive technologies (ART).
Materials and Methods: Equal number of rCHO cells were cultured in four DMEM media with different pH ranging from 6.8-7.7 (6.8, 7, 7.3 and 7.7). The cells were harvested at confluence and viable cell concentration was determined by trypan blue exclusion. The rhCG production was assessed using SDS-PAGE, Bradford and Western blotting techniques. ImageJ software was used for quantitating Western blotting results.
Results: Results demonstrated that maximum viable cell concentration was at pH=7. Bradford assay showed that total protein concentration reached its maximum level at pH=7 and its minimum at pH=6.8. hCG expression was confirmed by SDS-PAGE and western blotting methods. According to ImageJ analysis, maximum hCG expression level was found at pH=7, and minimun expression at pH=7.7 but no significant difference was found at pH=7.7 and pH=6.8. Overall, pH=7 was assessed as optimum pH for culturing rCHO cells.
Conclusion: Several strategies including culture condition optimization, have developed to improve productivity of a recombinant cell line. Findings suggest that optimizing simple parameters such as culture pH, had the potential to increase the viability and productivity of recombinant CHO cells.
Key words: Chinese hamster ovary (CHO) cell, Optimization, pH, Human chorionic gonadotropin (hCG), Assisted reproductive technology (ART).
P-43
Noscapine induced cell death in human endometriotic epithelial cells
Rashidi Z1, Khazaei M2, Khazaei MR2.
Introduction: Endometriosis is the presence of a functional endometrial tissue outside the uterine cavity and defines as cancer like model. Noscapine is a safe cough suppressant which has been introduced as cancer suppressor.
Materials and Methods: Human endometriotic biopsies (n=7) were digested by enzymatic method (collagenase, 2 mg/ml). Epithelial glands were collected by sequential filtration through nylon meshes (70 and 40 µm). The cells were divided to five groups: control and 10, 25, 50 and 100µM concentration of noscapine and cultured for 24, 48 and 72 hr.Viability of cells was assessed by MTT assay; cell morphological analyses with Acridine orange (AO)-ethidium bromide (EB) double staining also cell death was assessed by TUNEL assay.
Results: Viability of endometrial epithelial cells were decreased in 10, 25, 50 and 100 µM noscapine concentration (84.1%, 80.8%, 72.7%, 67.1%) compared to control group (91.0%) in 48 hr respectively. Cell death increased in high concentrations noscapine and were increased TUNEL positive cells in 10, 25, 50 and 100 µm noscapine concentration (9.04%, 13.07%, 15.15% and 17.82%) respectively compared to control group (4.96%) in 48 hr (p<0.05).
Conclusion: Noscapineas inducer cell death decreased endometriotic epithelial cells viability and decreased apoptotic index in dose dependent manner. It can suggest for endometriosis treatment.
Key words: Apoptosis, Epithelial Cell, Endometriosis, Noscapine.
P-44
The effect of chronic prenatal stress on insulin secretion from Langerhans isolated islets in male offspring rats
Rostamkhani F1, Zardooz H2, Shirvani H3.
Introduction: A large number of studies have reported associations between prenatal stress and offspring lifetime consequences. Chronic gestational stress alters maternal glucocorticids and subsequently disturbs intrauterine environment which may lead to metabolic disorders in the offspring.
Materials and Methods: In this study the effects of gestational 8 and 20 days foot-shock and psychological stress on body weight, plasma corticosterone, insulin, glucose, concentrations and also insulin secretion from isolated pancreatic islets of rats' offsprings were examined. Stress was induced by Communication Box twice a day (1 h/session) for 8 consecutive days beginning on E8 in 8-day stressed group and for 20 consecutive days beginning on E1 in 20-day stressed group.
Results: The results obtained from this investigation indicate that 8 and 20-day chronic foot-shock stress arises maternal plasma corticosterone concentration. Prenatal stress induces lower birth weight and body weight gain in offspring. Insulin secretion from isolated pancreatic islets of offspring in 8 and 20-day foot-shock stress groups in presence of 8.3 and 16.7 mM glucose increased as compared to the control and psychological groups. Furthermore, prenatal stressed offspring had significant elevation in plasma glucose concentration without marked alteration in plasma insulin and corticosterone concentrations.
Conclusion: These data suggest that prenatal stress could result in impaired glucose metabolism in the offspring which is independent of timing of the stress exposure.
Key words: Prenatal stress, Corticosterone, Insulin, Glucose, Langerhans isolated islets.
P-45
In vitro culture of vitrified mouse ovarian tissue derived preantral follicles in two and three dimensional systems
Sadr SZ1, Ebrahimi B1, Shahhoseini M2, Fatehi R1, Favaedi R2, Maroufizadeh S3.
Introduction: Cryopreservation is the best choice to preserve fertility in patients exposed to premature ovarian failure. Designing an appropriate system for in vitro follicle culture that resembles in vivo conditions is really valuable and could improve the usage of cryopreserved tissue.
Materials and Methods: Ovaries of 12-day-old female NMRI mice were exposed to EG and DMSO combination (7.5% and 15%) in two steps for 15 and 30 minutes respectively, then cryopreserved by needle immersed method (NIV). Afterwards, middle sized preantral follicles (110-130 µm) were mechanically isolated and were distributed into two different groups: two dimensional culture system (2D) and three dimensional culture system (3D; alginate encapsulation). Both groups were cultured for 12 days in α-MEM that supplemented with 10 mIU FSH, 1% ITS, 50 ng/ml activin A and 5% FBS. Finally, follicular morphology, survival, growth rate and also quantitative expression of oocyte maturation genes (Gdf9, Bmp15 and Bmp6) were studied on the first and last days of culture.
Results: At the end of culture period, although follicular morphology in 3D culture system was better preserved as compared to the 2D one, survival rate of cultured preantral follicles was significantly (p<0.05) lower than in 2D group. Expression analysis of oocyte maturation genes indicated a reduction process in both groups during 12 days of culture. In the last day of culture period, genes expression level in 3D culture system was significantly lower than 2D group.
Conclusion: Although the better survival rate was seen in 2D culture system, morphologic characteristics including antrum formation and follicle development was extremely better preserved in 3D culture system. Overall, as three dimensional culture system mimic in vivo conditions, so could be used as a more appropriate system for follicle culture.
Key words: Ovarian tissue, Preantral follicle, Two dimensional culture system, Three dimensional culture system, Vitrification.
P-46
Routine use of EmbryoGlue® as embryo transfer medium does not improve the ART outcomes
Safari S1, Razi MH1, Safari S2, Razi Y3.
Introduction: The assisted reproductive technology (ART) success rates have been significantly improved over last decades; however, embryo implantation still remains a major limiting factor. The composition of an embryo transfer (ET) medium is important for interaction between embryo and endometrium at the time of implantation. Some modifications to the embryo culture media are developed to mimic the in vivo conditions. One of the examples for that is supplementation of ET media with hyaluronan (HA, hyaluronic acid), a major glycosaminoglycan in uterine fluid, which assumed to improve the process of implantation.
Materials and Methods: A cohort of total 229 patients was retrospectively enrolled for the present study. They were subjected for ET on day 2 either in EmbryoGlue® (n=117) as study group or in conventional ET medium with low concentration of HA as control group (n=112).
Results: Patients in the both groups, in regards to the mean level of day 3 FSH, the etiology of infertility, the history of implantation failure and the rate of good quality embryos showed similar characteristics. There were no significant differences between two groups in terms of clinical and ongoing pregnancies, implantation, delivery and live birth rates. In spite of a decreased abortion and increased multiple pregnancy rates in the study group compared to the control group (15.8 vs. 19% and 20.6 vs. 15.6 respectively), the differences were not statistically significant.
Conclusion: Routine use of EmbryoGlue® as a HA enriched ET medium for cleavage stage embryos does not have advantage to the conventional one for infertile patients undergoing ART.
Key words: Hyaluronan, EmbryoGlue®, Embryo transfer, Pregnancy outcome.
P-47
The relation between vitamin C in follicular fluid with the morphology of oocyte and the quality of embryo in IVF patient
Saffari S1, Bahadori MH2, Sharami SH3, TorabZadeh P1, Goudarzvand M1.
Introduction: Oxidative stress and the inappropriate effect of reactive oxygen in the body or laboratory circumstances can decrease the quality of embryo and sexual cells. Ascorbic acid is a natural antioxidant which has a protective effect in the body.
Materials and Methods: This is a cross-sectional analytical study which was conducted on 50 women with IVF referred to Al Zahra Hospital, Rasht, Iran. 20-45 years old women with infertility caused by tubal dysfunction, ovarian factors or male factors were included. Patients underwent same protocol for stimulating; the ovulation and injection of 10,000 units of hCG. After 36 hr, follicles were suctioned and matured oocytes were separated for fertilization. Vitamin C was assessed biochemically and the morphology of oocyte and the quality of embryo were evaluated by by inverted optical microscope.
Results: Among patients 583 oocytes and 275 embryos were assessed. There was no significant relation between, age, BMI and duration of infertility with vitamin C (p>0.05). Also there was no significant relation between vitamin C, the maturity of oocyte and the quality of embryo (p>0.05). But MII oocytes were more in patients with vitamin C less than 1 in comparison with ≥1 (p=0.038). Also the mean 2PN embryos in 0-0.05 level of vitamin C was higher than other groups but no significant relation was noted (p=0.719).
Conclusion: According to our results vitamin C at special level can improve the morphology of oocytes and the quality of embryos.
Key words: Infertility, Follicular fluid, IVF, Vitamin C, Oocyte, Embryo, Antioxidants.
P-48
Polscope analysis of meiotic spindle and zona pellucida birefringence of metaphase ΙΙ oocytes in polycystic ovarian syndrome patients
Safian F1, Khalili MA1, 2, Ashourzadeh S2, 3, Omidi M2.
Introduction: Polycystic ovarian syndrome (PCOS) is one of the most common causes of infertility in women of reproductive age. Currently, one of the best therapeutic options for PCOS patients is ICSI. Moreover, IVM can be a useful technique for women with PCOS who are at the risk of ovarian hyperstimulation syndrome (OHSS). On the other hand, the oocyte quality can be a determining factor for outcome of ICSI cycles.
Materials and Methods: This prospective study included immature oocytes (30 GV and 5 MI) undergoing IVM, and MII oocytes obtained from PCOS patients (29.64±5.31 years) in ICSI program. Using a PolScope, the presence of MS and ZP birefringence was assessed in both in vivo-matured (n=32) and matured oocytes after IVM (n=24). Oocytes were classified as high birefringent (HB) ZP and low birefringent (LB) ZP. Furthermore, the rates of fertilization and embryo development were evaluated.
Results: The maturation rate was 68.5% after IVM. Analysis revealed that the percentage of a HB ZP was significantly higher in the IVM oocytes than in vivo-matured ones (58.3% vs. 31.2%, p=0.04). There was insignificant relationship between spindle detection and either in vivo-maturation or IVM oocytes (p=0.53). Likewise, there were similar outcomes for the rates of fertilization and embryo development after ICSI between two groups (p=0.80 and p=0.13, respectively).
Conclusion: Clinical IVM is a safe technology for the maturation and maintenance of oocytes integrity. Furthermore, the use of non-invasive PolScope is recommended for the detection of healthy oocytes in ICSI.
Key words: PCOS, IVM, PolScope, ZP birefringence, Meiotic spindle.
P-49
History and application of public bank of umbilical cord blood
Seyfipoor Naghne E1, Bolorytabar F1, Khoshravash S2.
Introduction: Umbilical cord blood is rich of hematopoietic stem cells. Today, umbilical cord blood constitutes about 20% of stem cells transplant. 75% of umbilical cord blood banks in the world are public and the remaining are private (trade). 27 years after the first successful umbilical cord blood transplant, more than 555,000 umbilical cord blood units have been made available around the world after performing qualitative study in public bank of umbilical cord blood in order to treat patients who need hematopoietic stem cells transplant. The first program of public umbilical cord blood bank was implemented by the New York in 1991 and in Asia in 1999 in Japan. In Iran in late 2008, the Stem Cells Association has started its activity by receiving financial supports. In this type of storing, umbilical cord blood does not only belong only to the family and public organizations are responsible for paying related costs. Cells in this banking are kept safe and number of their alive cells remains unchanged during the freezing process. Samples saved in bank in terms of health indices such as viral and microbial contaminations are evaluated by colony measurement method. With regard to high prevalence of blood malignancies and thalassemia in Iran, it has been very important to establish public bank of umbilical cord blood and support it spiritually and materially.
Materials and Methods: Subjects of this review study are derived from articles and sites related to collecting, maintain and transplanting umbilical cord blood.
Results: Development of umbilical cord blood banks to develop transplantation centers and treat patients is of unavoidable necessities in the society health system. In societies that the government has ability to establish public bank and supports this bank, development of public banks has preference to development of private banks.
Conclusion: In year 2011, research and development department established public bank of umbilical cord blood in order to scientific and practical promoting and proceeded to amniotic membrane, standardization of tissue adhesive, production and standardization of amniotic extract and platelet extract by establishing bank. Collecting this bio garbage and transforming it to biologic bands, eye drop to improve cornea wounds and mixing hydrogen with amniotic membrane extract to treat wounds for therapeutic consumptions is economical and is effective to decrease therapeutic costs. Also fibrin glue is produced only via umbilical cord blood. Fibrin glue as cell carrier and tissue adhesive is widely used in surgery and cell therapy. Transplant of stem cells of umbilical cord has opened bright and promising horizons for medical society to treat patients with different chronic and acute disorders. So, saving umbilical cord blood finds high importance and its necessity feels further.
Key words: Umbilical cord, Blood bank
P-50
Protective effect of alcoholic extract of Garden cress (Lepidium sativum) seeds on the histopathological changes of epididymis and fertility in diabetic rats
Seyed Hoseini ES1, Nikzad H1, Amini Mahabadi J1, Haddad Kashani H2.
Introduction: Diabetes mellitus (DM) is a frequent and serious metabolic illness all over the world and plants have been a desirable source of medicine recently. Diabetes has unpleasant effect on male reproductive system and it may lead to male infertility. It causes erectile dysfunction and reduces ejaculate volume by affecting the health of small blood vessels and the small nerves that control ejaculation and also decrease libido by decreasing testosterone levels. Current study evaluated the possible protective efficiency of Garden cress (Lepidium sativum) seed extract on fasting blood sugar (FBS) and then assessed epididymal histopathologic changes in streptozotocine (STZ)-induced diabetic rats.
Materials and Methods: 50 male adult Wistar rats were randomly categorized into five groups (each 10 rats). Groups 1 were control placebo group receiving only 0.1 ml normal saline via gastric gavages, Group 2 as control diabetic rats received an intraperitoneally injection of STZ 60 mg/kg body weight. Rats with FBS >250 mg/dl were considered as diabetic. Group 3 were diabetic rats receiving insulin in dose 3U/100 g body weight and Groups 4 and 5 were diabetic rats that received 0.1 cc of 200 and 400 mg/kg‚ ethanol extract of Lepidium sativum seed by gavages daily. One day after the last gavages, rats were anesthetized by chloroform. Epididymis duct was removed from abdomen and weighed with a digital scale. Afterwards, samples were putted in Bouin's solution for histological measurement.
Results: Administration of 200 and 400 mg/ml doses of Lepidium sativum seed extract increased epithelium height and decreased interstitial volume density and fibro muscular thickness significantly. Also, volume density of epithelium, fibro muscular, lumen and interstitial were decreased significantly. Tubular and lumen diameter did not change significantly in different groups.
Conclusion: It seems Lepidium sativum seed extract has benefit as a supplementary protective agent against bad effects of diabetes on reproductive system in diabetic male.
Key words: Diabetes‚ Lepidium sativum seed extract, Epididymis‚ Streptozotocine‚ Insulin.
P-51
New insights into the effect of aflatoxin on infertility of animal models
Sheikhpour R, Jabbari S.
Yazd Medical Science Branch, Islamic Azad University, Yazd, Iran.
Email: R.sheikhpour@yahoo.com
Introduction: Aflatoxins are produced via Aspergillus flavus and Aspergillus parasiticus, species of fungi. Toxic and carcinogenic properties of Aflatoxin are determined. After entering the body, Aflatoxins may be metabolized by liver to a reactive epoxide intermediate or hydroxylated to become the less harmful. Nowadays, infertility properties of Aflotoxine have been known in studies.
Materials and Methods: Literature review was conducted using the following databases: SciELO, Lilacs, Medline, PubMed, Scopus and science direct from 2000-2014. The key-words used were Aflatoxin, infertility, fertility, animal, human.
Results: When Aflatoxin level increased in diet of male goats, glucose and total protein concentration were decreased in the testes and testosterone level was significantly reduced. The study suggests that exposure of male goats to dietary Aflatoxin will reduced testicular biochemical and testosterone with resultant depression in sperm storage capability and daily sperm production in the animals. Chronic Aflatoxin exposure in animals can result in impaired reproductive efficiency. One study showed that Aflatoxin decreased fertility, abortion, lowered birth weights and caused disturbances in hormonal metabolism in sheep. Another study showed that the fertility of treated mice with Aflatoxin at a daily dose of 50 µg/kg body weight was reduced drastically. Sperm concentration in the epididymis and sperm motility decreased whereas sperm abnormalities increased.
Conclusion: The results of studies showed that Aflatoxin can cause infertility in animal models, but these results need to be evaluated by more clinical trials.
Key words: Aflatoxin, Infertility, Animal.
P-52
ART cycles outcome in donor oocyte recipient couples: A case-control study
Tahajjodi S, Khalili MA, Nabi A, Omidi M.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Science, Yazd, Iran.
Email: s.tahajjodi@yahoo.com
Introduction: Oocyte donation is an acceptable alteration in women infertility treatment for different indications. Several factors including the age of the oocyte donor and recipient, oocyte and embryo quality and characteristics of endometrium can influence on ART outcome.
Materials and Methods: Outcome of 202 cycles, consist of 113 DO cycles and 89 AO cycles, were compared. Both DO and AO groups were divided to two subgroups according to fresh and frozen embryo transfer. First DO and AO groups included the fresh embryo transfer (fDO, fAO) and another DO and AO groups included the frozen embryo transfer (fzDO, fzAO). Comparison was performed in regard to chemical pregnancy, clinical pregnancy, implantation rate, live birth and abortion rate.
Results: DO (mean=28.29 years) and AO (mean=29 years) groups were significantly matched by age. No significant differences were observed between fDO and fAO groups and also between fzDO and fzAO groups with regard to chemical pregnancy, clinical pregnancy, implantation rate, live birth and abortion rate. But, there was a significant increase in oocyte number, MII oocyte number and embryo number in DO compared with AO groups (p<0.001).
Conclusion: Despite of increasing in total number of oocyte, MII oocyte and embryo, our findings showed that oocyte donation program has no impact on ART pregnancy outcome.
Key words: Oocyte donation, ART outcome, Women infertility.
P-53
Dose stress always has a negative effect on sperm cell?
Vaseghi-Dodaran H1, Zhandi M2, Daghighkia H1, Sobhani A3.
Introduction: Cryopreservation of semen requires optimized condition to minimize the harmful effects of various stresses. Oxidative stressors are known to be major mediators of cell damages during cryopreservation. An important perspective is that free radicals are not exclusively beneficial or exclusively detrimental. Rather, they need to be maintained at appropriate levels to ensure physiological function, while preventing pathological damage. Recent studies showed that placing the cells in a controlled stress conditions could affect cell survival in later stages.
Materials and Methods: This article discusses some of the new approaches employed for improve spermatozoa before and after freezing-thawing. A review of recent bibliography was carried out in PubMed, Google scholar and SID by the use of relevant keywords.
Results: Osmotic, oxidative, hydrostatic and mechanical stressors before freezing induce expression a series of heat shock proteins (HSPs), decrease apoptotic sperm amount and increase live sperm during treatment and high mitochondrial activity observed in treatments. Cell's reaction to environmental stresses is depended on domain, concentration and acts time of this reactions.
Conclusion: Although cellular and sub-cellular mechanisms, supposedly contributing to these processes, require further research, the new principle may outline a completely new strategy in mammalian embryology, as well as cryopreservation of other cells and tissues with remarkable theoretical and practical consequences.
Key words: Cryopreservation, Sperm, Stress, Sublethal.
P-54
The effect of KIT ligand (KITL) and leukemia inhibitory factor (LIF) on maturation and apoptosis of follicles after vitrification of mouse ovary and three dimensional culture
Abdi Sh1, Salehnia M1, Hosseinkhani S2.
Introduction: In vitro ovarian follicle culture techniques not only provide a model for research into the mechanism of folliculogenesis but also, in combination with cryopreservation of ovarian tissue, it may have clinical applications in preserving of fertility.
Materials and Methods: This experimental study is carrying out on 7-days-old female mice (NMRI). In the first step, the ovaries were vitrified with a solution containing ethylene glycol and then their morphology, ultrastructure and apoptosis were compared with non-vitrified ovaries. In the second step, the non-vitrified and vitrified ovaries were cultured in base medium α-MEM supplemented with KL and leukemia inhibitory factor (LIF) for 7 days then their morphology, area, apoptosis, hormone assay were analyzed. In third step, mechanically isolated preantral follicles were cultured in two dimensional and three dimensional systems in α-MEM supplemented with 5% FBS, 100 mlU/ml, 1% ITS, 10 ng/ml rEGF and KL for 12 days then the ovulation were induced. At the end of culture survival and maturation rate, apoptosis and hormones were assayed. In the fourth step, the level of reactive oxygen species (ROS) and ATP, distribution of mitochondria and fertilization rate in MII oocyte were assessed.
Results: The results of first step showed that the morphology, ultrastructure and apoptosis of vitrified ovaries were similar to non-vitrified ovaries. The results of second step showed that the percentage of preantral follicles, ovarian area, production of hormone in the non-vitrified ovaries cultured in medium supplemented with KL were significantly higher and the level of caspase 3/7 was lower in comparision to other groups (p<0.001). The results of next step showed that the survival, maturation rates and production of hormone of follicles in KL supplemented in three dimensional system groups were higher than other groups. The level of caspase-3/7 activity was lower in three dimensional culture systems (p<0.05). The results of fourth step showed that the level of ROS and ATP in MII from in vitro culture of follicle had significant different with control in vivo group and the rate of embryo development were significantly higher in three dimensional culture system.
Conclusion: Our results demonstrated that the culture of ovarian tissue and three dimensional culture of ovarian follicle in combination of KL improved the growth and development of ovarian primordial follicle.
Key words: Vitrification, In vitro maturation, Apoptosis, Mitochondria, KIT Ligand (KITL).
P-55
Evaluating the levels of expression of TLR 9 in women with endometriosis and its comparison with normal endometria
Aflatoonian Ali1, Janan A2, Ramazanali F2, Moieni A1, 2, Aflatoonian R2.
Introduction: Endometriosis is a benign condition which endometrial glands and stroma appear outside the uterine cavity which presents by pelvic pain and infertility. Endometriosis is associated with changes in cellular and humeral immunity and impaired immune response, leading to inefficient removal of debris after a menstrual cycle. Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system. Based on recent studies TLRs are increased in endometriosis and initiate immune responses.
Materials and Methods: This case-control study assessed and compared expression of Toll like Receptor 9 in the endometrium of three type tissues: 1) Eutopic endometria of women with endometriosis 2) Ectopic endometria women with endometriosis 3) Endometrium of women without endometriosis. Eutopic and ectopic endometrial samples were taken from 10 patients with endometriosis (Case Group). Also endometrial samples were taken from 10 patients without endometriosis or infertility history whom operated for other gynecological cause. The level of TLR9 gene expression in ectopic samples, eutopic and controls were evaluated by Real Time-PCR. Patients were chosen randomly from Arash Hospital. Data was analyzed by SPSS.
Results: Real Time-PCR showed that TLR9 expressed in all three groups of eutopic, ectopic and control. According to statistical analysis, level of TLR9 was higher in ectopic group, but not significantly (p=0.13). There was no significant difference between eutopic and control groups.
Conclusion: Considering the role of TLR9 in the innate immune system, such as pathogen detection and set up a cascade of inflammatory response associated with cell proliferation and inhibition of apoptosis, this study approves the association between TLR9 and endometriosis. Because of lack of information about this new issue, we suggest further researches and more studies.
Key words: TLR9, Endometriosis, Inniate immune system.
P-56
Congenital uterine malformations: A widespread phenomenon among infertile women with recurrent miscarriages and IVF failure
Javam M, Ahmadi F.
Department of Reproductive Imaging at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: f_ahmadi@royaninstitute.org
Introduction: Uterine malformations are a various group of congenital uterine disorders found in women with recurrent abortions or IVF failure.
Materials and Methods: A narrative review was performed within articles published at “PubMed”, “Elsevier”, "EBSCO", original text books and etc. to reach the aim. Several unique high-quality images were provided in this article, using the archive of infertile patients referred to imaging department of Royan Institute.
Results: Congenital uterine anomalies are originated from development defects of mullerian ducts during fetal growth. They are also associated with higher incidences of infertility, preterm birth, intra uterine fetal death and etc. However, manifestations and severity of the obstetric/ gynecologic complications and treatment procedures vary depending on the type of anomaly. Thus, accurate diagnosis of uterine malformations and differentiation between various types of them, have a vital role in decision about treatment procedures and management of these patients. Several imaging modalities are used to investigate women suspected to have uterine anomalies. In this article, we described about various types of uterine malformations and imaging evaluation of them prior to IVF treatment cycles.
Conclusion: Congenital anomalies of the uterus are a major cause of recurrent miscarriage and pregnancy failure. Several imaging methods help midwives and obstetricians evaluate this group of infertile women to detect suspected uterine anomalies.
Key words: Congenital uterine malformation, Infertility, Recurrent miscarriages.
P-57
Study on the impacts of anti-histamine dimenhydrinate on testicular tissues and sperm production of male mature rat
Ahmadifar M1, 2, Vahidi-eyrisofla N3, Taghvaei F4, Jafarzade Rastin S4, Yousef Zadeh AR6, Daliri L5, Gharanfoli M1.
Introduction: In this study we did a research about the effects of AHDH on testicular tissues and sperm production of rats. Dimenhydrinate (DH) is used as anti-nausea as well as an anti-gastric reflux in cases of nausea caused during travelling. It is easily taken orally and it will be excreted by the kidneys within 24 hr.
Materials and Methods: This survey is conducted in laboratory on experiment basis among 30 mature male rats categorized in 3 different groups; two experimental groups and one control group. The control group was watered normally for 60 days and the first experimental group was watered with 200 mg DH blended in their water equal to one kg of their body weight for 60 days. In the end, after the rats were anesthetized, testicles were removed, sectioned and painted with Hematoxilin and Eozine and were investigated.
Results: The obtained results have revealed that in the experimental groups, testicles weight decreased substantially in comparison to those of the control group (p<0.001). The reduction of the number of sperm cells within the experimental group, in comparison to those of the control group was significant (p<0.001). The reduction of the Leydig cell was very substantial in the second experimental group (p<0.001). The difference of the Sertoli cells was not noticeable in neither of the experimental groups.
Conclusion: DH has impact on testicular tissues and weakens their mechanism.
Key words: Dimenhidranate, Testis.
P-58
Investigating association of G103T polymorphism of Coagulation factor XIII and recurrent pregnancy loss
Ahmadpour Nazm S1, Bonyadi M2, Taghavi S3, Ajamian F1.
Introduction: Recurrent miscarriage (RM) occurs in 1-3% of couples that attempting to bear children. Thrombophilia is one of the suspected cause of recurrent miscarriage. At the end of coagulation cascade there is Factor XIII that makes blot stable. The polymorphism G103T of factor XIII gene is the most common polymorphism that affects FXIII’s activity.
Materials and Methods: The study groups consisted 50 patients with two or more consecutive miscarriage. The control group included 50 women with at least two successful delivery and no history of pregnancy loss. By using PCR-RFLP, DNA from both groups analyzed for carrying mutation of FXIII.
Results: 4% in the case group were homozygote (TT) for 34 Leu mutation whereas no homozygote (TT) was found in control group (p<0.05). 28% patients in the case group and 26% women in the control group were found to be heterozygote for G103T polymorphism (p>0.05). No significantly differs was observed between patients with RPL and healthy women for G103T mutation.
Conclusion: No statistically differs between case group and control group was observed.
Key words: Recurrent miscarriage, Thrombophilia, F XIII.
P-59
Effect of pumpkin seed and ginger extracts on adult rat’s sperm characteristics, biochemical parameters and epididymal histology treated with cyclophosphamide
Amini Mahabadi J1, Nikzad H1, Aghaie S2, Taghizadeh M3, Azami-Tameh A2, Taherian SS2.
Introduction: Infertility is one of the problems of human society and 10-15% of couples have experienced some forms of infertility problems. Reproductive toxicity is one of cyclophosphamide (CP) side effects in cancer treatment.
Materials and Methods: Male adult Wistar rats were randomly divided into six groups. Group 1 as control received an isotonic saline solution injection intraperitoneally (IP). Group 2 was injected intraperitoneally with a single dose of CP (100 mg/kg) once. Group 3 and 4 received CP plus 300 and 600 mg/kg combined pumpkin seed and Zingiber officinale extract (50:50). Group 5 and 6 received only 300 and 600 mg/kg combined pumpkin seed and Zingiber officinale extract. Six weeks after treatment, sperm characteristics, histopathological changes and biochemical parameters were assessed.
Results: Results showed that in CP treated rat’s sperm characteristics were diminished significantly. Biochemical analysis showed that the administration of combined extracts could increase the Total Antioxidant Capacity (TAC) level significantly in groups 3, 4, 5 and 6. Also, in these groups, the sperm viability, motility, count, normal sperm morphology, epididymal epithelium and fibromascular thickness were improved compared to control and CP groups. Interestingly, the mixed extract could improve histopathological changes such as vacuolization, disorganization and separation of epididymal tissue in CP treated rats as well.
Conclusion: Overall our findings indicated that the combined extracts might be used as a protective agent against CP-induced reproductive toxicity.
Key words: Pumpkin seed extract, Zingiber officinale extract, Cyclophosphamide, Total antioxidant capacity (TAC), Sperm parameters, Rat epididymis.
P-60
Effect of vitamin D insufficiency treatment on fertility outcomes in frozen-thawed embryo transfer cycles: A randomized clinical trial
Aflatoonian A, Arabjahvani F, Eftekhar M, Sayadi M.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: F_arabjahvani@yahoo.com
Introduction: Frozen- thawed embryo transfer is an essential part of ART treatment and outcomes of this procedure are associated with several clinical factors. Several studies have showed an increase level of IVF outcomes in women with sufficient vitamin D. whether treatment of vitamin D insufficiency can improve pregnancy rates in frozen-thawed embryo transfer cycles.
Materials and Methods: This is an interventional, randomized clinical trial. Serum 25-(OH) vitamin D level of 128 women who had undergone IVF/ ICSI with cryopreservation of embryos was checked. One hundred fourteen infertile women with insufficient serum vitamin D (less than 30 ng/ml) were included in the study. Fifty seven women were treated with supplementary vitamin D, 50000 IU weekly, for 6-8 weeks and 57 women were received no supplementation. 106 women completed frozen thawed embryo transfer cycles and included in the final analysis. Primary and secondary outcomes were chemical and clinical pregnancy respectively.
Results: Our study did not show any significant difference between vitamin D insufficient and treated women in term of chemical (29.40% vs. 29.10% respectively, p=1.00) or clinical (25.50% vs. 21.80% respectively, p=0.81) pregnancy rates.
Conclusion: Vitamin D insufficiency treatment is not associated with higher pregnancy rate in frozen-thawed embryo transfer cycles.
Key words: Vitamin D, Embryo transfer, Pregnancy rate.
P-61
Association of rs10954213 polymorphism of IRF-5 gene with idiopathic recurrent miscarriage
Arjmand F1, Ashrafzadeh Mehrjardi HR2, 3, Ghasemi N3.
Introduction: Interferon regulatory factor (IRF) 5 is a transcription factor that can induce transcription of IFN-α mRNA. Recent studies have shown that IRF-5 promotes the proliferation of T cells and the activation of TH1 and TH17 cells but does not induce TH2 or Treg differentiation. TH17 cells produce inflammatory cytokine IL-17 which plays an important role in the induction of inflammation via neutrophil infiltration and stimulation of inflammatory cytokines. There are many reports that inflammatory processes play a major role in recurrent miscarriage in a manner that an uncontrolled and persistent inflammatory response during pregnancy can harm placental growth. We analyzed the rs10954213 polymorphism of IRF-5 gene in normal pregnancy and recurrent miscarriage patients in order to discover a possible mechanism for the genetic control of immune regulation in patients with recurrent miscarriage.
Materials and Methods: Genomic DNA from 100 recurrent miscarriage (RM) patients and 100 normal fertile control individuals using the routine salting out method were isolated. DNA fragments were then analyzed by real time PCR with SYBR Premix Ex Taq II master mix. Statistical analysis used SPSS 19 software. IRF-5 allele frequencies and genotypes in RM women and the fertile control group were compared using a Chi-square test.
Results: Our results so far indicate that there is a detectable relationship between rs10954213 polymorphism of IRF-5 and susceptibility to recurrent miscarriage.
Conclusion: IRF-5 gene plays a possible role in the induction of inflammation and therefore leads to recurrent miscarriage. However, additional studies are needed in this regard.
Key words: Rs10954213, IRF-5, Recurrent miscarriage.
P-62
Role of 45,X mosaicism in couples with fertility problems
Bahman I1, Bagherizadeh I1, Abdi A1, Shajarehpour L1, Hadipour F1, Hadipour Z1, Saremi A2, Shafaghati Y1, 3, Behjati F1, 3.
Introduction: Infertility is typically defined as the biological inability of a person to contribute to conception after one year of attempting to have a child. Genetic factors such as chromosomal abnormalities are one of the major causes of infertility and spontaneous abortions. The objective of this study was to establish chromosome X abnormality rate among women referred for infertility problems to Sarem Women's Hospital in Tehran between October 2007 and January 2015.
Material and methods: A total number of 1764 women with the age range of 12-60 years were referred for chromosomal investigation. Referral reasons included recurrent abortions, primary infertility, secondary infertility, Turner syndrome, premature ovarian failure, amenorrhea and ART failures. Cytogenetic analysis was performed on cultured peripheral blood lymphocytes stimulated with phytohaemagglutinin M, using standard techniques. Karyotype was done in all patients using high resolution GTG banding technique. For each patient, at least 15 chromosome spreads were examined by light microscopy, and extended to 100 in the case of mosaicism.
Results: Cytogenetic investigation was performed on 1764 patients. The overall chromosome X abnormality rate in infertile and subfertile women was 4% (74 out of 1764). The cytogenetic result for these patients with fertility problems is as follows: 74 patients had a numerical chromosome abnormality and 65 (85%) of them had mosaicism of 45,X with other different cell lines. The age range of women with 45,X mosaicism was 12-53 years old.
Conclusion: Accurate genetic diagnosis is the most important prerequisite for genetic counseling in patients with fertility problems. Finding of chromosome X numerical abnormality are of great value in better management of the patients. However, the attribution of chromosome X instability due to age should be considered in Genetic counseling of these couples.
Key words: Fertility problems, Chromosome X abnormality, 45,X cell line.
P-63
Effects of mitochondrial gene deletions on human sperm motility
Bahrehmand Namaghi I, Vaziri H.
Department of Biology, Faculty of Science, Guilan University of Medical sciences, Rasht, Iran.
Email: i_man_bahrehmand@yahoo.com
Introduction: Asthenozoospermia is an important cause of male infertility. It has been primarily characterized by reduced sperm motility owing to a variety of factors, including ultra-structural abnormalities, abnormal semen liquefaction, anti-sperm antibodies, varicocele and endocrine abnormality, etc. The mitochondrion is the major energy provider for sperm motility. Mitochondrial DNA contains several genes encoding for proteins that play an important role in oxidative phosphorylation and in ATP production. Mutations in sperm mtDNA result in either functionless or malfunctioning proteins, subsequently affecting sperm motility leading to asthenozoospermia.
Materials and Methods: To detect 4977 bp deletion in spermatozoa mtDNA, semen samples of 42 asthenozoospermic infertiles and 50 controls from northern Iran were collected. After extraction of spermatozoa total DNA, Gap-PCR was performed.
Results: 4977 bp deletion was observed in 73.80% of patients with asthenozoospermia, compared with 36% in controls (OR=5.0101, 95% CI: 2.0408-12.2998, p=0.0004).
Conclusion: Large-scale mtDNA deletions in spermatozoa may induce bioenergetic disorders and cause subfertility or infertility in men. It is concluded that there is a strong association between sperm mtDNA 4977 bp deletion and asthenozoospermia-induced infertility in the population examined. Nevertheless, to validate our results broader research may be needed.
Key words: Asthenozoospermia, mtDNA deletion, Male infertility, Motility.
P-64
Effects of Nano TiO2 on chromatin, apoptosis and parameters of sperm in mice
Danafar AH1, 2, Khoradmehr A1, Hosseini M3, Halvaei I1, Golzadeh J1, Anvari M1.
Introduction: Titanium dioxide nanoparticles (TiO2 NPs) are manufactured worldwide and used in a broad range of applications. In mammals, there are only limited reports regarding the effect of TiO2 NPs on male reproductive system. It has been demonstrated that TiO2 NPs taken up by mouse Leydig cells reduced the viability and proliferation of these cells. Also it is improved that titanium compounds can disrupt the mouse blood-testis barrier. In this study we decide to evaluate the effects of nano-TiO2 on chromatin, apoptosis and sperm parameters of male mice.
Materials and Methods: 35 NMRI male mice were classed in five groups: control, sham and three treated groups. Low, medium and high treated groups were gavaged 2.5, 5 and 10 mg/kg doses of TiO2 respectively for thirty five days. Sham group only received saline instead. The mice were weighted before and after administration. Sperm count, motility, morphology and viability were analyzed accordingly. Sperm DNA integrity and apoptosis were assessed using acridine orange, aniline blue, toluidine blue, CMA3 and TUNEL assay.
Results: Non-significant decrease in body weight gain was observed. Sperm analysis showed no count, motility and viability changes but morphological changes were significant. Most of the morphological abnormalities were observed in sperm neck and tail. Tail morphological changes in treated groups were significant in comparison to non-treated ones (p<0.001). Compared to non-treated groups, sperm neck changes in medium and high treated groups were significant but not in low treated group (p<0.001). Results of acridine orange, aniline blue, toluidine blue and CMA3 were non-significant in all groups. Also increasing apoptotic cells in treated groups were discovered by TUNEL assay but there were non-significant statistically.
Conclusion: TiO2 significantly may increase sperms with abnormal morphology especially in tail and neck part. Also apoptosis boosting occurred but the exact mechanism is still controversial.
Key words: Nano-TiO2, Sperm parameters, Apoptosis.
P-65
Protective role of vitamin C on hazardous effects of acrylamide in rat offspring
Dortaj H, Anvari M, Yadegari M.
Shahid Sadoughi University of Medical Scienses, Yazd, Iran.
Email: hengameh.dourtaj@gmail.com
Introduction: Acrylamide (ACR) is a substance chemical used in industrial and laboratory procedures. Acrylamide according to the method of cooking foods are increasingly used and its adverse effects on multiple organ systems have been described sporadically in the literature. The purpose of this study was to evaluate the effects of ACR during pregnancy and lactation on the development and changes in cortical layer of the cerebellum and cerebellar Purkinje cells in fetal and neonatal rat by using histological and morphometric and stereological technique.
Materials and Methods: In this study 20 adult female Wistar rats weighing 180 gr and aged two months were used. Animals were randomly divided into four groups. Female pregnant rats were orally administered 10 mg/kg ACR and/or 200 mg/kg vitamin C (vit C). Pregnant rats were sacrificed on the 15th day of gestation and mother’s weight was measured. After that, their fetuses were taken out and were evaluated for fetus number, weight, crown-rump length (CRL) and cerebellar development. To study the neonatal period, 6 infants at day 21 were randomly selected and placed under deep anesthesia and transcardial perfusion. The cerebellum was taken out and fixed and cerebellum changes were evaluated by crysel violet and Hematoxylin-Eosin staining method. The cerebellar cortex layers volume were investigated by Cavalieri's principle method. Data were analyzed by SPSS software and by ANOVA and LSD Test. P<0.05 considered as statistically significant.
Results: The results showed that ACR decreased fetal weight and CRL, but this reduction in weight and volume of the cerebellum p˂0.001 and the number of embryos with p<0.05 was significant. Histological and stereological examinations revealed that the cerebellar volume was decreased in ACR and ACR+vitC group vs control (p˃0.001). While in vitamin C group the cerebellar volume was inversed (p˃0.05). ACR in newborn decreases body weight, brain weight, thickness of cerebellum with p˃0.001. The extent of this reduction in the weight of the cerebellum was significant with p˂0.05.
Conclusion: ACR exhibits a harmful effect on the development of the cerebellar cortical layers, which may be prevented by administration of vit C as an anti oxidant.
Key words: Acrylamide, Vitamin C, Cerebellum, Development, Rat.
P-66
Robust stem cell isolation from human dental pulp
Ebrahimi B, Banaei M, Namiranian G, Tabatabaei SA.
Yazd Cardiovascular Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: bhnmebrahimi@yahoo.com
Introduction: Dental pulp stem cells (DPSCs) are a population of clonogenic and highly proliferative cells derived from enzymatically digested Dental pulp tissue. Providing scaling-up of stem cells at early passages is of importance for regenerative medicine purposes. Typically, two protocols are employed to isolate stem cells from human dental pulp: tissue explants culture and enzyme digestion of pulp tissue and culture of released cells.
Materials and Methods: Dental pulp was extracted from third molars of 60 healthy subjects. In the first method pulp was digested with 1 mg/mL collagenase/ dispase (Roche) for 30 min and released cells obtained using a 70-µm cell strainer for culture, in the second method intact pieces of pulp were cultured and in the third method digested pulp pieces were immobilized and cultured. The cells and tissues maintained in alpha-MEM supplemented with 20% fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, and 25 ng/mL amphotericin B and incubated in humidified incubator with 5% CO2 at 37oC. In each group cells and colonies were counted and compared.
Results: Results showed that treating pulp segments with enzymes and culturing them (combinatory method) increased the efficiency of cell isolation up to 60% significantly in 3-4 days of culture compared with other methods which this value was <20% in 10-15 days.
Conclusion: According to the small size of pulp tissue and its low stem cell contents, acquiring substantial quantities of cells in primary culture will facilitate the in vitro expansion and providing adequate production of the stem cells at early passages with minimum risk of losing their ‘stemness’ and aberrant genetic changes for use in research, tissue engineering and regenerative medicine. Optimized method increase efficiency of cell isolation and provides significant quantities of stem cells in primary culture more than other methods.
Key words: DPSCs, Cell isolation, Cell culture.
P-67
Evaluation of exon 8 of DPY19L2 gene in total globozoospermic patients referred to Royan Institute
Ebrahimi Nasab M1, Totonchi M2, Sabbaghian M3.
Introduction: Lack of acrosome decreases the capacity of the sperm for penetrating in the oocyte which consequently results in infertility. Globozoospermia is a rare and severe teratozoospermia characterized by round-headed spermatozoa lacking acrosomes. DPY19L2 is one of the genes which are dominantly expressed in the testis and it has been shown that is involved in the cause of this phenotype. Recent studies have shown that in large majority of globozoospermia patients a 200 kb deletion including DPY19L2 gene occurs. Different mutations in exon 8 of this gene have been also observed.
Materials and Methods: In total 24 men with total globozospermia and 24 men with normal spermogram referring to Royan Institute were selected. Then we sequenced exon 8 and intron boundaries in the non-deleted patients using specific primers and PCR technique.
Results: In our results no mutations were detected in exon 8 of the patients without the gene deletion, which were 29.17% of the total globozospermia patients.
Conclusion: According to our data, exon 8 mutations or polymorphisms has no effect on globozospermia but whereas 70.83% of globozospermia patients had whole DPY19L2 deletion and on the critical role of DPY19L2 protein in acroplaxome attachment to the nucleus, it can be concluded that the absence of this protein is one of the major causes of globozoospermia in Iranian infertile men. In addition, exploring other coding exons of this gene is our next aim.
Key words: DPY19L2 gene, Globozoospermia, Male infertility.
P-68
Anti-oxidant effects of herbal supplements on seminal parametmeters
Eghbal Eftekhaari T, Fallahi S, Malekzadeh K.
Molecular Medicine Research Center, Hormozgan University of Medical Sciences, BandarAbbas, Iran.
Email: tasnim.eftekhaari@gmail.com
Introduction: Varicocele is one of the leading causes of male infertility, causing a decrease in sperm parameters such as motility, count, concentration, and abnormal sperms such as azoospermia and asthenospermia.
Materials and Methods: A herbal compound capsule containing 4 herbs was prescribed 5 times perday (3 times during morning and evening and 2 times at night), and because of the warm nature of these herbs, 3 herbal extracts including Cichorium intybus L, Salix Aegyptiaca and Fumaria Parviflora 3 cups/day were added to this medication.
Results: After 4 weeks of consuming these medications, the body warmness decreased , ejaculated sperm volume and other sperm parameters increased including motility, concentration and morphology of sperm changed form astenia and oligospermia to normal morphology.
Conclusion: The herbal compound containing antioxidant supplements and enriched in minerals and fat soluble vitamins showed promising effects on improving sperm parameters and increase the chance of male fertility.
Key words: Varicocele, Increased sperm motility, Increased sperm count, Sperm paramteters, Antioxidants.
P-69
The effect of brown algae Sargassum extract on freezing of sperm
Fardmanesh H1, Fallahi S2, Shakerizadeh S3, Masoudi M3, Fadaei- Arsanjan M4.
Introduction: Oxidative stress process resulting from freezing and thawing of sperm, influences parameters of sperm and decreases its fertility. Antioxidants play a conservative role against oxidative damages during freezing of sperm. Using an appropriate freezing bank effects on fertility and motility of sperm, so we decided to evaluate effect of antioxidant extract of brown algae Sargassum on reactive oxygen species (ROS) and parameters of frozen sperm.
Materials and Methods: 11 normal semen samples were divided in to 3 groups, including A treated with 250 µg/ml and B with 500 µg/ml of extract of algae Sargassum and C group without any treatment (as a control group) then froze throughout rapid freezing method. Using CASA software and oxisperm kit, the motility/morphology and ROS levels of sperm were measured, respectively. Finally data were analyzed with SPSS software assumed significant level of p<0.05.
Results: The analysis of sperm parameters demonstrated that general motility (p=0.006) and advanced motility (p=0.007) significantly increased in both A and B groups, after treatment compared with C group. Moreover, the level of ROS notably declined in both treated group. However this treatment revealed any changes in sperm morphology.
Conclusion: The brown algae Sargassum extract is rich in antioxidant component that leads to decrease oxidative damage via neutralizing of ROS and improve sperm motility. Our results suggested that the extract can be used as a potential antioxidant factor in sperm freezing bank.
Key words: Sperm motility, Antioxidants, Reactive oxygen Species, Brown algae sargassum.
P-70
Immunohistochemical evaluation of a testis-specific histone demethylase, JMJD1A, in tissue samples of infertile men referred to Royan Institute
Favaedi R1, Shahhoseini M1, Kordestani shargh E2, Sodeifi N3, Sadighi Gilani MA3.
Introduction: Post-meiotic stages of mammalian spermatogenesis require unique and dynamic epigenetic events leading to histone removal followed by chromatin condensation. Through these events, histone demethylases such as JMJD1A play an important role in compaction of sperm chromatin due to regulation of histone methylation dynamics and alteration of chromatin structure. As “histone methylation” is one of the best-characterized modifications in the study of germ cell development, evaluation of presence/ absence of JMJD1A protein as in impaired spermiogenesis were aimed in this study.
Materials and Methods: For this respect, consent was obtained from azoospermic infertile men referred to Royan Institute according to local ethical approval, and then testis tissue samples were collected from three groups including complete maturation arrest, Sertoli cell only syndrome, and hypo spermatogenesis as positive control. Immunohistochemical analysis of paraffin embedded tissue samples was performed qualitatively using anti-JMJD1A antibody to elucidate presence/ absence of this protein in nucleus of germ cells.
Results: Immunohistochemical analysis data showed absence of JMJD1A protein in nucleus of germ cells in groups with spermatogenesis impairment (complete maturation arrest and Sertoli cell only syndrome groups) compared to control group.
Conclusion: It can be concluded that there is an obvious association between absence of histone demethylation as a chromatin condensing state with impairment of spermatogenesis and male infertility.
Key words: Spermatogenesis, Male infertility, Epigenetic, JMJD1A, Demethylation.
P-71
Pregnancy outcome of intracytoplasmic injection with epididymal and testicular sperm retrieval in azoospermic patients
Moein MR, Ghasemi-EsmailabadS, Ghasemzadeh J, Fesahat F, Tabibnejad N.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: qasemisaeed@yahoo.com
Introduction: Intra cytoplasmic sperm injection (ICSI) is an assisted reproductive technique for treatment of infertility in azoospermic men. It seems that pregnancy outcome in ICSI following percutaneous epididymal sperm aspiration (PESA) is better than using testicular sperm retrieving from testis due to greater motility and better morphology of epididymal spermatozoa. To determine clinical pregnancy outcome following ICSI with epididymal sperm comparing to that of testicular sperm in men with azoospermia.
Materials and Methods: 60 men with azoospermia who were candidate for ICSI have been selected. Sperm retrieval was performed using PESA (n=30) or testicular sperm extraction (TESE) (n=30). The number of embryos and live births were analyzed and evaluated between two mentioned groups and also thawed sperms after freezing in TESE group (n=30).
Results: No difference was seen in age and duration of infertility between groups. The number of embryos were not different significantly between TESE and PESA groups (p>0.05), but the different was significant between PESA and TESE freeze groups (3.67±2.89 vs. 2.10±1.53 respectively; p<0.05). The live birth rate was higher in the PESA group compared with the TESE group p<0.05).
Conclusion: Intracytoplasmic sperm injection using sperm from epididymis is more effective than testicular sperm injection and can successfully be performed to treat men with azoospermia.
Key words: ICSI, TESE, PESA, Freeze, Live birth rate.
P-72
Prevalence and risk factor of congenital malformation in Kashan
Haddad Kashani H1, Seyed Hoseini ES2, Dadkhah M3, Nikzad H2.
Introduction: Congenital malformation (CM) is a major childhood health difficulty. Treatment and rehabilitation of children with congenital malformations is costly and complete recovery is usually impossible. The prevention and treatment of congenital malformations are basic concerns for child health. The purpose of this study was to define rate of CM in Beheshti Hospital in Kashan, Iran to find out if there has been any variation in the rate and types of CM in this area.
Materials and Methods: This descriptive-observational study carried on 2700 births delivered from Beheshti Hospital in Kashan during 2012 and determines the prevalence of CM and type of it in this city.
Results: Prevalence of CM was 1.851% (2% in male and 1.61 % in female). Out of the 50 cases, 28 (56%) were males and 21 (42%) were females and 1 with uncertain genitalia. 13 members of the family were CM positive in musculoskeletal system (26%). Overall, anomalies of the cardiovascular system were second in frequency which involved 10 out of 50 patients (20%), also Down syndrome and skin malformation were the lowest rate anomaly in this study in Kashan (4% and 2% respectively). There wasn't statistical difference between prevalence of CM and neonatal gender and mother’s age, father’s age, mother’s number of abortion, and mother’s number of live children. But there was statistical difference between prevalence of CM with gestational age and Apgar number.
Conclusion: In this study the overall prevalence of congenital malformation among the newborn was less than those previous reported in Tehran and Yazd and higher than the rate of malformation in Gorgan and Arak. This difference determining the needs of more extensive studies.
Key words: Congenital malformation, Newborn, Beheshti Hospital, Kashan.
P-73
Relevance between testicular tissue vitrification and short term culture with degeneration and apoptosis genes expression
Hajiaghalou S1, 2, Ebrahimi B2, Shahverdi AH2, Sharbatoghli M2.
Introduction: Cryopreservation of testicular tissue has been recommended as a promising technique for fertility preservation in pre-pubertal boys who scheduled to undergo gonadotoxic treatment. Our aim was finding the quota of apoptosis genes involved in extrinsic pathway in testicular cell death after vitrification and during short term culture.
Materials and Methods: Testes were obtained from 7 days old NMRI male mice, divided and randomly distributed into control and vitrification groups. Vitrification was performed in 3 step by increasing concentration of vitrification solution (DMSO, EG). Both fresh and vitrified-warmed testes was cultured in RPMI and 10% KOSR for 20 hr. Real-time PCR, flow cytometery and light microscopy were used respectively for evaluation of gene expression, cell death and tissue integrity at 0, 3 and 20 hr of culture.
Results: Decreasing of tissue integrity was obvious in vitrification group as compared to the control one at all times of culture. Mean percentage of cell death was significantly (p<0.05) higher in vitrification group in comparison with control during culture period. Although expression of Fas was significantly (p<0.05) higher in vitrification group at 0 and 3 hr of culture, it was significantly lower at 20 hr of culture as compared to the control group. Significant (p<0.05) increase of Fas ligand was found in vitrification group at 3 and 20 hr of culture. Mean percentage of Caspase 3 was significantly (p<0.05) higher in vitrification group than control group.
Conclusion: Concurrent increment of cell death and apoptosis genes expression in vitrification group during culture period, could be a reason for extrinsic pathway involvement in degeneration of testis tissue after vitrification and also during culture period.
Key words: Extrinsic pathway of apoptosis, Tissue degeneration, Vitrification, Short term culture.
P-74
Magnetic activated cell sorting and its application for selection of human non apoptotic spermatozoa in ART
Hassani Bafrani H1, Catt S2, Pangestu M2, Smith Peter T2.
Introduction: Hydrogen peroxide has been implemented in literature to induce a significant increase in caspase. Activation of caspase 9 triggers a cascade of caspase activation, including caspase 3, which promotes cellular apoptosis. Magnetic cell sorting (MACS) using annexin V-conjugated microbeads eliminates apoptotic spermatozoa with annexin V-positive.
Materials and Methods: A total of 20 semen samples were obtained from male partners of couples for analysis. One aliquot (0.5ml) of the sperm suspension was subjected to MACS. Motility and concentration was checked, and a sample taken for tunnel before and after MACS. The remaining sperm suspension was divided into 6 tubes (2x control, 2x peroxide, 2x peroxide/ melatonin). DNA fragmentation was evaluated using the TUNEL assay (Roche, Indianapolis, IN, USA) (14), with some modifications. Data were analyzed using Graph Pad Instat Ver. 3.10.
Results: Results of the TUNEL assay in pretreatments of human spermatozoa with 100uM peroxidase for 24 hrs revealed that the percentage of sperm with fragmented DNA was significantly lower on the sored sperm after sorting MACS (p˂0.001 vs. control). Following the pretreated human spermatozoa with peroxidase for 24 hr, percentage of sperm motility and progressive motility were significantly reduced (p˂0.001 vs. control). Pretreatments of human spermatozoa for 24 hr revealed that the percentage of sperm motility and progressive motility were significantly reduced by 100 µM peroxidase and peroxidase with MACS, versus zero hour in control group (p<0.001).
Conclusion: The sperm was treated with 100 µM peroxidase sorting using MACS retain appropriate spermatozoa and select sperm good quality. The use of MACS will select only sperm with intact this result in high percentage motility and progressive motility.
Key words: Apoptosis, Human spermatozoa, MACS.
P-75
Bibliometric mapping and clustering analysis of Iranian-based research on reproductive medicine
Bazm S1, Ramezani A2.
Introduction: Nowadays infertility is a major problem in the world. Increasing the rate of infertility in the world and Iran led our study to assess the trends in Iranian research output related to reproductive medicine through the year of 2010-2014.
Materials and Methods: We used bibliometric mapping and clustering analysis method to visualize representing bibliographic data of Iranian production. All publication data from Scopus was retrieved for 2010-2014.
Results: Analysis of data showed that a total number of 3035 papers had been indexed in Scopus through the period of study.
Conclusion: The study indicated that scientific production in the field of reproductive medicine had increased in the past 5 years.
Key words: Reproductive medicine, Bibliometric analysis.
P-76
Menstrual pattern following tubal ligation: A historical cohorts
Jahanian Sadatmahalleh Sh1, Ziaei S1, Kazemnejad A2, Mohamadi E3.
Introduction: Tubal ligation (TL) is recommendable for women completed their family. The existence of the menstrual disorders following this procedure has been the subject of debate for decades.
Materials and Methods: A historical cohort study was carried out on 140 women undergoing tubal ligation and on 140 women who used condom as the main contraceptive method. They aged between 20 and 40 years and were selected from a health care center, during 2013-2014. The two groups were comparable in demographic and personal characteristics. Data collection tool was a questionnaire including questions regarding demographic, menstrual and obstetrical characteristics. A validated Pictorial Blood Loss Assessment Chart (PBLAC) was also used to measure the menstrual blood loss. All statistical analyses were carried out using software package used for statistical analysis (SPSS) version 20 (SPCC Inc., Chicago, IL, USA). Student’s t-test and chi-square test were carried out to reveal the statistical differences between the groups. Logistic regression was done to build a prediction model in menorrhagia.
Results: Women with TL had more menstrual irregularity than those without TL (24.3% vs. 10% respectively, p=0.002). Women with TL had more polymenorrhea (9.3% vs. 1.4%; p=0.006), hypermenorrhea (12.1% vs. 2.1%; p=0.002), menorrhagia (62.9% vs. 22.1%; p<0.0001) and menometrorrhagia (15.7% vs. 3.6%; p=0.001) than those without TL. There is a significant difference in the PBLAC score between women with and without TL (p<0.0001). According to logistic regression, age (OR=1.08, CI:1.07-1.17; p=0.03), TL (OR=5.95, CI:3.45-10.26; p<0.0001), and cesarean section (OR=2.72, CI:1.49-4.97; p=0.001) were significantly associated with menorrhagia.
Conclusion: We found significant differences in menstrual disorders between women with and without TL. Therefore, women should be informed by the health providers regarding the advantages and disadvantages of TL before the procedures.
Key words: Historical cohort study, Tubal ligation, Menstrual disorders, PBLAC.
P-77
Effect of nano silver on morphological and chromosomal abnormality of NMRI mouse fetus
Mozafari M1, Kalantar SM2, Khoradmehr A2, Danafar AH2, 3, Miresmaeili M4.
Introduction: Development of nanotechnology caused the use of the materials in nano sizes. Nowadays, special biological properties of nano silver are playing an important role in our life. Some studies suggest a potential for adverse effects of nano silver on fetal development of mammals, but additional research is needed. In this study we decide to evaluate the effects of nano silver on fetus chromosomal structure and its development.
Materials and Methods: 24 pregnant mice were divided into four groups. Nano silver (1 mg/kg, 70 nanometer) were gavaged to the first, second and third group, from the 1th-7th, 8th-14th and 1th-14th gestational days respectively. Nothing was gavaged to the control group. On 14th day the pregnant mice were dislocated and the liver of fetus was used for karyotyping analysis. The fetuses were weighed and their crown-rump length and head circumference were measured by caliper.
Results: According to the effects of nano silver, the results indicated that body weight of embryos were significant decreased in third group in comparison with the others (p<0.001). Also there were significant reduction of crown-rump length of fetus in third and second group compared to other groups (p<0.05). There was lower fetus head circumference among nano silver treated and controls (p<0.05). Karyotyping analysis of fetal liver was normal in all groups.
Conclusion: It seems that abuse of nano silver during pregnancy can reduce weight, crown-rump length and head circumference in mice fetus, but its mechanism is not completely clear.
Key words: Pregnant mice, Nano silver, Karyotype.
P-78
Neuronal markers expression of induced human adipose-derived stem cells in alginate hydrogel
Khosravizadeh Z1, Khosravizadeh Z1, Razavi Sh2.
Introduction: Hydrogels provide appropriate three-dimensional environment for cell cultures and cell encapsulation in hydrogels is a promise plan for tissue engineering applications. Alginate is a biocompatible hydrogel that provides a supportive system for the encapsulated cells. Moreover human adipose derived stem cells (hADSCs) might be a suitable source of cells for use in autologous cell therapy; also these cells can differentiate into neuron-like cells. Therefore, in this study we evaluated effect of alginate hydrogel on the neural potential of induced hADSCs.
Materials and Methods: Isolated hADSCs were induced in neural medium and encapsulated in alginate hydrogel. Using Immunocytochemical and real-time RT-PCR analysis, some neural markers were evaluated in differentiated hADSCs.
Results: The expression of Nestin, GFAP and MAP2 markers significantly increased in alginate cell cultures relative to monolayer induced cells (p<0.001).
Conclusion: These findings showed that alginate hydrogel can provide a suitable environment for neural differentiation of hADSCS.
Key words: Human adipose-derived stem cells, Alginate hydrogel, Neural differentiation, Tissue engineering.
P-79
Protective effect of N-acetyl-L-cysteine on testicular tissue in mice treated with Para-Nonylphenol
Malmir M, Soleimani Mehranjani M, Naderi Noreini S.
Faculty of Science, Arak Univercity of Medical Scinecs, Arak, Iran.
Email: mehhdi66@yahoo.com
Introduction: Para-Nonylphenol (p-NP) is an alkylphenol considered as an environmental pollutant with estrogenic and toxic effects. It can also cause morphological and functional alterations in the male reproductive system inducing infertility. The purpose of this study was to investigate the protective effects of N-acetyl-L-cysteine (NAC), as a powerful antioxidant, on p-NP-induced testicular toxicity in mice.
Materials and Methods: Adult male NMRI mice (32±4 gr) were divided randomly into 4 groups (n=6), control, NAC (150 mg/kg/day), p-NP (250 mg/kg/day) and p-NP+NAC, and they were treated orally for 35 days. Finally, mice were killed, their right testis were removed and fixed followed by sectioning, tissue processing and Heidenhain azan staining. Testicular tissue sections were then evaluated using stereological method. The level of Malondialdehyde (MDA), a lipid peroxidation index, was also measured in the testis of mice. Data were statistically analyzed using one way ANOVA and means were considered significantly different at p<0.05.
Results: A significant decrease in the mean total volume of testis, diameter, length and the volume of the seminiferous tubules, height of the germinal epithelium and the thickness of the basement membrane along with a significant increase in the MDA level was observed in the p-NP group compared to the control (p<0.01) while the above parameters were compensated to the control level in the p-NP+NAC group (p<0.01).
Conclusion: Our findings indicate that N-acetyl-L-cysteine can protect the testicular tissue against the tissue damage and oxidative stress induced by para-Nonylphenol in mice.
Key words: Para-nonylphenol, N-acetyl-L-cysteine, Stereology, Testis.
P-80
Effects of dietary canola oil on sperm quality parameters at Afshari Ram Breed
Mansouri S, Pirestani A.
Islamic Azad University, Khorasgan Branch, Isfahan, Iran.
Email: nmansorrin@yahoo.com
Introduction: In mammalian and non-mammalian spermatozoa, there are natural fatty acids, cholesterol, phospholipids (mainly lecithin, cephalin and sphingomielin) and glycolipids. Phospholipids of mammalian sperm cell membranes characteristically contain very high proportions of long-chain polyunsaturated fatty acids, particularly n-3 series
Materials and Methods: 18 Kurdish rams were selected with weight average 54.47±2.58 kg and with the age of 3-4 years approximately. They were divided to two experimental groups randomly. Experimental groups were control and canola oil (2.5% of DMI). Before of study, 10 day was considered as adaptation period and then Sperm was collected by electro ejaculation at 6 week and 11 week after begging of adaptation period and sperm motility was analyzed by using CASA software at zero time and 24 hours post ejaculation. Also, percentage of live/dead sperm and morphology was evaluated by staining of Eosin-Nigrosin and Papanicolaou, respectively.
Results: The results showed that motility parameter wasn’t significantly different between whole experimental groups at first time (week 6) but PM% and TM% was significantly different in canola oil group compare to control group at second time (week 11), separately. Percentage of live/dead sperm and morphology was higher significantly in canola oil group compare to control group.
Conclusion: It was concluded that canola oil can improve sperm motility, morphology and viability in Afshari ram.
Key words: Canola oil, Motility, Afshari Ram, Sperm.
P-81
Mutation analysis of bone morphogenetic protein-15 gene in Iranian patients with polycystic ovarian syndrome
Mehdizadeh A, Kalantar SM, Sheikhha MH.
Department of Genetics, International Campus, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: anahita.mz@gmail.com
Introduction: With the prevalence on the order of 6-10%, polycystic ovarian syndrome (PCOS) is considered the most common endocrinologic disorder affecting women in their reproductive age. Major criteria that have been proposed for the diagnostic characteristics of PCOS are clinical and/ or biochemical hyperandrogenism, ovulatory dysfunction and polycystic ovary in ultrasound. It has been suggested that genetic factors participate in the development of PCOS. Follicular development has been considered as one of the impaired processes in PCOS. BMP-15 gene is a candidate gene in follicular development and its variants may play role in pathogenesis of PCOS. Previous investigations have revealed controversial results on BMP-15 mutations in PCOS women among various racial groups.
Materials and Methods: A cross-sectional study was carried out on 70 PCOS patients. Following taking the informed consent, 5ml venous blood was taken from each participant. Genomic DNA was extracted from the blood sample by salting out Method. Then a set of PCR reactions for BMP15 gene was performed using specific primers followed by genotyping with direct sequencing.
Results: As for exon1 in BMP-15, 20 heterozygote (G/C) and 2 homozygote (G/G) cases were found in 70 PCOS patients. Also, one -9 C>G polymorphism in 5’UTR and 3 cases of A308G mutation (A/G) were discovered in our patients.
Conclusion: Result of this study indicates that variants of BMP-15 gene could be related to susceptibility of development of PCOS and may be used as genetic markers for detecting PCOS in the future.
Key words: Polycystic ovarian syndrome, Bone morphogenetic protein 15.
P-82
Effect of adding human chorionic gonadotropin to frozen thawed embryo transfer cycles with history of thin endometrium
Davar R1, Farid Mojtahedi M1, Miraj S1, 2.
1. Department of Obstetrics and Gynecology, Research and Clinical Center for Infertility, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Department of Obstetrics and Gynecology, School of Medicine, Shahrekord University of Medical Sciences, Shahrekord, Iran.
Email: miraj.sepideh@gmail.com; dr.s.miraj@gmail.com
Introduction: Embryo implantation process is a complex phenomenon and depends on fetal and maternal factors interaction. Endometrial thickness is needed for successful implantation. Increasing endometrial thickness, raise the chance of clinical pregnancy. The triple line pattern, with thickening more than 7mm, is indicating the greater chance of successful implantation. We designed this study to assess adding HCG to the conventional protocol in endometrial preparation in women with thin endometrium and history of IVF-ET failure.
Materials and Methods: This non-randomized clinical trial study (quasi experimental design) was performed in Yazd Research and Clinical Center for Infertility on 28 patients. Participants were women who candidate for frozen thawed embryo transfer and had 2 previous failed ET cycle because of thin endometrium. All patients received 8 mg estradiol valterate on second day of menstrual cycle and continued during the study. HCG was administrated (150 IU, IM) from 8th days of cycle. In 12th-13th day Trans-vaginal sonography was done, when endometrial thickness reached at least 7mm, HCG was discontinued and frozen thawed embryo transfer was done.
Results: Totally 28 patients were included. The mean age of participants was 30.39±4.7 years. The mean of endometrial thickness before and after HCG were 5.07±0.43 and 7.85±0.52 mm which were significantly different (p=0.00). After HCG administration 100% patient's endometrial thickness reached more than 7mm. The frequency of 20% improvement after HCG was 89.3% (25 patients). Also there were 5 (17.8%) clinical and chemical pregnant women after HCG.
Conclusion: Our study findings suggest that adding HCG to the conventional preparation method is an effective protocol and significantly improved endometrial thickness and pregnancy outcomes in women with previous embryo transfer failure because of thin endometrium.
Key words: Human chorionic gonadotropin, Frozen thawed embryo transfer, Thin endometrium.
P-83
Glycyrrhiza glabra and vitamin C can reduce toxic effects of acrylamide on sperm parameters in rat
Mirjalili A1, Talebi AR2, Mangoli E2, Ghasemzadeh J2.
Introduction: Acrylamide (AA) is a chemically reactive substance used in various industries. Recently, acrylamide was discovered in a variety of human foods including heat-processed starchy foods such as potato chips and bread. AA is able to induce sperm damage in male mice.
Materials and Methods: In this experimental study, 30 male Wistar rats of 28 days of age were divided in to five groups: Acrylamide, Acrylamide+ Vitamin C, Acrylamide+ Glycyrrhiza glabra, Acrylamide+ Vitamin C+ Glycyrrhiza glabra and Control. All treatments were administered (oral Acrylamide 10 mg/kg, Vitamin C 200 mg/kg and Glycyrrhiza glabra 150 mg/kg) daily for two months. Thereafter, the cauda epididymis of each rat was dissected and placed in 1 mL of pre-warm Ham’s F10 culture medium for 30 min. The swim-out spermatozoa were analyzed for count, motility, morphology and viability.
Results: The results showed that almost all of the sperm parameters except non progressive motility were significantly different between groups (p=0.001). Also, the mean of sperm parameters in Acrylamide+ Vitamin C+ Glycyrrhiza glabra group was higher than other groups.
Conclusion: This study showed that the co-administration of vitamin C and Glycyrrhiza glabra as antioxidant can reduce the detrimental effects of acrylamide on sperm parameters in rats.
Key words: Glycyrrhiza glabra, Vitamin C, Acrylamide, Sperm, Rat.
P-84
Infertility in male by neurological disorders
Mollaei AR, Seihei E.
Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
Email: ptalireza@yahoo.com
Introduction: Normal sexual and reproductive functions depend largely on neurological mechanisms. Neurological defects in men can cause infertility through erectile dysfunction, ejaculatory dysfunction and semen abnormalities.
Materials and Methods: Science Direct, PubMed, Cochrane, CINAHL, Embase, ProQuest Dissertations, Scopus, (2000-2015) were searched for English-language studies using a list of keywords. The books about physical therapy and medical and neurological were studied too.
Results: Among the major conditions contributing to these symptoms are pelvic and retroperitoneal surgery, diabetes, congenital spinal abnormalities, multiple sclerosis and spinal cord injury. Erectile dysfunction can be managed by an increasingly invasive range of treatments including medications, injection therapy and the surgical insertion of a penile implant. Retrograde ejaculation is managed by medications to reverse the condition in mild cases and in bladder harvest of semen after ejaculation in more severe cases.
Conclusion: An ejaculation might also be managed by medication in mild cases while assisted ejaculatory techniques including penile vibratory stimulation and electro ejaculation are used in more severe cases. If these measures fail, surgical sperm retrieval can be attempted. Ejaculation with penile vibratory stimulation can be done by some spinal cord injured men and their partners at home, followed by in-home insemination if circumstances and sperm quality are adequate. The other options always require assisted reproductive techniques including intrauterine insemination or in vitro fertilization with or without intracytoplasmic sperm injection. The method of choice depends largely on the number of motile sperm in the ejaculate.
Key words: Ejaculation, Electro ejaculation, Infertility, Nervous system diseases.
P-85
Adenosine deaminase G22A polymorphism and recurrent spontaneous abortions
Mohammadi A1, Bonyadi M1, Khalaj kondori M1, Taghavi S2.
Introduction: Adenosine deaminase (ADA) is an enzyme of purine salvage pathway and has two important isoenzymes ADA1 and ADA2. The adenosine deaminase G22A polymorphism (ADA*2) increases the level of adenosine. Adenosine may play a protective role against recurrent spontaneous abortions (RSA), since it regulates blood flow into the uterus and placenta.
Materials and Methods: A total of 100 women were classified in two groups: First group, with a history of recurrent spontaneous abortions (n=50), and second one, without a history of abortions (n=50). Genomic DNA was extracted from peripheral blood with a commercial kit and PCR-RFLP analysis was used to identify the G22A genetic polymorphism.
Results: The frequency of homozygotes (AA) was 2% in control group, whereas no homozygote (AA) was found in the case group (p>0.05). The frequency of heterozygotes (AG) was 20% in the control group and 8% in the case group (p<0.05). The frequency of homozygotes (GG) was 78% in the control group and 92% in the case group (p<0.05). A significant increase in the frequency of AG genotype in controls (p<0.05, OR=0.348) relative to women with the history of RSA demonstrates the protective effect of AG genotype in controls.
Conclusion: The data suggest that women carrying the ADA*2 allele which is associated with the lower enzymatic activity are better protected against recurrent spontaneous abortions. Because decreased enzymatic activity increases adenosine levels. High adenosine levels may play a protective role against recurrent spontaneous abortions, since it regulates blood flow into the uterus and placenta.
Key words: PCR-RFLP, Recurrent spontaneous abortion, Adenosine deaminase.
P-86
Seminal bacterial contaminations: Probable factor in unexplained recurrent pregnancy loss
Nabi A, Khalili MA, Halvaei I, Ghasemzadeh J, Zare E.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: ali.nabi67@yahoo.com
Introduction: It is estimated that about 50% of causes of recurrent pregnancy loss (RPL) cases remain unknown. Sperm factors are suggested to have probable role in cases with RPL. The goal was to determine the possible relationship between semen bacterial contaminations with unexplained RPL. Also, the correlation between number of bacterial colony and sperm chromatin condensation was examined.
Materials and Methods: This study consisted of 30 fertile men (group A) and 30 infertile men (group B) with unknown RPL in their wives. Semen collection and analysis were done according to WHO manuals. Sperm count and motility were evaluated by Makler chamber. Eosin-Nigrosin and Papanicolaou staining methods were applied for viability and morphology assessment, respectively. The semen samples from both groups were cultured for aerobic bacteria. Aniline blue (AB) and toluidine blue (TB) staining methods were applied for evaluating sperm chromatin condensation.
Results: The numbers of colonies were significantly higher in group B when compared to group A. Also, S. aureus and E. coli contaminations showed significant differences between two groups. Both AB+ and TB+ sperm cells showed significant increase in group B compared to group A. There was a significant negative correlation between colony number and progressive motility (p=0.01), and sperm viability (p=0.007). In addition, positive correlations were found between colony number and AB+ (p=0.001) and TB+ (p=0.004) as well.
Conclusion: Bacterial contaminations in semen of men from RPL couples had significantly higher levels when compared to fertile controls. Presence of microorganisms in semen may be correlated with irregular sperm parameters and quality.
Key words: Recurrent pregnancy loss, Bacteria, Semen.
P-87
Precedent infertility and attitudes towards preferred Cesarean
Naeimi N.
Department of Biology, Faculty of Science, University of Sistan and Baluchestan, Zahedan, Iran.
Email: nnaeimi@rocketmail.com
Introduction: Prevalence of natural delivery and caesarean section in pregnant women of a country is one of the indicators of health system performance. Currently, we are exposure to increasing elective Cesarean with different reasons and attitudes.
Materials and Methods: In this descriptive-analytical study, 271 pregnant women completed a researcher-made questionnaire including, demographic characteristics (age, precedent of infertility) and 10 attitude questions about the influencing factors on choosing Cesarean, based on five Likert scale which was a score of 10-30 negative attitude (opposite preference for Cesarean) and 31-50 positive attitude (positive preference for Cesarean). Therefore, to analyze the obtained data, Chi-square test was used through applying SPSS19 software.
Results: Overall 74 subjects (28%) had negative attitudes, while 192 subjects (72%) had positive attitudes. In total 7 subject of pregnant women group (2.6%) had a precedent of infertility and 259 pregnant women had not (95.6%). During the study, 5 subjects did not answer questions. In addition, 5 subjects with precedent infertility (73.4%) vs. 69 subjects (26.6%) with no precedent, showed negative attitudes. 2 subjects (26.6%) vs. 190 pregnant women (73.4%) had positive attitude toward preference of Caesarean section. There was a significant relationship between precedent infertility and attitude.
Conclusion: According to the results, women with precedent infertility who are using assisted reproductive techniques, are more aware of advantages and disadvantages of different methods of termination of pregnancy and most of them opposed natural delivery. Therefor giving them information through related groups of women's health, can change their attitudes.
Key words: Precedent infertility, Attitude, Preference cesarean.
P-88
Protective effects of BDNF against oxidative damage and apoptosis in human spermatozoa
Najafi A1, Sobhani AGH1, Amidi F1, Sadighi Gilani MA2, Moawad AR3, Mikaeili S1, Fallah P2, Rezaiian Z2.
Introduction: Oxidative Stress (OS) is a condition when there is an improper balance between the amount of pro-oxidant substances and the amount of seminal plasma antioxidant factors that can be induced by repeated cycles of centrifugation and seminal plasma antioxidants, removal during the sperm preparation in Assisted Reproductive Technology (ART) Reactive Oxygen Species (ROS) can cause decrease of motility, increase of apoptosis and impaired sperm function, and ultimately, influence on fertilization and pregnancy rate. Therefore, currently there is a great interest to the use of antioxidants to prevent ROS generation and ROS-induced apoptosis during the sperm preparation processes. Brain-derived neurotrophic factor (BDNF) is member of neurotrophin family that has anti- oxidant and anti-apoptotic effects on nervous system. Recent researches show that it also plays key role in male and female reproductive system such as spermatogenesis, nuclear and cytoplasmic maturation and embryo development.
Materials and Methods: Liquefied semen samples obtained from normozoospermic men (n=25) after preparation by double wash (400× gr, 5 min) swim-up technique were divided in to two groups of control and treated group (with BDNF). Then motility was evaluated by means of CASA. Reactive oxygen species (ROS) and apoptosis were assessed by flowcytometry.
Results: Addition of BDNF to the sperm media significantly increased mean total motility and progressive motility (75.54±2.63% vs. 66.81±2.48%, p<0.001, and 46.18±2.2 vs. 39.631±1.97%, p<0.002, respectively). Also BDNF treatment caused decrease of H2O2 (50.01±6.08 vs. 38.31±5.250, p<0.02), necrosis (23.26±2.71 vs. 27.52±2.27, p<0.001) and apoptosis (3.26±0.4 vs. 3.76±0.55, p<0.49).
Conclusion: Considering this result, BDNF treatment can be a potential tool against oxidative damage and apoptosis in human spermatozoa.
Key words: Brain-derived neurotrophic factor (BDNF), ROS, Apoptosis, Flowcytometry.
P-89
Iranian couple’s sexual compatibility during the time: A qualitative study
Nekoolaltak M1, Keshavarz Z1, Simbar M2, Nazari AM3, Baghestani AR4.
Introduction: Since sexual compatibility is facilitators for marital compatibility and the root of many divorces is the sexual incompatibility, and due to the limited data available in this topic, this qualitative study was designed to exploring the changes of Iranian couple’s sexual compatibility during marriage years.
Materials and Methods: In-depth interview and written narrative methods were used. Purposive sampling with maximum variation (age, education, years of marriage, number of children, etc.) was conducted. Ale Yasin Clinic in Tehran and Fatemieh Clinic in Varamin were the setting of research. Times of interviews were almost 40 min. Data reached to saturation after 14 semi-structured depth interviews and 54 written narratives with the 68 married men and women. Conventional content analysis was done in MAXQDA10 software. Lincoln and Guba criteria were used to validate qualitative data.
Results: Emerged categories included: “duration to reach sexual compatibility" and “mainly increasing sexual compatibility during the time" and "rarely decreasing sexual compatibility during the time". Couple’s sexual compatibility increases over time due to “Getting to know each other more", “increasing sexual awareness and experience”, “talking about sex with spouse”, “skill acquisition for mutual pleasure and satisfaction”. Couple’s sexual compatibility may decrease over the time because "children growing and conflict parental role with spousal role" and “financial difficulties”.
Conclusion: With knowing the impact of passing the time on couple’s sexual compatibility many divorces can be prevented by suitable planning and counseling.
Key words: Sexual compatibility, Iranian couples, Qualitative study.
P-90
Effect of non-polar extract of Phaleria macrocarpa (Mahkota Dewa) on spermatogenesis in rats
Parhizkar S1, Aziz Dollah M2, Ferdaus Binti N2.
Introduction: Fertility is the natural capability of giving live. In male, fertility requires the production of large numbers of normal and mature spermatozoa by testes through a complex process call spermatogenesis. Evidence shows increase male age is associated with the decline of mature sperm in seminiferous tubule. Aqueous extract of Phaleria macrocarpa (PM) has been shown to improve fertility in rats by increasing the spermatogenesis.
Materials and Methods: Hexane extract of Phaleria Macrocarpa was prepared by extracting grinded dried slices of fruit followed with drying of the extract using rotary evaporator. A total of 30 male rats were divided into 5 different groups and subjected to daily treatments with PM extract at one of the following concentration: 0, 6, 12, 60 mg/kg of hexane extract, or commercial testosterone hormone for seven consecutive weeks. They will be fed with pellet once a day in the morning and drinking water will be given ad libitum. Body weight was measured once a week. On the last day of supplementation period, the rats were sacrificed and the testis was isolated for histological evaluation.
Results: The result showed that no significant effect of non-polar extract of PM on testes size and volume, thickness of seminiferous tubules and spermatogonia cell number. The mean thickness of seminiferous tubules were 62, 68, 62, 60, 60 µm for 60, 12, 6, 0 mg/kg of PM extract and commercial testosterone respectively. The mean for spermatogonia cell count in rats treated with 60, 12, 6 and 0 mg/kg and commercial testosterone were 52.8, 43.8, 47.0, 52.6, 52.0 cells respectively.
Conclusion: The study concludes that the non-polar extract of PM has no effect on the fertility of male.
Key words: Phaleria macrocarpa (PM), Spermatogenesis, Rat.
P-91
The functional SNP analysis of CYP2D6 gene in patients with Polycystic ovary syndrome (PCOS)
Peirosabet M1, 2,Khosravifar M2, Chekini Z2, Rahimi A1, 2, Shiva M3, Afsharian P2.
Introduction: PCOS is a common ovulatory disturbance, which influents about 5-10% women of reproductive age. Clomiphene citrate is used for ovulatory induction in PCOS, that is metabolized by Cytochrome P450 2D6 (CYP2D6). More than 120 variable alleles have been reported for CYP2D6 that some of them are poor metabolizer and cause resistance to drugs that are substrates of CYP2D6.
Materials and Methods: Whole blood was collected from drug control, PCOS and fertile women (60 in each group). RFLP method was used to determine CYD2D6*34 (2850C˃T). Patients should have the Rotterdam criteria. Inclusion criteria’s were age under 35 years old, candidates for IUI, not using other drugs and not have ovary cautery.
Results: Genotype distribution of CC, CT and TT in PCOS group was 47.4%, 40.4% and 12.3% respectively (p=0.479), and frequency of these genotypes in drug control group was 36.7%, 50% and 13.3% respectively (p=0.479), which was very close to results in fertile group: 33.3%, 56.7% and 10% respectively. The number of antral follicles (follicle diameter≥15mm) was measured as drug response which was 1.21 (p=0.118) in clomiphene citrate administered PCOS patients (64%) while this was 1.64 (p=0.09) in 36% of patients treated with letrozole (follicle diameter≥15mm). Same results were observed in drug control group: antral follicles were 1.66 in 80% of clomiphene citrate treated whereas it was 1.77 when they received letrozole (20% of patients).
Conclusion: Although genotype frequencies of 2850C˃T polymorphisms were not significant between three groups, we observed cases bearing this polymorphism, showed resistance to clomiphene citrate, in both groups, which is the first time to report.
Key words: CYP2D6, Polycystic ovary syndrome, Clomiphene citrate.
P-92
Does L-carnitine therapy add any extra benefit to standard inguinal varicocelectomy in terms of deoxyribonucleic acid damage or sperm quality factor indices: A randomized study
Pourmand GH1, Movahedin M2, Dehghan S1, Mehrsai A1, Ahmadi A1, Pourhosein M1.
Introduction: Varicocelectomy and anti-oxidant therapy are both documented to have positive effect on spermatogenesis and improving semen quality.
Materials and Methods: 100 patients enrolled in this study and were randomly divided into 2 groups (50 patients in each group). In group 1, standard inguinal varicocelectomy and, in group 2, standard inguinal varicocelectomy plus oral antioxidant therapy (oral L-carnitine, 250 mg 3 times a day) were performed for 6 months. For all patients, routine semen analysis and DNA damage test of spermatozoa (by 2 methods of terminal deoxynucleotidyl transferase dUTP nick end labeling and protamine damage assay) were performed at baseline and at 3 and 6 months postoperatively.
Results: In both groups, the improvement in semen analysis parameters and DNA damage was observed, but there was not any statistically significant difference between the 2 groups in these parameters, although the slope of improvement in DNA damage was slightly better in group 2 (that was not statistically significant).
Conclusion: We observed that addition of 750 mg of L-carnitine orally daily to standard inguinal varicocelectomy does not add any extra benefit in terms of improvement in semen analysis parameters or DNA damage.
Key words: DNA damage, Varicocelectomy, L-carnitine therapy.
P-93
Different haplotype frequency in AKAP3 gene in Iranian patients with short tail sperm
Poursafari E1, Mohseni Meybody A2, Sabbaghian M2, Modarresi T2, Hosseini H1, Gourabi H2.
Introduction: One kind of sperm abnormalities that leads to men infertility is short flagella of sperms. In this defect, fibrous sheath and axoneme are disorganized, the sperms tail is short, the numbers of sperms in the semen fluid reduce and the sperms are immotile. A Kinas anchoring protein 3 (AKAP3) gene encodes a protein that is involved in the fibrous sheath structure, regulation of sperm motility and head-associated functions such as capacitation and the acrosome reaction. AKAP3 interacts with the regulatory subunit of Protein Kinas A via its dimerization/ docking domain. In the present study, 30 patients with short tail sperm defect and 40 males with normal spermogram referred to Royan Institute were enrolled as case and control groups respectively. The genetic variation in exon 5 of AKAP3 gene which encodes the functional domain of this protein was studied.
Materials and Methods: PCR- sequencing was done on extracted DNA from blood samples of control and patient groups.
Results: According to the results, four haplotype polymorphisms 1378 T>C, 1391 C>G, 1437 T>C and 1573 G>A were observed in all samples studied. These polymorphisms were all observed as the mutant alleles. 92% C allele and 8% G allele of 1391 C>G polymorphism has been reported in East Asia. In the present study, 100% G allele was observed in this polymorphism.
Conclusion: This difference in frequency of mutant allele can be due to different ethnic of Asian population and ours. 1391 C>G alternation is located on the outside of the AKAP3 binding domain to AKAP4 and seems that it cannot have a role in the interaction between these two proteins.
Key words: Short tail sperm defect, Fibrous sheath, AKAP3 gene, PKA, Polymorphism.
P-94
Study of genetic alterations of STK11 in patients with polycystic ovary syndrome (PCOS) and their response to ovarian stimulation
Rahimi A1, 2, Khosravifar M2, Peirosabet M1, 2, Chekini Z2, Shiva M3, Afsharian P2.
Introduction: PCOS is the main reason of anovulatory caused infertility. Metformin use for PCOS patients to reduces hyperinsulinemia and androgens production through activation of STK11. We aimed to study the associations between c.842C>T and c.996G>A polymorphisms and risk of PCOS susceptibility in women plus the relations of two polymorphisms and response to ovarian stimulation in PCOS patients.
Materials and Methods: Blood samples were collected from patients and drug control group (male infertility) and fertile group (60 in each group). For genotype analysis, we used RFLP method. We used Rotterdam criteria for patient selection. Inclusion criteria's were age below 35 years old, candidates for IUI, not using other drugs and not have ovary cautery. Our data shown that frequency distribution of CT, TT,CC genotypes in exon6, in PCO Patients was 1.7%, 0%, 98.3% respectively (p=0.236), these frequencies in fertile group was 5%, 0%, 95% respectively and in drug control group was 0%, 1.7%, 98.3% respectively (p=0.236). We have not detected c.996 G>A polymorphism in any groups.
Results: The mean of age and BMI were 26.31±3.68 years and 27 kg/m2 in PCOS and control group. Most of PCOS patients (64%) were received clomiphene citrate and 36% used letrozole whereas 80% of drug control was treated by clomiphene citrate and 20% used letrozole. In both groups their responses to drugs was measured by the number of antral follicles (follicle diameter ≥15mm) by ultrasonography that was 1.64 in PCOS, p=0.09 and 1.77 in drug control treated with letrozole that were better than clomiphene citrate (1.21 in PCOS, p=0.118 and1.66 in control).
Conclusion: Our data did not find any significant differences between these polymorphisms and response to ART medicines in PCOS women. According to our study, PCOS patients had higher response to letrozole rather than clomiphene citrate.
Key words: STK11, Polycystic ovary syndrome, Polymorphism.
P-95
Effects of electromagnetic fields of magnetic resonance imaging on male mice fertilization
Rostamzadeh A1, 2, Anjamrooz SH1, Fathi F1, Rezaie MJ1,3.
Introduction: Currently, the use of electromagnetic waves in medicine, especially in diagnostic devices such as magnetic resonance imaging (MRI) has increased and many of its biological effects have been reported. The aim of this study was to investigate the adverse effects of 1.5T MRI on fertility and reproductive parameters of male mice.
Materials and Methods: 40 NMRI adult male mice were randomly divided into two groups of control and experimental. The mice in the experimental group were exposed to MRI at 1.5T for 36 min once a week for a period of 3 weeks. Then, in the 1st day and 35th day after the final exposure, 10 mice were used for IVF and 10 mice for In vivo studies. MRI effects on testis weight, the duration of pregnancy, the number of newborns, sperm count, and fertility were evaluated. The obtained data were analyzed using ANOVA and Tukey’s tests.
Results: According to the present study, one day after MRI exposure, testis weight, sperm count, and the number of born children were significantly decreased in the experimental group (p<0.05). Moreover, a significant number of the embryos failed to develop to the blastocyst stage. In contrast, 35 days after exposure, no statistically significant difference was found (p>0.05).
Conclusion: Based on the results of the present study, it seems that although the MRI at 1.5T has adverse effects on fertility and reproductive parameters of the adult male mice, these side-effects are reversible.
Key words: Electromagnetic fields, MRI, Sperm, Fertility.
P-96
Interference of histone modification with aberrant expression of HOXA10 gene in eutopic and ectopic endometrial tissues of women with endometriosis during menstrual cycle
Samadieh Y1, Shahhoseini M1, Favaedi R1, Mahdian S3, Ramezanali F2, Ashrafi M2, Afsharian P1, Aflatoonian R2.
Introduction: Disruption of the balance of epigenetic networks, which involves DNA and histone modifications, can cause several pathologies, including reproductive disorders such as endometriosis. HOXA10 gene expressed in endometrium plays an important role in uterine receptivity at the time of implantation, uterine organogenesis and functional endometrial differentiation.
Materials and Methods: Epigenetic analysis were assayed by Chromatin Immunoprecipitation (ChIP), using anti- H3K9ac, H3K9Me2, H3K27Me3, H3K4Me3 antibodies and quantitative expression analysis was performed by real-time PCR technique. For this respect, eutopic and ectopic endometrial samples were collected using laparoscopy from 36 women with documented endometriosis, and also endometrial biopsies were obtained from 22 healthy women with male factor problem as a control group. Ethical approval and informed patient consent was gained for the use of tissue samples.
Results: Data showed a harmonious pattern between mRNA expression of HOXA10 and epigenetic state of its promoter region, in the way that, in secretory phase the activating epigenetic marks, H3k9ac and H3k4me3 were higher in ectopic endometrial tissues and H3K9ac itself were lower in eutopic endometrium. In contrast, H3K9me2 and H3k27me3, the epigenetic marks, known to be associated with gene repression, showed a different pattern in which that, H3K9me2 were higher in eutopic endometrium and H3K27me3 were lower in ectopic endometrial tissues.
Conclusion: Our findings suggest epigenetic might be greatly responsible for aberrant expression of HOXA10 gene in eutopic and ectopic endometrial lesions of patients with endometriosis.
Key words: HOXA10 gene, Endometriosis, Menstrual cycle, Histone modifications, Epigenetics.
P-97
Evaluation of action and mechanism of L-Carnitine in improved sperm quality in male infertility
Sheikhpour R.
Yazd Medical Science Branch, Islamic Azad University, Yazd, Iran.
Email: R.sheikhpour@yahoo.com
Introduction: About 15% of married couples are infertile and that approximately 50% of this is due to male factor infertility. A number of drugs have been proposed as being possible causes of male factor infertility. Estudies showed that a diet supplemented with L-carnitine can improve sperm quality in some mammalian species. In humans, 75% of carnitine derives from diet, while 25% is synthesized from lysine and methionine, although the enzyme that catalyses the hydroxylation of the 4-butirrobetain in L-carnitine is present in few tissues.
Materials and Methods: This review was conducted using the following databases: Medline, Pubmed, Scopus and Science direct from 1990-2014 for better understanding of the mechanism and action of L-Carnitine in male infertility.
Results: L-Carnitine can increase the concentration of this component in the epididymal tubules and spermatozoa. This molecule increases their motility and fertilizing abilities. Both free L-carnitine and acetylated L-carnitine can be accumulated in spermatozoa. In sperm cells, L-carnitine transports fatty acid to mitochondria for oxidation and production of energy for epididymal spermatozoa. Also L-Carnitine has antioxidant property; it via deleting excess acyl-CoA (formyl-CoA), due to toxic effect have important role in cellular detoxification. L-carnitine as antiaging protects cellular membranes against oxidative damage. It prevents protein oxidation, pyruvate and lactate oxidative damage. During sperm maturation, a reduction in total lipid content has been seen due to the changing composition of fatty acids in sperm cells. This leads to an increase in the fluidity of spermatozoa’s plasmalemma. Moreover, L-carnitine reduces lipid availability for peroxidation which guards against potential peroxidative damage.
Conclusion: The result of studies showed that addition of L-carnitine to the diet of male infertility is effective in increasing semen quality, but these results need to be confirmed by more clinical trials.
Key words: L-Carnitine, Sperm quality, Male infertility.
P-98
Effects of cell phone radiofrequency electromagnetic waves (RF-EMW) on human spermatozoa
Soltani S1, Khalili MA1, Omidi M1, Nabi A1, Shamsi F2.
Introduction: Cellular phones emit radiofrequency electromagnetic waves (RF-EMW) in the low frequency microwave. The detrimental effects of RF-EMW on the reproductive system and human fertility have been debated in recent years. The goal was to assess the influence of cell phones RF-EMW on different parameters and DNA integrity of human spermatozoa.
Materials and Methods: 50 semen samples were categorized in two groups: a) normospermia (n=24); and b) asthenozoospermia (n=24). After liquefaction, each sample was divided into two aliquots. One aliquot (experimental) was exposed to mobile radiation for 1 hr, and the second aliquot (unexposed) served as control. The sperm parameters and chromatin/ DNA integrity were examined and compared between groups.
Results: Normal samples exposed to RF-EMW showed insignificant differences in sperm motility, viability, and chromatin/DNA integrity. Regarding sperm morphology, normal morphology was significantly decreased in experimental group (p=0.04). Moreover, sperm parameters as well as DNA structure showed no significant differences in astenozoospermic samples.
Conclusion: Data did not show any noticeable impact of RF-EMW on human sperm quality. Further studies should be considered for confirmation of the results.
Key words: Cell phone, Electromagnetic radiation, Sperm parameters, DNA integrity.
P-99
Assessment of genetic variations in intron4 and exon5 of RABL2B gene in Iranian infertile men with immotile short tail sperm defect
Hosseini H1, Sabbaghian M2, Mohseni Meybodi A2, Hosseinifar H2, Poursafari E1, Sadighi Gilani MA2.
Introduction: The immotile short tail sperm (ISTS) defect, is a syndrome which causes male infertility. Patients with ISTS disorder have immotile short-tailed sperm with disorganized axonem, and a significant decrease in sperm counts too. Numerous proteins are involved in sperm tail formation. One of these proteins is RAB Like 2B (RABL2B), which recently its essential role in sperm intra-flagellar transport and fertility in male mouse has been demonstrated. So its gene, which called RAB Like 2B (RABL2B), is an appropriate candidate gene in human studies. RABL2B protein has 4 GTP binding domains which have important roles in protein function. Exon 5 of RABL2B gene, codes one of these main domains and intron 4 is the location for binding to some important transcription factors. The purpose of this study was to evaluate the genetic variations of exon 5 and intron 4 of RABL2B gene in infertile men with ISTS defect and controls
Materials and Methods: In this study, 30 infertile men with ISTS defect and 30 normozospermic men as controls were recruited. Remarkably it took 2 years to collect patients samples. To study the genetic variations, DNA was extracted from peripheral blood, then PCR sequencing was done.
Results: Sequence analysis results did not identify any mutations or single-nucleotide polymorphisms (SNPs) in exon 5, but an intronic variant (rs:144944885), was found in heterozygote form in one patient. No mutations or SNPs was identified in controls.
Conclusion: Although our data just revealed an intronic variations and no mutations or SNPs was detected in exon 5, due to the high expression of RABL2B gene in testis, and considering the fact that RABL2B is evolutionarily conserved and not many studies have been conducted about the exact role of this gene in human male fertility, evaluation of other exons and regulatory areas of this gene is strongly recommended.
Key words: RABL2B gene, ISTS, Male infertility.
P-100
Localization of septin 14 protein in sperm
Vahabi Barzi N, Hosseinifar H, Nickhah Kalashami Z, Sadighi Gilani MA, Sabbaghian M.
Department of Andrology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: nastaranbarzi@yahoo.com
Introduction: In mammals, 14 septin genes have been identified so far. Disruption of septin functions has been implicated in the pathology of many diseases, including male infertility. Here, we study about one of the new members of the septin family called septin14 that is specially expressed in testis. This gene has two transcripts but only one of them transcript into proteins.
Materials and Methods: In this study, semen obtained from subjects attending Royan Institute that had normal spermogram. After that, immunocytochemistry, which is a common laboratory technique, was used to anatomically localize presence of a specific protein in cells by a specific primary antibody. The primary antibody allowed visualization of the protein under a fluorescence microscope when it was bound by a secondary antibody with a conjugated fluorescence.
Results: The protein expression was detected in the sperm from head to tail and highly localized in the front of the acrosome and the neck.
Conclusion: This is the first report on the localization of septin 14 in sperm. Septin 14 plays an important role in sperm morphology. Regarding the presence of this protein in sperm acrosome and neck, it can be concluded that the probable decrease of septin 14 protein expression in sperm may be associated with the pathogenesis of male infertility.
Key words: Septin 14, Immunocytochemistry, Sperm, Male infertility.
P-101
Molecular studying of sperm surface proteins and their cellular functions
Vaseghi-Dodaran H1, Daghighkia H1, Atshan M2, Jafari S3, Sobhani AGH4.
Introduction: Fertilization in mammalian is a complicated process with some connections among wide range of glycolipid, glycoprotein and antigenic indexes on sperm and egg surface of every species. We can improve our understanding in fertilization mechanisms and also get some information on molecular or immunological defects responsible for infertility by recognizing the antigens on sperm surface and investigating their role on cell physiological performance. The key challenge is to move from lists of identified proteins to informed understanding of biological function. Studying the sperm surface molecules and investigating their biochemical, biophysical and physiological properties to understand fertilization process.
Materials and Methods: A review of recent bibliography collected from internet database as PubMed, Google scholar, SID by the use of relevant keywords on different markers of cell surface and Sperm cell proteomic published in 2000-2014.
Results: Sperm membrane proteins contain an important part of membrane and are considered as special antigenic index to each species. Every change, increase or decrease in these proteins alters sperm performances and abilities. The important events which occurs during the interaction of sperm and egg and the reactions between them for fertilization relates to identification of these two cells by connecting receptors to ligands which exist on their cell membrane. Discovering the proteins on sperm surface which act as fertility biomarkers and participate in fertilization leads to more apprehension of this cell physiology and performance, so understanding molecular events like sperm acrosome reaction, capacitation and motility.
Conclusion: Sperm surface proteins can use as fertility biomarkers, therefore identification and supplementation these proteins to cryopreservation medium can improve post-thaw parameters of sperm and prevent sperm quality during cryopreservation.
Key words: Proteomics, Sperm, Marker, Fertilization.
P-102
Frequency of chromosome inversion (pericentric and paracentric) in recurrent abortions in patients referred to cytogenetics laboratory of Sarem Hospital
Abdi A1, Bagherizadeh I2, Bahman I2, Shajarehpour L1, Hadipour F3, Hadipour Z3, Shafagh Y4, Behjati F5.
Introduction: Recurrent abortion affects almost 15% of diagnosed pregnansis. More than 50% of miscarriages in the 1st trimester are due to chromosome abnormality. The aim of this study is to present the role of chromosomal inversions in recurrent miscarriages in patients referred to the Cytogenetics Laboratory of Sarem Hospital in Tehran. One group of inversions is pericentric inversion of chromosome 9, which is usually regarded as a normal population variant.
Materials and Methods: The samples were studied using high resolution GTG banding technique. For each patient, a minimum of 15 metaphases was examined by light microscopy.
Results: Pericentric inversion around centromere of chromosome 9 was observed in 29 patients (1.37%) and pericentric inversion in hetrochromatin region was found in chromosome 1 in one patient and chromosome Y in another patient, and one patient had pericentric inversion of chromosome 2. Chromosomal inversion involving other autosomal chromosomes included pericentric inversion of chromosomes 1, 5, 11 and 12 (1.9%), and paracentric inversion of chromosomes 3, 6, 7, 8, and 12 (0.23%).
Conclusion: The chromosomal imbalance of gametes may produce spontaneous abortions and malformed offsprings. This suggests that such inversions should not be ignored and they can play an important role in reproduction failure. However, we have shown that the rate of pericentric inversion of chromosome 9 is similar in different referral groups (1-2%) and similar to normal population and thus of no clinical significance.
Key words: Pericentric and paracentric chromosome inversions, Recurrent abortions, inv (9).
P-103
Kit Ligand (KL) promotes the primordial follicle growth in mouse vitrified ovaries
Abdi Sh1, Salehnia M1, Hosseinkhani S2.
Introduction: Following cryopreservation of ovarian tissue, the primordial follicles are well preserved with minimal damage. An alternative technique to improve the development of follicles within ovarian tissue is ovarian organ culture following cryopreservation. Kit ligand (KL) known as stem cell factor, steel factor or mast cell growth factor is involved in the activation of primordial follicles, oocyte growth and proliferation of granulosa and theca cells.
Materials and Methods: One week old mouse ovaries were collected and divided into vitrified and non-vitrified groups. Then they were cultured in the presence or absence of KL for 7 days. The development of ovarian follicles was evaluated by histology and also the mean area and hormonal level was analyzed during culture period. Apoptosis assessment was done using DNA laddering, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) method and caspase -3/7 activity assay.
Results: The proportion of preantral follicles and the level of hormones were increased in all cultured groups and it was significantly higher in KL treated groups than their control (p<0.001). DNA fragmentation and TUNEL positive signals were seen in vitrified cultured ovaries. The level of caspase -3/7 activity was higher in vitrified cultured ovaries.
Conclusion: KL could improve the development of follicles in vitrified cultured ovaries also it could act as anti-apoptotic factor during culture of vitrified samples. The development potential of follicles in vitrified groups was lower than fresh ovaries.
Key words: In vitro culture, Kit Ligand, Vitrification, Caspase -3/7.
P-104
Association between rs1264457 A/G polymorphism of HLA-E gene and recurrent pregnancy loss
Zarezade Z1, 4, Ashrafzadeh Mehrjardi HR2, 4, Zaimy MA3, Ghasemi N4.
Introduction: Recurrent pregnancy loss (RPL) is defined as two or more consecutive miscarriages before twentieth week of pregnancy. The cause of 50% recurrent miscarriages is unexplained. The goal of this research was to investigate the association between RPL and rs1264457 A/G polymorphism of HLA-E gene.
Materials and Methods: In this case-control study we used Polymerase Chain Reaction Restriction Fragment Length Polymorphism (RFLP-PCR) to determine frequency of this polymorphism in 105 women with RPL in comparison with 109 healthy controls. The collected data were analyzed by the statistical package for the social sciences software (SPSS) and the chi-square test.
Results: The finding showed clear correlation between this polymorphism and RPL. The results will be presented in the congress.
Conclusion: If distinctive data increase frequency of this polymorphism in RPL, it can exponent influence of this polymorphism in generating RPL. We hope result of this investigation to be useful for finding some causes of unexplained recurrent pregnancy loss.
Key words: Recurrent pregnancy loss, HLA-E, Polymorphism.
P-105
Evaluation of the meiotic spindle and zona pellucida after vitrification of mouse MII oocytes
Akyash F, Khalili MA, Safari S, Ashourzadeh S, Agha-Rahimi A, Khoradmehr A.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: fakyash@gmail.com
Introduction: Oocyte vitrification is used as fertility preservation method for young aged females with cancer. The oocyte is one of the biggest mammalian cells with large volume of cytoplasm, compares with other cells. Also, oocyte has less permeability to cryoprotectants (CPAs), since high concentration of CPAs used in vitrification procedure with possible toxic effects. Evaluation of the effect of vitrificaiton on meiotic spindle (MS) and zona pellucida (ZP) of mouse MII oocytes using Polyscope technology was the subject of this study.
Materials and Methods: Ovulation induction performed for 6-8 weeks old NMRI mice by injection of 10 IU PMSG (IP) and 48 hr later with 10 IU HCG. After oviduct removal, COCs were denuded and MII oocytes were retrieved aseptically. The equilibration (7.5% EG, 7.5% DMSO) and vitrification (15% EG, 15% DMSO) solutions were prepared in Hams F10 supplemented with 20% HSA as base medium. For 3 steps of thawing, base medium plus 1, 0.5 and 0.25 M sucrose were used respectively. Assessment of ZP and MS performed at different time intervals after thawing.
Results: After warming, vitrified oocytes showed a significantly fast ZP digestion timing compared to the control group (p<0.05). Presence of MS was detected in 69.23% of vitrified oocytes 1 hr after warming, and presence of MS was showed in 11.11% of vitrified oocytes 2 hr after warming and 26.92% of vitrified- warmed oocytes showed sign of degeneration.
Conclusion: ZP digestion was faster in vitrified group rather than control and recovery of MS structure after vitrification/ warming at 37oC. Vitrification technique still needs more modification before its application in fertility preservation program.
Key words: Oocyte, Meiotic spindle, Zona pellucida, Vitrification.
P-106
Protective effect of vitamin E on testicular germ cells and serum Malondialdehyde concentration in rats fallowing exposure to bisphenol A
Amjadi M, Soleimani Mehranjani M, Naderi Noreini S.
Department of Biology, Faculty of Sciences, Arak University, Arak, Iran.
Email: m.amjadi1369@gmail.com
Introduction: Bisphenol-A (BPA), used in plastic industries, has toxic effects on the reproductive system. It can cause testis injury through the production of Reactive oxygen species (ROS).
Materials and Methods: 24 adult male rats (220±15 gr) were divided into 4 groups (n=6): control, BPA (250 mg/kg/day), Vit E (150 mg/kg/day) and BPA+ Vit E. All groups were orally treated for 56 days. By the end of the treatment, animals were killed, their right testis were taken out, fixed, sectioned, processed and stained with heidenhain azan method. The total number of germ and Sertoli cells was estimated using the optical dissector technique. Serum MDA levels were also measured with the spectrophotometric method. Data were statistically analyzed using one way ANOVA and Tukey's test and means were considered significantly different at p<0.05.
Results: A significant decrease in the total volume of testis, number of long and round spermatids, spermatocytes and Sertoli cells (p<0.05) and a significant increase (p<0.03) in the serum MDA level were found in rats treated by BPA compared to the control group. Histopathology observations revealed morphological changes in BPA-exposed rats including atrophy and vacuolation in the germinal epithelium. The above parameters were compensated to the control level in the BPA+ Vit E group.
Conclusion: Due to the antioxidant activity of vitamin E, it may ameliorate the damaging effects of bisphenol A on spermatogenesis.
Key words: Bisphenol A, Vitamin E, Sertoli and germ cells number, Optical disector.
P-107
Evaluation of follicular genes pattern and growth of preantral follicles after culture in alginate hydrogel following vitrification of the mouse ovarian tissue
Asgari F1, Valojerdi MR1, 2, Ebrahimi B 2.
Introduction: This study was set up to evaluate the effect of ovarian tissue vitrification on the in vitro growth and pattern of follicular genes expression in mouse preantral follicles encapsulated within alginate hydrogel.
Materials and Methods: Ovaries of 12-14 days old female NMRI mice allocated into fresh control and vitrification groups. For cryopreservation, ovaries equilibrated with a solution (ES) that composed of 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 15 min and vitrification was performed by a solution (VS) that composed of 15% EG and 15% DMSO for 30 min then ovaries loaded to nitrogen with needle. Descending concentrations of sucrose (1, 0.5, 0.25 M) used for warming step. After histologic assessment of ovaries, in the next stage, pre-antral follicles was mechanically isolated from control and vitrified ovaries and was cultured for 12 days in 0.7% sodium alginate. Preantral follicles survival rate, growth, antrum formation and relative expression of oocyte- specific genes (Bmp15, Gdf9،Fgf8, Igf1, Kit, Kit ligand) was assessed after 1, 8 and 12 days of culture and finally maturation rate of oocytes was studied.
Results: Preantral follicles in vitrified group showed a lower survival rate on 8 and 12 days of culture (p<0.05) but could retain a comparable morphological appearance, growth and antral formation with the control group. Reduction of Bmp15, Gdf9, Fgf8, Kit, Kit-l showed during 12 days of culture (p<0.05). Although the expression of Gdf9, Kit, Kit-l in vitrification group was more than control group at 1st day of culture but all genes in both groups showed same expression after 12 days of culture (p<0.05).
Conclusion: Although vitrification of ovarian tissue reduces the survival rate, it is a safe method for preservation of preantral follicles and could not modify the relative expression of follicullar genes and oocytes maturation capacity.
Key words: Follicular genes, Preantral follicle, Vitrification of ovarian tissue, Alginate.
P-108
Effects of morphine on sperm parameters, protamine deficiency and DNA integrity in mice
Bahari H1, Talebi AR1, 2, Nahangi H1, Anvari M1, 2, Abbasi Sarcheshmeh A1, Khoradmehr A2, Danafar AH2.
Introduction: Morphine as a natural alkaloid (opiate) is the most effective pain-relieving drugs and can be abused because of its high addictive potential. Opiate abuse is considered as one of the problems associated with poor semen production and sperm quality. Therefore, for the first time, this experimental study was carried out to evaluate the effect of intraperitoneal injection of morphine on sperm parameters, protamine deficiency and DNA integrity of spermatozoa aspirated from cauda epididymis of mice.
Materials and Methods: Totally 24 adult male Balb/c mice (8 weeks old. 30g) were equally divided into 3 groups each containing 8 mice. Mice of group 1 served as control fed on basal diet, group 2 received basal diet and normal saline and group 3 received basal diet and morphine (15 mg/kg/daily, intraperitoneal) for 35 days. Finally right tail of epididymis of each mouse was cut and placed in Ham’s F10 for 30 min. Released sperm were used to analyze count, motility, viability (eosin-nigrosin staining), morphology (Papanicolaou), protamine deficiency with chromomycin A3 (CMA3) and apoptosis via TUNEL assay.
Results: In morphine-treated mice a significant decrease was found in sperm viability, normal morphology, count and motility compared to other groups (p<0.05). In relation with protamine deficiency the rates of CMA3-reacted spermatozoa were similar in groups (p>0.05). In addition, in morphine-treated mice there was a significant increase in apoptosis compared to other groups (p<0.05).
Conclusion: The results showed that morphine abuse disturbs sperm parameters and DNA integrity but not protamine content of the sperm nucleus.
Key words: Mice, Morphine, Sperm parameters, Apoptosis, Protamine deficiency.
P-109
Study the protective role of jujube extract on growth index of mice fetuses exposed to Carbamazepine
Doostabadi MR1, Afshar M1, Nahangi H2, Hasanzadeh Taheri MM1, Hassanpour M1.
Introduction: Carbamazepine (CBZ) is an anticonvulsant drug that is widely used, primarily to treat seizures. One of the side effects of this medication during pregnancy is the reduced fetal growth. The jujube has been widely used in traditional medicine because of its special compound. The purpose of this study was to evaluate the preventive effects of aqueous extract of jujube on growth index of mice fetuses exposed to Carbamazepine.
Materials and Methods: In this study, 100 Balb/C mice (28-30 gr; 8-9 weeks-old) were divided into eight experimental and two control groups. Experimental groups (I, III, V) and (II, IV, VI) received daily intraperitoneal injections of 50 and 100 mg/kg of CBZ, respectively. Experimental groups (III, IV) and groups (V, VI) were gavaged doses of 200 and 400 mg/kg of aqueous extract of jujube respectively. Groups VII and VIII received only doses of 200 and 400 mg/kg of aqueous extract of jujube respectively. Control groups I and II were gavaged to normal saline and tween respectively. Injections were done on 0-15 gestational days (GD), and aqueous extract was gavaged along with CBZ starting 10 days prior to gestation. On GD 18, fetuses were removed for weighing and crown-rump measuring. Data were analyzed by ANOVA and Tukey tests using SPSS (version 18) at 0.05%.
Results: Average weight and body length of fetuses in experimental groups I and II, that received CBZ, significantly reduced compared with control groups. In the fetuses of experimental groups V (1.20±0.38) and VI (1.21±0.21 gr) that recieved jujube extract (400 mg/day) with CBZ, the mean weight of these fetuses increased meaningfully in comparison with the experimental groups I (0.93±0.19 gr) and II (0.92±0.26 gr) respectively.
Conclusion: This study showed that aqueous extracts of jujube can have preventive effects on reducing effect of CBZ on birth fetal weight.
Key words: Growth index, Jujube, Carbamazepine, Mice.
P-110
Effectiveness of mindfulness-based cognitive therapy on improvement of perceived stress and infertility in infertile women undergoing IVF irrational cognitions
Dortaj A1, Mehdizadeh A1, Dortaj H2.
Introduction: Effectiveness of mindfulness-based cognitive therapy on improvement of perceived stress and infertility in infertile women undergoing IVF irrational cognitions.
Materials and Methods: This clinical trial with pre-test post-test was done in 1331 infertile women referred to the Reproductive Health Research Center, Crescent Hospital, UAE who were treated with IVF. In total, 24 infertile women filled an irrational cognitions questionnaire related to childbearing and infertility. They were divided into experimental and control groups. The experimental group received mindfulness-based cognitive therapy for 8 sessions of 2 hr each. The control group did not receive any mental health services. Those two questionnaires were completed before and after the intervention. Data were analyzed by SPSS and p<0.05 was considered significant.
Results: There were no significant differences between the experimental and control groups in terms of reduction of perceived stress of infertility. But, the improvements in the recognition of irrational for childbearing between two groups was significantly different.
Conclusion: Teaching mindfulness-based cognitive therapy on improvement of perceived stress and irrational cognitions related to fertility in infertile women undergoing IVF treatment is effective.
Key words: Perceived stress, Infertility, IVF treatment, Mindfulness-based cognitive therapy, Irrational cognitions of parents.
P-111
Genotype and phenotype frequencies of paraoxonase 1 in fertile and infertile men
Fattahi A1, Tavilani H1, Latifi Z1, Esfahani M2, Khodadadi I2, Karimi J2,Bahrayni E2, Vatannejad A3, Vaisi-Raygani A4, Ghorbani M2.
Introduction: Paraoxonase1 (PON1) is a glycoprotein associated with high density lipoprotein and has antioxidant activity. The impact of PON1 in various stages of spermatogenesis has also been suggested.
Materials and Methods: Q192R variants of PON1 were determined in 150 fertile and 150 infertile men using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Plasma arylesterase and paraoxonase activities were detected by spectrophotometry and malondialdehyde (MDA) level was measured using thiobarbituric acid.
Results: Our results showed no significant difference in the distribution of PON1 genotypes and alleles between fertile and infertile groups. However morphology and motility of sperm were associated with various genotypes of PON1. The number of fertile males with the BB phenotype (high activity) was significantly higher than that of infertile males, whereas the number of individuals with the AB phenotype (moderate activity) was statistically higher in infertile men compared with the fertile group. Additionally, MDA and arylesterase activity levels were significantly higher in infertile subjects compared with fertile men.
Conclusion: We speculate that the low activity of PON1 can be a risk factor for male infertility probably due to a decrease in antioxidant activity of PON1 and increase in lipid peroxidation.
Key words: Genetic polymorphism, Male infertility, Paraoxonase 1, Reproduction.
P-112
Saccharin consumption increases sperm DNA fragmentation and apoptosis in mice
Rahimipour M, Talebi AR.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: marziehrahimipour@yahoo.com
Introduction: Saccharin is an artificial non-caloric sweetener that used to sweeten products such as drinks, candies, medicines, and toothpaste, but our bodies cannot metabolize it. Sodium saccharin is considered as an important factor in tumor promotion in male rats but not in humans.
Materials and Methods: Totally 14 adult male mice were divided into 2 groups. Group 1 served as control fed on basal diet and group 2 or experimental animals received distilled water containing saccharin (0.2% w/v) for 35 days. After that, the left cauda epididymis of each mouse was cut and placed in Ham’s F10. Swimmed-out spermatozoa were used to analyze count, motility, morphology (Pap-staining) and viability (eosin-Y staining). Sperm DNA integrity, as an indicator of apoptosis, was assessed by sperm chromatin dispersion (SCD) and terminal deoxynucleotidyl transferase (TUNEL) assay.
Results: Following saccharin consumption, we had a reduction in sperm motility with respect to control animals (p=0.000). In addition, the sperm count diminished (17.70±1.11 in controls vs. 12.80±2.79 in case group, p=0.003) and the rate of sperm normal morphology decreased from 77.00±6.40 in control animals into 63.85±6.81 in saccharin-treated mice (p=0.001). Also, we saw a statistically significant increase in rates of sperm DNA damage and apoptosis in experimental group when compared to control one (p=0.001, p=0.002 respectively).
Conclusion: Saccharin consumption may have negative effects on sperm parameters, and increases the rate of sperm DNA fragmentation and apoptosis in mice.
Key words: Sperm, Saccharin, Apoptosis, Mice.
P-113
Increased telomeric repeat containing RNA (TERRA) levels in cumulus cells of infertile polycystic ovary syndrome patients
Ghobadi N1, 2, Shahhoseini M2, Favaedi R2, Hassani F3, Movaghar M3, Karimian L3, Eftekhari Yazdi P3.
Introduction: One of the important reasons of anovulation based infertility is polycystic ovary syndrome (PCOS) which occurs in 5-10% of women in reproductive age. It seems that the length of telomere, TTAGGG tandem repeats, is related to proliferation and differentiation events during follicular development including the mechanisms which regulate successful reproduction. It is widely recognized that bi-directional communications exist between oocytes and the surrounding cumulus cells which are essential for the production of competent oocytes. During the past few years, study on cumulus cells showed that the length of telomere is longer in cumulus cells of mature oocytes than immature ones. Until now, telomeres have been considered to be transcriptionally silent but recent studies have clearly shown that transcription of subtelomere regions produces telomeric repeat containing RNA, named TERRA. In this study, we tried to identify the correlation of TERRA levels with oocytes maturation by evaluating of TERRA transcripts in cumulus cells of PCOS patients.
Materials and Methods: For this respect, cumulus cells were collected from 6 PCOS patients and 9 healthy women with male factor infertility through ICSI/ IVF procedure. TERRA transcripts were measured by using quantitative real- time PCR.
Results: Our data showed that the level of TERRA transcripts was increased significantly in PCOS group vs. control group (p˂0.05).
Conclusion: This finding implies a considerable association between TERRA transcript levels and PCOS, so it can be concluded that any changes in levels of TERRA transcripts can be judged as a potential marker for the quality of oocytes in ART procedure.
Key words: TERRA, Telomere, PCOS, Cumulus cells.
P-114
Epigenetic modification profile of endometrium in endometriosis patients
Hosseini E1, Afsharian P2, Ashrafi M3, 4, Ramazanali F2, Mehraein F1, Shahhoseini M2, Aflatoonian R3.
Introduction: Endometriosis defined as the presence of endometrium-like tissue outside of the uterine cavity, is a common gynecologic disorder. Although endometriosis is a multifactorial disease and the exact etiology is not clearly understood, recently, some evidence suggests that epigenetic is associated with the molecular features of endometriosis. Two main epigenetic regulatory mechanisms, DNA methylation and histone modifications regulate expression of genes and recognize the states of diseases. The aim of this study was to investigate alterations of DNA methylation and histone acetylation and methylation levels in eutopic and ectopic endometrium of endometriosis patients.
Materials and Methods: Informed consents were gained from all patients then eutopic and ectopic endometrium samples (n=5) were collected from endometriosis patients undergoing surgery and biopsy, as well as endometrial tissues from healthy fertile women (n=5) during the proliferative phase of menstrual cycle. Chromatin extracts from samples were prepared following fixation and then shearing into fragments by sonication. Nucleosome ELISA was performed on chromatin extracts, in order to identify global histone H3K9 acetylation/ methylation and DNA methylation, using antibodies against H3K9ac, H3K9me and MeCP2, respectively.
Results: We have identified global histone H3K9 hypermethylation in ectopic and eutopic endometrium, compared with controls. A significant hyperacetylation at histone H3K9 was observed in eutopic samples compared to ectopic and control groups. Furthermore, eutopic endometrial samples were globally DNA hypermethylated in comparison with controls.
Conclusion: These results clearly show an epigenetic switch in endometrial and endometriotic tissue of patients with endometriosis, in the way that aberrant DNA methylation and histone acetylation/ methylation status may play a dynamic role in occurrence of endometriosis and support the opinion that epigenetic abnormalities have causative functions in endometriosis.
Key words: Endometriosis, Endometrium, Epigenetic, Methylation, Acetylation.
P-115
The effects of chronic and acute ethanol administration on sperm chromatin parameters in mice
Hosseini A, Borzouei Z, Faramarzi A, Ghasemi-Esmailabad S, Talebi AR.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: hosseinia30@yahoo.com
Introduction: Sperm chromatin can be damaged by effects of some toxic materials. Among this, ethanol is one of the reproductive toxins that consumes abuse in men and may be associated with poor sperm quality. The purpose of this investigation was to examine the acute and chronic effects of ethanol consumption on sperm parameters and chromatin assay through testing the effects in mice.
Materials and Methods: Animals were randomly divided into 3 groups consisting of 10 mice each. Group 1 and 2 served as alcohol group and were received a daily dose of (3 g/kg body weight as 25%, v/v) ethanol by I.P. for four and eight weeks respectively. Group 3 (control group) was given normal access of food and water. The subdivisions and dosages were based on past works. At the end of treatments, laparotomy was conducted to expose the reproductive system, cauda of epididymis were immediately dissected out for evaluations of sperm parameters and various tests were used to analysis of the chromatin integrity of sperm.
Results: Various tests such as aniline blue (AB), chromomycin A3 (CMA3), toluidine blue (TB), and acridine orange (AO) were used to analysis the sperm quality. We did not find any statistically significant differences in acute and control groups. Although sperm count and motility were increased but viability, normal morphology, DNA and chromatin integrity were unaffected by ethanol in acute group and were identical with control group. Our results demonstrated that count, motility and viability of sperms were dramatically decreased in chronic treated animals. Besides of that, DNA and chromatin integrity were significantly decreased.
Conclusion: Therefore, ethanol abuse can induce abnormality in sperm structure and function, and this may be one possible cause of male infertility.
Key words: Sperm, Ethanol, Chromatin integrity.
P-116
Comparison of oral dydrogesterone with vaginal progesteronefor luteal support in IUI cycles: A randomized clinical trial (RCT)
Khosravi D1, Taheripanah R1, Khosravi R1, Taheripanah A2, Tarighat Monfared V1, Defaee S1, Mehdighalb S1, Hosseini Zijoud SM3.
Introduction: Progesterone supplementation is the first line of treatment in ovarian stimulation. This study was conduct to compare the effect of oral dydrogestrone with vaginal cyclogest on luteal phase support in intrauterine insemination (IUI) cycles.
Material and methods: This prospective, randomized, double blind study was performed in a local infertility center from May 2013-2014. It consisted of 150 infertile women younger than 35 years old undergoing ovarian stimulation for IUI cycles. They underwent ovarian stimulation with oral dydrogesterone (20 mg) as group A and vaginal cyclogest (400 mg) as group B in preparation for the IUI cycle. Clinical pregnancy and abortion rates, mid luteal progesterone (7 days after IUI) and patients' satisfaction were compared between the two groups.
Results: The mean serum progesterone levels was significantly higher in group A compared to group B (p=0.001). Pregnancy rate in group A was not statistically different from group B (p=0.58). Abortion rate in group B was higher than this rate in group A, but the difference was not statistically significant (p=0.056). Satisfaction rates were significantly higher in group A compare to group B (p<0.001).
Conclusion: We concluded that oral dydrogestrone is as effective as vaginal progesterone for luteal-phase support in woman undergoing IUI cycles. Moreover, the mean serum progesterone levels and satisfaction rates in dydrogestrone group were higher than cyclogest group.
Key words: Dydrogesterone, Cyclogest, Luteal-phase, Infertility.
P-117
The detrimental effects of alcohol on sperm parameters and DNA integrity in diabetic mice
Mangoli E1, Pourentezari M2, Talebi AR1, Rahimipour M3, Taheri F1.
Introduction: Diabetes mellitus (DM) is a chronic disease that can affect male reproductive function at multiple levels. Ethanol can suppress reproductive function and sexual behavior in laboratory animals and humans. Alcohol also is considered as one of the problems associated with poor semen production and sperm quality. The main goal of this study is to examine the effect of alcohol on sperm parameters and DNA integrity in diabetic mice.
Materials and Methods: Totally 32 adult male Syrian mice (10 weeks old, 35 gr) were divided into 4 groups, mice of group 1 served as control fed on basal diet, group 2 received streptozotocin (STZ) (200 mg/kg, single dose, intra peritoneal) and basal diet, group 3 received alcohol (10 mg/kg, water-soluble) and basal diet and group 4 received streptozotocin and alcohol. After 35 days, the cauda epididymis of each mouse was dissected and placed in 1 mL of pre-warm Ham’s F10 culture medium for 30 min. The swim-out spermatozoa were analyzed for count, motility, morphology and viability. Also the sperm chromatin quality and DNA integrity, was evaluated with Aniline blue (AB), Toluidine blue (TB), Acridine orange (AO) and Chromomycin A3 (CMA3) staining.
Results: In this study all of the sperm parameters were significantly diffrent between groups (p=0.001). Also, regarding the sperm DNA integrity tests, the results from four tests showed significant differences between groups. Also, in diabetes+ alcohol mice, a significant increase was found in mean tests compare to other groups.
Conclusion: Although the DM may have bad effects on sperm fertility potential and DNA integrity, but, on the other hands, consumption of alcohol in diabetic mice, may have detrimental effects on sperm parameters, sperm function and also sperm chromatin condensation in experimentally-induced diabetic mice.
Key words: Mice, Sperm parameters, DNA integrity, Diabetes, Alcohol.
P-118
Protective effect of Silymarin on plasma membrane integrity, acrosome integrity and mitochondrial membrane potential in cadmium-treated mice
Ali Asgarnejad P, Momeni HR.
Department of Biology, Faculty of Sciences, Arak University, Arak, Iran.
Email: asgarnejad3@gmail.com
Introduction: Cadmium as a heavy metal and environmental pollutant is able to exert numerous undesirable effects on human reproduction by inducing oxidative stress. Silymarin, a polyphenolic flavonoid extracted from the seeds of Silybum marianum, has effective antioxidant properties.
Materials and Methods: Adult NMRI mice were divided into four groups: 1) control 2) cadmium chloride (5 mg/kg, sc) 3) Silymarin (100 mg/kg, ip) and 4) Silymarin + cadmium. Treatment period was 24 hours. After treatment mice were dissected and their epididymis was cut into small pieces in HTF medium in order to swim out spermatozoa. The spermatozoa from different groups were used to evaluate sperm parameters. Sperm plasma membrane integrity, acrosome integrity and mitochondrial membrane potential assessed by Hoechst and propidium iodide, coomassie blue and rhodamin staining respectively. Data were analyzed with one way ANOVA and p<0.05 was considered significant.
Results: In cadmium-treated mice, the percentage of intact membrane and acrosome were significantly decreased compared to the control. In addition, mitochondrial membrane potential was significantly decreased in cadmium-treated mice in comparison with the control. In Silymarin + cadmium group, Silymarin could significantly compensate the adverse effects of cadmium compared to cadmium group.
Conclusion: The present study showed that cadmium induces toxic effects on mice sperm plasma membrane integrity, acrosome integrity and mitochondrial membrane potential and Silymarin can compensate the toxic effects of cadmium on these sperm parameters.
Key words: Cadmium, Silymarin, Sperm parameters.
P-119
Evaluating quality of well-being, marital adjustment and sexual dysfunction between infertile women
Mousavi Nik M1, Saberi Zafarghandi MB2, Birashk B3, Assari A4, Eshaghi Farahman SR1.
Introduction: Infertility is a stressful experience and has a high impact on the infertile women’s psychological status. Infertility and it is treatment create a major and prolonged crisis for the women and create a heavy psychological trauma for the women. The effectiveness of infertility treatment depends on success rate of the treatment facility, the emotional well-being and sexual health of the women seeking treatment.
Materials and Methods: For the propose of the study, 50 fertile and 50 infertile women were selected randomly (n=100) from Midwife IVF and Fertility Research Hospital in Mysore-India. All subjects were assessed using Quality of Well-Being Scale-Self Administered (QWB-SA), Marital Adjustment Test (MAT) and Massachusetts General Hospital Sexual Functioning Questionnaire (MGH) questionnaire were administered.
Results: The results showed significant difference between fertile and infertile women assessment on Quality of Well-Being and Marital Adjustment score. Compare with the fertile women, the patients with infertility had significantly lower scores in the sexual interest and sexual arousal domains and lower frequency of intercourse. The patients with infertility retrospectively reported an orgasm, lubrication and sexual satisfaction score that was similar to that of the fertile women.
Conclusion: Women with a diagnosis of infertility were found to be at higher risk for sexual dysfunction when compare with fertile women. Further infertile women reported poor marital adjustment and quality of well being compare with controls. The results suggest to apply counseling and psychotherapy services in the infertility centers to reduce the psychological problems, increase Quality of Well-Being, Marital Adjustment and identify the factors that can contribute to the development of sexual dysfunction on women facing infertility to help them to enhance fertility chances.
Key words: Quality of well-being, Marital adjustment, Sexual dysfunction, Infertile women.
P-120
Assessment of aerobic bacterial and fungal contaminations in liquid nitrogen tanks
Nabi A, Khalili MA, Torki AR, Fesahat F, Anbari F.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: ali.nabi67@yahoo.com
Introduction: Cryopreservation technologies play an important role in assisted reproduction for both medical and research purposes. But, there are some concerns about the technical and biological safety of this technology because of pathogen transmission during cryopreservation and germplasm banking. The aim of this study was to evaluate the presence of microbial contaminations in liquid nitrogen (LN2) and carrier tanks used in different laboratories of our reproductive science institute.
Materials and Methods: In this descriptive study, different parts of 20 carrier and storage LN2 tanks, such as LN2, vapors, cryocans, and inner surface were qualitatively evaluated for microorganism contaminations through the assessment of bacterial and fungal growth in minimal and selective Petri dishes.
Results: Two fungal (Candida albicans and aspergillus flavus) and two aerobic bacterial microorganisms (Ecoli, Pseudomonas aeruginosa) were the most common findings in bottoms, vapors, and inner surfaces of 2 carriers and 11 storage LN2 tanks. There were Pseudomonas aeruginosa growths in 8 cryocans from contaminated tanks. Also, there were staphylococcus aureus and acinetobacter baumannii contaminations in one storage tank.
Conclusion: Theoretically, LN2 tanks can be considered as a possible source of pathogen transmission. Therefore, performing quality control assays, like microorganism detection tests for LN2 and supplementary equipments should be considered as a routine practice in IVF clinics. Sterilization of tanks should be performed on regular basis.
Key words: Bacterial growth, Contamination, Liquid nitrogen.
P-121
Effect of non-polar extract of Phaleria macrocarpa on sperm quality in adult rats
Aziz Dollah M1, Parhizkar S2, Baharudin N1.
Introduction: Diminish production of testosterone hormone could affect sperm quality; hence contribute to the infertility in man.
Materials and Methods: 25 Sprague Dawley adult male rats were randomly divided into five groups. Rats were supplemented orally once a day with 0.25 ml Tween 20 solution containing non-polar hexane extract of PM at one of the following concentrations: 0, 6, 12, and 60 mg/kg for seven weeks. Commercial testosterone hormone was used as positive control. On the last day of supplementation period, all rats were sacrificed and orchidectomy technique was performed to collect semen sample from cauda epididymis of the testis. The sperm count, motility, viability and morphology were determined.
Results: The results showed that the sperm motility was significantly improved by non-polar PM extract (p<0.05) with the value of 66.98% (6 mg/kg), 63.96% (12 mg/kg) and 65.21% (60 mg/kg) compared to 44.08% for 0 mg/kg group. The supplementation of PM extract however did not improved the sperm count, sperm viability and sperm morphology. The sperm count was 852 (0 mg/kg), 918 (6 mg/kg), 906 (12 mg/kg), 1030 (60 mg/kg) and 1027 million cells/ml (commercial testosterone). The sperm viability was 85.90 (0 mg/kg), 85.26 (6 mg/kg), 85.45 (12 mg/kg), 87.88 (60 mg/kg), and 89.32% (commercial testosterone). The sperm morphology was 62.5% (0 mg/kg), 63.7% (6 mg/kg), 72.2% (12 mg/kg), 67.0% (60 mg/kg) and 64.5% (commercial testosterone). The results showed that non-polar PM extract only improved the sperm motility and did not have any effect on the sperm count, morphology and viability.
Conclusion: The study concludes that non-polar extract of PM has no compound that can improve sperm quality except on the motility of the sperm.
Key words: Phaleria macrocarpa, Sperm count, Sperm motility, Sperm viability, Sperm morphology.
P-122
Protective effect of ascorbic acid on testicular tissue in mice exposed with sodium arsenite
Pirozmanesh H, Soleimani Mehranjani M, Naderi Noreini S.
Department of Biology, Faculty of Sciences, Arak University, Arak, Iran.
Email: hp457@yahoo.com
Introduction: Arsenic is one of the major environmental contaminants with carcinogenic and toxic effects. Ascorbic acid as an antioxidant is able to restrict oxidative stress. The aim of this study was to investigate the adverse effect of sodium arsenite on the mice testicular tissue as well as to examine whether vitamin c is able to ameliorate this effect.
Materials and Methods: 24 adult male NMRI mice were randomly allocated into four groups (n=6) including control, sodium arsenite (7 mg/kg/day, orally), ascorbic acid (150 mg/kg/day, orally) and finally sodium arsenite+ ascorbic acid. Mice were treated for 35 days. At the end, mice were sacrificed and their right testis were taken out, fixed, processed and stained with Heidenhain azan method. The total volume of testis, volume of interstitial tissue, volume of seminiferous tubules, diameter and of length of seminiferous tubules, germinal epithelium height and basement membrane thickness were estimated using stereological methods. Data were analyzed using one way ANOVA and Tukey's test and means difference were considered significant at p<0.05.
Results: A significant reduction in total volume of testis, volume of seminiferous tubules and its diameter and germinal epithelium height, was found in sodium arsenite group compared to control group (p<0.04). In ascorbic acid + sodium arsenite group, the above parameters were significantly increased compared with sodium arsenite group (p<0.02). In addition, germinal epithelium height and diameter of seminiferous tubules were increased in mice treated with ascorbic acid alone compared to the control group (p<0.002).
Conclusion: The results indicate that ascorbic acid may be useful in reducing the sodium arsenite- induced toxic effects on testicular tissue.
Key words: Sodium arsenite, Ascorbic acid, Stereology, Testis.
P-123
The comparison of anxiety and depression rate between medical staff of Infertility Centers and Obstetrics and Gynecology Centers of Yazd, Shiraz, Isfahan and Kerman Hospitals
Pourmovahed Z1, Yassini SM1, Khalili MA2, Halvaei I2, Nabi A2, Ghasemi M3, Fesahat F2.
Introduction: Mental health is an important issue in personal, social and occupational function of anyone in the life; moreover, mental problems have many negative consequences on the quality of professional work. Regarding the close and continuous interaction of infertility staff with hopeless infertile couples and in the contrary the atmosphere of happiness especially in Obstetric Wards it make sense that anxiety and depression rates would be different between them.
Materials and Methods: This study was a descriptive-correlation study based on cross-sectional method. 199 individuals who were the staff of Infertility Centers and Obstetrics and Gynecology Wards in four provinces enrolled in this study through stratified sampling. Data collection was done by demographic questionnaire, Spiel Berger and Beck depression inventory tests. Data were analyzed by SPSS software and ANOVA test.
Results: The results showed that there is statistically significant higher rate of anxiety in staff of Obstetrics and Gynecology Wards in Isfahan (54.69±13.58) while there was a higher rate of depression in staff of Infertility Center in Shiraz (14.94±10.87). Overall, correlation between anxiety, depression and work place was statistically significant (p=0.047 and 0.008 respectively). According to ANOVA test, the mean value of anxiety level was higher in the staff of Obstetrics and Gynecology Centers of Isfahan, Shiraz, Shahid Sadoughi and Madar Hospitals in Yazd (p=0.03, 0.006, 0.008, 0.012 respectively) and Infertility Center of Yazd (p=0.00).
Conclusion: Comparison of the mean value of anxiety in staff of Obstetrics and Gynecology Wards and Infertility Centers was not significant in these four centers except for Isfahan Center. As long as we know that infertile couples have little chance for success rate and Obstetrics and Gynecology patients have little risk of failure in treatment it could be mentioned that the anxiety and depression in the staff are not correlated with the client illness.
Key words: Anxiety, Depression, Infertility centers, Obstetrics and gynecology centers.
P-124
Developmental competence of immature oocytes aspirated from ovarian antral follicles as a method for fertility preservation
Safian F1, Khalili MA2, Karimi Zarchi M3, Mohsenzadeh M1, Ashourzadeh S1, 4.
Introduction: Advances in the treatment of cancerous women in reproductive age have markedly been increased recently. Also, in vitro maturation (IVM) of immature oocytes collected from ovary has been proposed for fertility preservation. In addition, the morphology of oocytes post IVM is one of the factors determining its developmental competence. By using the non-invasive PolScope system, the meiotic spindle and zona pellucida (ZP) can be assessed in living oocytes.
Materials and Methods: The ovarian cortex from 26 cancer patients (21-45 years old), were obtained directly from collaborating hospitals, and transported to the IVF center on ice. 61 immature oocytes were aspirated, of which 18 (29.5%) were degenerated and discarded. The remaining 43 (70.5%) healthy oocytes were cultured for 48 hr IVM culture media. The rate of maturity was assessed and the ZP birefringence and meiosis spindle (MS) were imaged with Polscope technology.
Results: 43 immature oocytes underwent IVM technology, of which 30.2% reached viable metaphase II (MII) oocytes. The ovarian tissues of 9 (34.6%) women were lacking oocytes at any stage. There was a positive correlation between the recovered number of oocytes and the ovarian volume. During polarized light microscopy examination, MS could be visualized only in one of the MII oocytes, but high ZP birefringences were observed in most of the oocytes post IVM (61.5%).
Conclusion: Oocytes maturation post IVM from unstimulated ovaries showed a good developmental competence in cancerous patients. Further studies should be performed to advance the oocyte maturation program, such as co-culture system, for fertility preservation.
Key words: Fertility preservation, Ovarian tissue, IVM, Polscope.
P-125
Effect of Heracleum persicumm oil and alcoholic extracts on sperm parameters and chromatin quality in mice
Taghizabet N1, Mangoli E2, Anbari F2, Talebi AR2, Masoodi A1.
Introduction: Seminal plasma from infertile men has lower antioxidant levels than that of fertile men, particularly of patients whose semen have poor sperm motility. Evaluation of the importance and the effects of plant derived drugs on fertility of laboratory animals have long been recognized. Antioxidant activity has been reported from Heracleum persicum (Golpar).
Materials and Methods: 80 adult male mice (10 weeks old, 35 gr) were divided to 3 groups: group1 received hydroalcoholic extract (1000 mg/kg, ip), group 2 received oil extract (200 ml/kg, ip) and group 3 serving as the sham control group that received water. After 35 days, the cauda epididymis of each mouse was dissected and placed in 1 mL of pre-warm Ham’s F10 culture medium for 30 min. The swim-out spermatozoa were analyzed for count, motility, morphology and viability. The sperm chromatin quality and DNA integrity, was evaluated with Aniline blue (AB), Toluidine blue (TB), Acridine orange (AO) and Chromomycin A3 (CMA3) staining.
Results: In sperm analysis, progressive and non-progressive motility were significantly differences between groups. Regard to sperm chromatin quality, the results of TB and AO tests showed statically significant differences between groups, but in AB and CMA3 staining, we didn’t see any differences between them. So we can say that the Heracleum persicum extracts doesn’t have any detrimental effects on histone-protamines replacement during the testicular phase of sperm chromatin packaging.
Conclusion: According to our results, Heracleum persicum extracts as an antioxidant although improved sperm motility but may influence the male fertility potential via affecting DNA/ chromatin quality in mice.
Key words: Heracleum persicum extracts, Sperm, Mice, Chromatin.
P-126
Effect of vitrification on high magnification morphology, chromatin condensation, and fertility potential of human spermatozoa
Taherzadeh S2, Khalili MA1, 2, Anvari M1, 2, Ghazali Sh1, Agha-Rahimi A1, Anbari F1, Ghasemi-Esmailabad S1.
Introduction: Sperm vitrification is a technique of ice- and CPA- free cryopreservation by direct plunging of a sperm suspension into liquid nitrogen (LN). Sperm characteristics of motility and morphology are important for normal spermatozoa-oocyte interaction. However, defects on chromatin condensation can cause vacuolization on sperm morphology. Motile sperm organelle morphology examination (MSOME) is an unstained real time high magnification that analysis viable sperm morphology. This study investigated the influence of vitrification on human sperm structure by MSOME technique, and fertility potential by zona binding assay (ZBA) and chromatin condensation by toluidine blue (TB) and aniline blue (AB) assessment.
Materials and Methods: 30 normozoospermic ejaculates were prepared by swim up technique, and supernatants were divided into two parts: fresh and vitrified groups. For vitrification, sperm suspension was mixed with equal volume (1:1) of Hams F10 +5% HSA +0.5 M sucrose. Then, 30 µl sperm suspension was dropped into LN. Warming was performed by quick submerging spheres into pre-warmed 5ml Hams F10 with 5% HSA at 37˚C. Sperm motility, stained morphology, MSOME and ZBA were evaluated for each sample. Three classes were considered for MSOME analysis: high quality sperm with a score of 4-6 (Class 1); medium-quality sperm with a score of 1-3 (Class 2); low-quality sperm with a score of 0 (Class 3). 2×10⁶ spermatozoa in each 25µl droplet containing 4 Oocytes were performed for ZBA. In addition, samples were fixed for TB and AB assessments.
Results: Cryopreservation significantly reduced both progressive motility and normal morphology of spermatozoa. There was no significant differences between the rates of MSOME in class 1 (14.93±14.66 vs. 13.56±11.34), class 2 (53.53±13.99 vs. 55.63±12.16), class 3 (31±20.81 vs. 30.80±16.03) pre and post vitrification, respectively. However, vitrification reduced the fertility potential of spermatozoa from normozoospermic samples (13.40±22.73 vs. 9.00±13.87) and chromatin condensation (TB: 60.32±16.60 vs. 57.28±17.19) (AB: 38.32±8.14 vs. 34.35±6.87).
Conclusion: Vitrification had adverse effects on sperm parameters of motility and morphology. However, this technique did not increase the rate of vacuolization of sperm head or severe alteration in fertility potential. Since, the majority of human spermatozoa contained vacuolization in head region, it is highly recommended to use MSOME technology for assessment of sperm fine morphology during clinical microinjection procedure.
Key words: Sperm vitrification, Motile sperm organelle morphology examination (MSOME), Zona binding assay (ZBA).
P-127
Vitamin C attenuates detrimental effects of diabetes mellitus on sperm parameters, chromatin quality and rate of apoptosis in mice
Talebi AR1, Mangoli E1, Nahangi H2, Anvari M2, Pourentezari M2, Halvaei I1.
Introduction: Diabetes Mellitus (DM) may affect male reproductive functions at multiple levels. It is shown that intake of antioxidants such as vitamin C and E can reinforce the stability of testicular blood barrier and protect sperm DNA from oxidative stress.The main goal was to examine the protective effects of vitamin C on sperm parameters, sperm chromatin condensation and apoptosis in experimentally-induced diabetic mice.
Materials and Methods: 28 adult Syrian mice were divided into 4 groups. In group 1, the mice were diabetic that received a single dose of Streptozocin (STZ) (200 mg/kg) intra-peritoneally (ip). Group 2 included diabetic mice that received vitamin C (10 mg/kg/daily, ip). Mice in group 3 received vitamin C and group 4 was considered as control. After 35 days, sperm analysis was done accordingly. To asses sperm chromatin and DNA quality, we used aniline blue (AB), toluidine blue (TB), chromomycin A3 (CMA3), acridine orange (AO) and terminal transferase mediated deoxyuridine triphosphate biotin end labeling (TUNEL) tests.
Results: All of the sperm parameters (count, motility, morphology and viability) had significant reduction in diabetic mice but, the data showed a significant increase in all of the sperm parameters in diabetic + vitamin C when compared with diabetic and control animals (p<0.05). There were significant differences (p<0.001) between groups regarding TB staining (48.8±5.92 vs. 34.3±4.13), AO test (35.9±6.11 vs. 20.8±2.89) and TUNEL test (39.42±7.18 vs. 22.00±3.65) in diabetic and diabetic + vitamin C groups, respectively. Nevertheless, in CMA3 and AB staining assays, there were not any significant differences between different groups.
Conclusion: Vitamin C, as a potent antioxidant, can attenuate detrimental effects of diabetes mellitus on the sperm parameters, chromatin quality and apoptosis in an experimental model.
Key words: Sperm, Diabetes mellitus, Mice, Vitamin C, Chromatin, Apoptosis.
P-128
DNA fragmentation in mammalian spermatozoa and its relationship with male infertility
Vaseghi-Dodaran H1, Daghighkia H1, Atshan M2, Sobhani A3, Sobhani AGH4.
Introduction: It has been suggested that altered nuclear chromatin structure or damaged DNA in spermatozoa is implicated as a possible cause of increased infertility in males. Since the first reports on sperm DNA integrity, this subject has become the focus of numerous studies. Up to 8% of infertile men have been shown to have high levels of sperm DNA fragmentation despite a normal semen analysis. Currently, there are eight major tests of sperm DNA fragmentation, including the comet assay, sperm chromatin structure assay (SCSA), acridine orange test (AOT), tritium-labeled 3H-actinomycin D (3H-AMD) incorporation assay, terminal TdT-mediated dUTP-nick-end labeling (TUNEL) assay, in-situ nick translation (ISNT) assay, DNA breakage detection-fluorescence in-situ hybridizations (DBD-FISH) assay and sperm chromatin dispersion (SCD) test. New studies suggest that sperm with certain levels of DNA fragmentation serve as a strong predictor of reduced male fertility. Diagnosis of the fertilizing ability of a semen sample is important for consistently high reproductive efficiency. This paper aimed at investigating the potential use of sperm DNA fragmentation (SDF) to improve the routine screening of male infertility.
Materials and Methods: This article discusses some of the current techniques employed for evaluating chromatin structure or DNA damage in spermatozoa. A review of recent bibliography was carried out in PubMed, Google scholar and SID by the use of relevant keywords, in order to evaluate the possible correlation between the conventional seminal parameters and sperm DNA fragmentation assessment as diagnostic tools in male infertility evaluation.
Results: A negative correlation was found between sperm characteristics and the proportion of sperm showing DNA fragmentation. For fragmentation >30%, a significant decrease of the fertilization rate was observed.
Conclusion: The proportion of sperm with DNA fragmentation appears to be potentially useful as a predictor of infertility. Sperm DNA fragmentation is a parameter worth integrating in routine clinical practice. However, additional large scale studies based on the optimization of sperm DNA integrity are needed.
Keywords: Spermatozoa, Male, DNA damage, Infertility.
P-129
Establishment of spermatogonial stem cell line in mouse testicular culture
Azizi H1, Amirian M2, Skutella Th2.
Introduction: In the seminiferous tubules of the testis spermatogonial stem cells (SSCs), Sertoli cells and differentiating cells during spermatogonesis are present. Materials and Methods: Here we report the establishment of a new VASA/ DAZL/ PLZF/-, Sox9/ Nanog/ GFRa1 low and KLF4, SOX2, VIMENTIN, N-MYC, OCT4+ cell line both from neonate and old mice, which could be expanded on SNL feeders for more than a year.
Results: Unlike undifferentiated SSCs they have a smaller nuclear/cytoplasm ratio in EM. FACS analysis showed the expression of CD49, CD29, CD9, GFRa1 and E-CAD. In Fluidigm analysis the cells expressed pluripotency markers OCT4, NANOG, SOX2 and KLF4 but only partially expressed the typical germ cell profile of SSCs. One month after transplantation in busulphan treated NOD SCID mice we observed localization of GFP labelled cells in the basal compartment of the seminiferous tubule.
Conclusion: These more differentiated SSCs could provide an ideal cell system for studying both pluripotency and in vitro differentiation of SSCs to sperm and also provide a new strategy for isolation of SSCs from neonate and old mice by morphology based selection.
Key words: Germ cells, Spermatogonial stem cells, Testicular culture.
P-130
The association between TNF-alpha -308 polymorphism and susceptibility to spermatogenic failure
Khademi Bami M1, Dehghan Tezerjani M2, Vahidi Mehrjardi MY2, Ashrafzadeh Mehrjardi HR1, Ghasemi-Esmailabad S2, Montazeri F2, SabbaghNejad Yazd S2, Shadram Y2, Golzade M2, Karimi R2, Sheikhha MH2, Kalantar SM2.
Introduction: Approximately half of infertility reasons are related to male factors among which genetic etiology is the main cause. Signaling molecules can regulate spermatogenesis during regulation of germ cells maturation. Tumor necrosis factor-α (TNF-α) is one of the prominent cytokines that supports signaling molecules and it can affect spermatogenesis.
Materials and Methods: This case-control study includes 50 azoospermic men who referred to Yazd Research and Clinical Center for Infertility and 50 healthy controls. After sperm analysis, DNA was extracted and Restriction Fragment Length Polymorphic-Polymerase Chain Reaction (RFLP-PCR) was carried out for TNF alpha -308 polymorphism.
Results: The frequencies of A allele and G allele were 58% and 42% in azoospermic group, and 34% and 67% among controls, respectively. Among azoospermic patients, 42% presented AA homozygous genotype, 32% AG heterozygous genotype, and 26% GG homozygous genotype. In the control group, 20% presented AA homozygous genotype, 28% AG heterozygous genotype, and 52% GG homozygous genotype. According to our findings, A allele (p=0.001, OR=2.681) and AA genotype (p=0.030, OR=2.89) have a positive association with spermatogenic failure in the cases.
Conclusion: Our data suggest that the polymorphism might be associated with the risk of spernatogenic failure in Iranian azoospermic and it can be used as an infertility marker for screening as well as further treatment.
Key words: Azoospermic, Cytokines, TNF-α, Polymorphism, RFLP-PCR.
P-131
The role of FSH receptor gene alterations in poor ovarian response to gonadotropins
Masoudi N1, Zari Moradi S1, Mohseni Meybodi A1, Mansouri Z1, Totonchi M1, Mirghavam-Aldin N2, Ashrafi M2, Gourabi H1.
Introduction: Poor ovarian response is an infertility disorder in which women’s ovaries don’t have proper response to gonadotropins. Follicle stimulating hormone (FSH) has a critical role in the maturation of the ovarian follicles from the antral to the graffian stage. FSH will start a signaling cascade in the granulosa cells after sitting on its receptor (FSHR). Alteration of this receptor may change follicle maturation and therefore result in improper response to gonadotropins. We investigated the association of FSH receptor gene alteration in ovarian response of poor responder patients.
Materials and Methods: The presence of P.Ala307Thr, P.Ser680Ala, P.Ala665Thr and Mut.Val341Ala were analyzed in a case control study. 70 Iranian poor responder patients were selected as the case group. 60 Iranian fertile women were enrolled as the control group. The patients DNA were extracted from their peripheral blood and amplified by relevant primers. For determining allelic variant status all PCR products were analyzed by Sequencing.
Results: The results showed that the homozygous Ser680 and Ala307 variants seem not to be significantly associated with poor response to gonadotropins. The FSHR P. Ala665Thr genotype frequency was similar in all patients and controls. The number of oocytes retrieved was comparable between patients with different FSHR genotype.
Conclusion: Although data are accumulating with evidence suggesting that the ovarian response to gonadotropins may be mediated by different genetic alterations, the optimal biomarkers and the efficacy of the tests still remain to be evaluated.
Key words: Female infertility, Poor ovarian response, Follicle stimulating hormone receptor (FSHR).
P-132
A review of studies about vascular endothelial growth factor gene polymorphisms as genetic cause of infertility
Nabat E1, Farahzadi R2.
Introduction: Infertility is defined as an inability of couples to achieve pregnancy after one year of regular, non-contraception intercourse. Diverse factors such as environmental, immunological, endocrine and genetic predispositions are thought to be involved in the development of the infertility. Infertility may be due to male or female factors or both. Causes of female infertility may include ovulation disorders, uterine abnormalities and fallopian tube blockage. Recurrent miscarriage (RM) and endometriosis are the common cause of infertility in female. Genetic factors can play an important role in susceptibility to RM and endometriosis, such as polymorphisms in vascular endothelial growth factor gene (VEGF). Recently researchers have found that endothelial dysfunction and placental ischemia/hypoxia are important factors in miscarriage. VEGF plays a pivotal role in the formation of blood supply of the fetus through the placenta and development of endometriosis. Many recent studies have indicated that a reduced VEGF serum concentration was associated with increased risk of RM and endometriosis. Therefore, the aim of this study was to investigate the association of VEGF polymorphisms such as +405 G/C, -460 C/T in 5’-UTR, +936 C/T in 3’-UTR and -1154 G/A in promoter region, with RM and endometriosis.
Materials and Methods: This review is focused on the most recent laboratory and clinical findings to investigate the association of VEGF polymorphisms with RM and endometriosis.
Results: -1154A and +93 6T alleles are increased in RM, while only in the study of Hsieh et al +405 C/C and -460 T/T genotypes are highest in patients suffering from endometriosis.
Conclusion: According to many functional studies, A allele of -1154G/A, T allele of +936C/T, C/C genotype of +405 G/C and T/T genotype of -460 C/T are associated with reduced VEGF transcriptional levels. These could lead to inadequate angiogenesis thus, lower endometrial vascularization, impaired placentation and subsequent predispose to RM and risk of endometriosis.
Key words: Infertility, Endometriosis, Recurrent miscarriage, Polymorphism, VEGF.
P-133
Association study of GSTO1 gene polymorphism (E155del) among patients with recurrent miscarriage from NorthWest of Iran
Yasari S1, Bonyadi M1, Taghavi S2.
Introduction: Recurrent miscarriage (RM) is the occurrence of three or more consecutive pregnancies that end in miscarriage of the fetus before viability. About 1-5% of couples trying to conceive are affected by recurrent miscarriage. RM is considered as a multi-factorial disease and nearly half of RM cases cannot currently be explained clinically. Several studies have indicated that genetic polymorphisms could be factors for causing susceptibility of RM with unknown etiology. GSTO1 protein has a crucial role in detoxification metabolism and it is believed that GSTO1 gene could be involved in RM. In this study the possible association of GSTO1 gene polymorphism [E155del (rs11509437)] with recurrent miscarriage in NorthWest of Iran was evaluated.
Materials and Methods: In this case-control study, forty-eight patients with a history of RM and forty-eight referent women, who had at least two live births without other pregnancy complications, were included. Glutathione S-transferase Omega 1 (GSTO1) gene polymorphism (E155del) was screened using polymerase chain reaction with confronting two-pair primers (PCR-CTPP). Subsequently, PCR products were electrophoresed in 2% agarose gel and visualized by ethidium bromide staining.
Results: This study revealed that there was a significant association with the allelic frequency of del155 between the patient and control groups (p=0.04). However, analysis of genotype’s frequencies between patient and control showed no significant association (p=0.14).
Conclusion: Although some studies have indicated that the genetic polymorphisms of detoxification genes, encoding related enzymes, could be involved in RM diseases, current study showed the weak association of GSTO1 gene polymorphism (E155del) with susceptibility to develop RM in this population.
Key words: NorthWest of Iran, Recurrent miscarriage, GSTO1 gene, E155del polymorphism, Cellular detoxification.
P-134
Effects of in vitro maturation (IVM) on epigenetic changes in oocytes and early embryos
Allahveisi A1, Rezaei MJ2, Nikkhoo B3, Yousefian E4.
Introduction: In vitro maturation (IVM) of oocyte is an effective technique for avoiding ovarian hyperstimulation syndrome in patients with polycystic ovaries (PCOS) during in vitro fertilization (IVF). Pervious study indicated that more than 1000 children have been born from IVM technique, especially in patient with PCOS. However, IVM remains questions even today. Epigenetic reprogramming occurs in the periods of gametogenesis and embryogenesis, which regulates the gene activity without alteration of DNA sequences. This review discuss about effects of in vitro maturation (IVM) on epigenetic changes in oocytes and early embryos.
Material and methods: This article presents result of a systematic review about effects of in vitro maturation (IVM) on epigenetic changes in oocytes and early embryos.
Results: Different studies showed that IVM down regulated the protein expression of enzymes controlling histone acetylation such as histone acetyltransferase GCN5 (GCN5) and histone deacetylase 1 (HDAC1), as well as their common target, acetyl-histone H3 (Ac-H3), in metaphase II (MII) oocytes and two-cell embryos. The significantly decreased HDAC1 mRNA levels in oocytes and early embryos from IVM strongly suggested that IVM technique down regulated the transcription of HDAC1 gene in oocytes before and after fertilization.
Conclusion: Our studies indicated that IVM could affect the protein and gene expression level related to histone acetylation in oocytes and early embryos. Therefore, the impacts of IVM on epigenetic changes may be reasons for the lower rates of fertilization and early embryos in IVM.
Key words: IVM, Oocyte, Epigenetic.
P-135
Association of 14-bp insertion/deletion polymorphism of HLA-G gene with idiopathic recurrent miscarriage
Arjmand F.
Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: fateme.arjmand@yahoo.com
Introduction: HLA-G is supposed to play a pivotal role in tolerance of the semi-allogeneic graft in pregnancy by inhibiting the cytotoxic functions of T- and NK cells. A 14-bp insertion/deletion polymorphism in exon-8 has a possible role in HLA-G expression.
Materials and Methods: Genomic DNA from 200 recurrent miscarriage (RM) patients and 200 normal fertile control individuals were isolated using the routine salting out method. Exon-8 of HLA-G gene of the two groups were amplified using polymerase chain reaction and analyzed by electrophoresis on 10% non-denaturing polyacrylamide gel electrophoresis containing ethidium bromide and visualized under ultraviolet light. Statistical analysis used SPSS 19 software. HLA-G allele frequencies and genotypes in RM women and the fertile control group were compared using a Chi-square test.
Results: There was a difference in allelic frequencies of 14-bp insertion polymorphism between fertile controls and RM patients; frequency of +14/-14 bp heterozygotes was significantly increased in RM patients as compared with fertile controls.
Conclusion: A 14-bp insertion/deletion polymorphism in exon 8 plays a possible role in HLA-G expression in certain cases of recurrent miscarriage. However, additional studies are needed in this regard.
Key words: 14-bp insertion, Deletion polymorphism, HLA-G, Recurrent miscarriage.
P-136
Mutation analysis of exone 2 of Tnp2 gene in varicocele patients
Danafar AH1, Heidari MM2, Khatami M2.
Introduction: Varicocele is one of the main causes of male infertility that caused by inflammation of the veins of the pampiniform plexus. Several studies have given great evidences that revealed the relationship between sperm DNA damage and varicocele. Because of the crucial role of Transition Nuclear Proteins (TNPs) and Protamines in sperm DNA condensation and integrity, the mutations in these genes can increase the risk of sperm DNA damage and infertility in varicocele condition.
Materials and Methods: DNA was extracted from total blood of 78 infertile patients with varicocele and 75 fertile control men for PCR amplification and SSCP analysis. DNA from samples with altered band pattern in the SSCP was then sequenced to search for mutations.
Results: The results of sequencing showed one variant at position IVS1-26G>C (rs8043625) in the intronic region of this gene. Comparison of the genotypes between cases and controls showed significant differences in frequencies of GG and CC (p=0.002, p=0.01), but not in GC genotype of this polymorphism (p=0.41). Also it was found that varicocele risk in men who have the CC and GC genotypes is respectively 3.07 and 1.37 fold higher than those who don't have these genotypes (OR=3.07, OR=1.37).
Conclusion: High conservation of this SNP position during evolution can represent the effects of this nucleotide in some important processes associated with the expression of this gene like mRNA splicing; but the exact mechanism is not clear.
Key words: Varicocele, Infertility, Tnp2, SSCP.
P-137
Assessing the polymorphism frequency rate of insulin receptor gene in PCOS
Khazamipour A1, Nejatizade A1, Kalantar SM2.
Introduction: Polycystic ovarian syndrome (PCOS) is a common endocrine disorder with prevalence of 5-10% globally among women of reproductive age. PCOS is being characterized by chronic anovulation, polycystic ovaries, hyperandrogenism, hirsutism, overweight, insulin resistance and infertility. It is well-known that PCOS is a complex trait like type-2 diabetes where both genetic and environmental factors play a crucial role in pathogenesis of the disease. Some type-2 diabetes susceptibility genes including those for insulin secretion and action such as insulin receptor showed considerable contribution to genetic predisposition of PCOS. Since insulin receptor gene seems to be a strong candidate gene to PCOS, we aimed to investigate the role of insulin receptor gene polymorphism (T/C) located in a transcription enhancer element of the gene to the disease susceptibility.
Materials and Methods: We carried out a cross-sectional case-control study. Using simple random sampling 90 healthy women were selected. All 90 patients fulfilled the 2003 Rotterdam criteria of PCOS. The subjects were genotyped for insulin receptor gene polymorphism (T/C) using PCR-Sequencing. Differences in genotype distributions between case and control subjects were examined via a chi-square test.
Results: In the present study 180 women between 18-40 years old age were assessed. Clinical, biochemical and metabolic characters of women were compared. Among them, obesity (Waist round more than 85 centimeters), hirsutism and irregular menarche (monthly cycles more than 35 days), LH/FSH >2, BMI>25, (mean: 28.19) in the PCOS group were significantly higher than control group (p<0.05). There was no significant associated between two groups for diabetes mellitus type II, hypertension (>140/90 mmHg) and hormonal level (TSH >5 and prolactin >29.2). Similarly in PCOS and control group, 95% of women had TT genotype and 2.2% had CC genotype. Frequency of C allele in PCOS and control group were 3.3% and 2.2% respectively. Frequency of TC genotype in PCOS and control group were 2.2% and 4.4% respectively. Noted polymorphism had no significant difference between PCOS and control women (p=0.99).
Conclusion: Although PCOS showed high prevalence among women in Bandar Abbas, Iran, insulin receptor gene polymorphism (T/C) didnot show significant influence on susceptibility to PCOS.
Key words: Polymorphism, Insulin receptor gene, Polycystic ovarian syndrome.
P-138
Association of SNP rs.2414096 of CYP19 gene with polycystic ovarian syndrome in Iranian women
Mehdizadeh A, Kalantar SM, Sheikhha MH.
Department of Genetics, International Campus, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: anahita.mz@gmail.com
Introduction: Polycystic ovarian syndrome (PCOS) is the most common endocrinologic disorder of women in their reproductive age. Major characteristics of PCOS are clinical and/ or biochemical hyperandrogenism, ovulatory dysfunction and polycystic ovaries. The etiology of PCOS is unknown, however, it has been suggested that genetic factors play major role in development of PCOS. Of those the ones implicating in metabolism of androgens are important as they are impaired in PCOS. CYP19 gene encodes aromatase which has a crucial role in androgen synthesis and variants of this gene might implicate in the pathogenesis of PCOS. Investigations have indicated the role of single nucleotide polymorphism of CYP19 in hyperandrogenism and PCOS in some racial groups. There is no data on the variants of this gene in PCOS population in Iran.
Materials and Methods: In a case-control study 70 PCOS women and 70 normal controls were selected. Following informed consent, 5 ml blood was taken from each woman of which genomic DNA was extracted by salting out method. Then a set of PCR reactions for CYP19 gene was carried out using specific primers for SNP rs.2414096 followed by a subsequent enzyme digest (RFLP) with HSP92II.
Results: Genotype frequencies of SNP rs.2414096 in PCOS women were as follows: AA (14.4%), AG (44.3%) and GG (41.4%) while in normal group, these genotypes were 24.3%, 52.9% and 22.9%, respectively. While allele frequencies in PCOS group were 49.3% for A and 50.7% for G, normal group had a different percentage of A (36.4%) and G (63.6%). The calculations for both genotype and allele frequencies showed statistical significance.
Conclusion: Variants of SNP rs.2414096 in CYP19 could play a role in development of PCOS in Iranian women.
Key words: Polycystic ovarian syndrome, CYP19, Single nucleotide polymorphism.
P-139
Exposure to tertiary-butyl hydroperoxide (TBHP)- down regulates the expression of Ddx3y in mature mice testis
Mokhtari P, Fatemi N, Golkar Narenji A, Mohseni Meybodi A, Gourabi H.
Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email:gourabi@royaninstitute.org
Introduction: Approximately 40% of infertility is associated with male factors. Two major causative factors of male infertility are oxidative stress (OS) and genetic factors. OS damages the sperm plasma membrane, the genome integrity and alter the expression profile of genes involved in spermatogenesis. Ddx3y gene is one of the important azoospermia factor (AZF) genes in Y chromosome. AZF deletion causes a severe block in spermatogenesis which affects the proliferation of spermatogonia, and consequently male infertility. The expression profile of Ddx3y gene was evaluated in testis tissues of Balb/c mice after OS induction.
Materials and Methods: A model of oxidative stress in adult male Balb/c mice testis by injection of the 1:10 concentration of tertiary-butyl hydroperoxide (TBHP) was created. Case group included treated mice by TBHP for 2 weeks and control group treated only by injection of dH2O. Induced ROS levels in testes tissue samples of all mice were measured by flow-cytometry. Consequently the expression of Ddx3y gene was quantitatively measured in samples of both groups by real-time PCR.
Results: According to flow-cytometry results, an increase of oxidative stress in TBHP treated mice in comparison to control group was observed. The gene expression of Ddx3y in testis was significantly down regulated in OS-exposed and ROS induced mice.
Conclusion: Our results indicated that Ddx3y may be a major target gene of OS and the down regulated expression of Ddx3y can be closely related to male reproductive toxicity induced by TBHP.
Key words: Male infertility, Oxidative stress, TBHP, Ddx3y.
P-140
Association of +331G/A polymorphism and differential expression of PR-B isoform in endometriosis
Mousazadeh S1, Afsharian P1, Shahhoseini B1, Aflatoonian R2.
Introduction: It is well accepted that endometriosis is a progesterone resistance disease. The effects of progesterone are mediated by its two progesterone receptor (PR) isoforms, namely PR-A and PR-B. These isoforms are functionally different. The most widely studied polymorphism in the promoter region of PGR is the G to A substitution at position +331 (rs10895068). Recently, many studies have investigated the role of +331G/A polymorphism in the etiology of various types of cancers.
Materials and Methods: Blood samples were recruited from 98 women undergoing laparoscopy for endometriosis and 102 healthy fertile women at Royan Institute, Tehran, Iran in 2013-2014. After DNA extraction, allele and genotype frequencies were determined by PCR-RFLP. Then, RNA was extracted from selected eutopic tissue samples of endometriosis patients. Analysis of PR-B mRNA expression was performed using Real-time PCR.
Results:Our data showed the frequency distribution of GG, G/A and AA genotypes in +331G/A polymorphism was 98.04%, 1.96% and 0.0% in patients and 97.96%, 2.04% and 0.0% in control groups respectively (p=0.968). Although our data didn’t show any significant association with +331G/A in our groups, however, we were able to demonstrate higher expression level of PR-B in patients with G/A compared to patients with GG genotypes.
Conclusion: Many studies have been shown that the expression level of PR-B reduced severely during endometriosis, which can affect the function of progesterone. Our findings support this observation, patients with G/A genotypes have high expression level of PR-B compared to patients with GG genotypes. Therefore, +331G/A have been found to lead increased transcriptional activity of PR-B in patients by favoring G/A or AA genotypes that may be able to influence the function of progesterone and reduce the susceptibility and symptoms of endometriosis.
Key words: Endometriosis, PGR, Polymorphism.
P-141
Association between GSTT1 null mutation and endometriosis in an Iranian population
Rastegar M1, Seifati SM1, Sheikhha MH2.
Introduction: Endometriosis is a common gynecological disease that involves growth of endometrial tissue outside its normal location. It seems that genetic factors have an important role in the pathophysiology of endometriosis. In addition, there are reports suggesting a role for environmental pollutants in developing endometriosis. GSTTl is one of the phase II enzymes involved in detoxification of some environmental carcinogens.
Materials and Methods: Genomic DNA from 95 patients with endometriosis and 141 healthy controls was collected. GSTT1 null genotyping was performed using polymerase chain reaction (PCR) method. Results: Frequency of GSTT1 null deletion in both patients and controls was 22%, which shows no significant difference between two groups (p=0.983).
Conclusion: These results suggest that GSTT1 null mutation is not associated with endometriosis in our population.
Key words: Endometriosis, Glutathione s-transfers, GSTT1.
P-142
The low frequency of breast and ovary cancer protective allele (D302H) of CASP8 gene among Iranian patients
Rasti A1, Farashahi Yazd E2, Shiryazdi SM3, Nematzade H4, Rastegar M1, Sheikhha MH1.
Introduction: Suppression of apoptosis is one of the major mechanisms underlying the origin and progression of cancer. The CASP8 plays a key role in the initiation of apoptosis. Previous case-control studies have indicated that the D302H genotype of CASP8 is associated with a reduced risk of breast and ovarian cancer. However, the frequency of this polymorphism in the Iranian people has not been reported yet. Therefore, this polymorphism was genotyped among patients with breast cancer and healthy women.
Materials and Methods: PCR-RFLP technique was used to genotyping of CASP8 D302H polymorphism among 100 female patients with breast cancer and 100 healthy women in this case-control study. Fisher's exact test and SPSS software were used to analyze the data.
Results: The mean age of patients and healthy groups was 51.37±1.27 and 52.57±1.21 years respectively. The frequencies of the GG and GC genotypes in case group were 91% and 9%, respectively, and among the control group, the frequencies were 97% and 3%, respectively )p=0.2). CC genotype was not found in any of the groups. Furthermore, the C allele frequency was 4.5% among the cases and 1.5% among the control (p=0.14). There was no significant difference between cases and controls. In total, the frequency of the C allele was observed in about 3% of people in our study (breast cancer patients and healthy women).
Conclusion: Previous case-control studies have indicated that the D302H genotype of CASP8 was associated with a reduced risk of breast and ovary cancer. In the present study the frequency of allele C was detected 3%. Indeed, the frequency of C allele in Iranian study population is less than one-fourth the frequency of this allele in Caucasian populations (13.29%). According to the low frequency of this allele in Iranian population and the protective effect of allele C, one of the conclusions that can be raised is that the Iranian population is more prone to breast and ovarian cancer than Caucasian populations in relation to this polymorphism. Therefore further studies would be needed with appropriate sample size to investigate this matter.
Key words: CASP8 D302H, Polymorphism, Apoptosis, Breast cancer.
P-143
“Cell-free fetal DNA” a novel promising biomarker for prenatal diagnosis and complicated pregnancies
Sadeghi F1, Khalaj kondori M1, Sadeghi M2.
Introduction: Cell free fetal DNA (cff DNA) is a novel promising molecular biomarker that has been applied in various aspects of obstetrical research, notably in prenatal diagnosis and complicated pregnancies.
Materials and Methods: Data base was browsed using key words “cell-free fetal DNA” and “noninvasive prenatal diagnosis” for obtaining related reports. Articles were screened for relatedness and the recent progresses in the field.
Results: Cell-free fetal nucleic acids can be detected in the maternal circulation during pregnancy, potentially offering an excellent method for early non-invasive prenatal diagnosis (NIPD) of the genetic status of a fetus. Using molecular techniques, fetal DNA and RNA can be detected from 5 weeks gestation and are rapidly cleared from the circulation following birth. Cell-free fetal DNA comprises only 3-6% of the total circulating cell-free DNA, therefore diagnoses are primarily limited to those caused by paternally inherited sequences as well as conditions that can be inferred by the unique gene expression patterns in the fetus and placenta. Broadly, the potential applications of this technology fall into two categories: first, high genetic risk families with inheritable monogenic diseases, including sex determination in cases at risk of X-linked diseases and detection of specific paternally inherited single gene disorders; and second, routine antenatal care offered to all pregnant women, including prenatal screening/diagnosis for aneuploidy, particularly Down syndrome (DS), and diagnosis of Rhesus factor status in RhD negative women. Already sex determination and Rhesus factor diagnosis are nearing translation into clinical practice for high-risk individuals.
Conclusion: In conclusion, the discovery of Cell free fetal DNA (cff DNA) has revolutionized the field of non-invasive prenatal diagnosis (NIPD) and has opened a new avenue in the field of obstetrical research and may in future form part of national antenatal screening programmes for DS and other common genetic disorders.
Key words: Cell free fetal DNA, Noninvasive prenatal testing (NIPT), Prenatal diagnosis, Pregnancy complications.
P-144
Identification of a time window for spontaneous establishment of pluripotency in mouse spermatogonial stem cells in vitro
Azizi H1, Conrad S2, Hajizadeh Moghaddam A1, Baharvand H3, Skutella T4.
Introduction: Although testis-derived embryonic stem cell-like (ES-like) cells have been obtained in several studies, the time window for the shift to pluripotency is not clear yet.
Materials and Methods: Here we describe, that only during a special time window (41 until 125 days) after initiation of germ line stem cell (GSCs) cultures from neonate and adult promoter-reporter Oct4-GFP transgenic mouse the spontaneous appearance of germline-derived pluripotent stem (gPS) cells from both neonate and adult GSCs occurred.
Results: The isolated and long-term cultured (more than one year) GSCs which were isolated by a morphology based selection procedure expressed germ cells markers and exhibited a similar morphology with a high nucleus/cytoplasm ratio in comparison to undifferentiated SSCs (spermatogonial stem cells) in vivo. The generated gPS cells expressed pluripotency marker, in-vitro differentiated into all three germ lineages, formed complex teratoma after transplantation in SCID mice and produced chimeric mice.
Conclusion: Although the exact mechanism of the development of gPS cells from GSCs is still unclear, this new information could provide an ideal strategy for scheduling natural conversion mechanisms of ES-like cells from mouse testis.
Key words: Germ cells, Spermatogonial stem cells, Pluripotency.
P-145
Association between MspI polymorphism of CYP1A1 gene and endometriosis in an Iranian population
Babaki A, Seifati SM.
Department of Biology, Ashkezar Branch, Islamic Azad University, Yazd, Iran.
Email: babakiaz@yahoo.com
Introduction: Endometriosis is defined as the presence of endometrial tissue outside the uterine cavity. It is associated with dyspareunia, dysmenorrhea, pelvic pain and infertility and about 3-10% of women suffer from it in reproductive age. It seems that genetic and environmental factors play a role in the development of endometriosis. The relationship between genetic polymorphisms and endometriosis has been investigated in several genetic studies. CYP1A1 gene is a member of the cytochrome P450 gene family. It is involved in estrogen metabolism and in detoxification.
Materials and Methods: In a case-control study, DNA extraction was performed from the blood specimens of 93 cases of endometriosis and 139 healthy controls. The polymorphism was determined by PCR-based restriction fragment length polymorphism (RFLP) method.
Results: There was no significant difference in frequency of TT, TC, and CC genotypes of MspI polymorphism of CYP1A1 gene between case and control groups (p=0.961).
Conclusion: Results of this study revealed no association between the MspI polymorphism of the CYP1A1 gene and susceptibility to endometriosis in the studied population.
Key words: Endometriosis, MspI polymorphism, CYP1A1 gene.
P-146
Preimplantation genetic screening: Which stage? Which technique?
Bazrgar M.
Department of Genetics at Reproductive Biomedicine Research Centre, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: bazrgar.masood76@gmail.com
Introduction: Preimplantation genetic screening (PGS) techniques have a wide range of resolution, complexity and costs. Additionally each of biopsy stages has advantages and disadvantages.
Materials and Methods: Recent publications certainly systematic reviews and randomized clinical trials were considered in details.
Results: Polar body biopsy (PB) is the least invasive however provides the least capture of abnormalities. PB focuses on female meiotic errors, fails to identify approximately 40% of aneuploidies from postzygotic and paternal origins. PB is suitable in countries with legal restrictions for embryo biopsy. Cleavage stage biopsy (CB) of few cells (1 or 2) does not affect differentiation potential, however removal of 2 cells can result in an impaired implantation potential while mosaicism is not detectable in one cell biopsy. In trophectoderm biopsy (TB) several cells are available for analyses. In day 5 TB when the genetic results cannot be obtained within 24 hours, cryopreservation should be considered. The least and the most aneuploidy rates has been reported for TBs and CBs, respectively (1 versus more than 3). TB increases absolute implantation rate about 20% in comparison with CB. Blastocoel fluid aspiration is a new source of DNA for PGS with few publications. FISH-based PGS is low resolution due to limitation in number of both chromosomes and probes per chromosome which could be tested. Therefore it has not been effective in improvement of clinical outcomes while PGS methods with ability for detection of all 24 chromosomes (PGS-24) result in increased implantation and pregnancy rates and diminished effect of advanced maternal age. Concerning PGS-24 methods, digital PCR is mostly applied for PBs; real time qPCR is only applicable to TBs and limited by number of samples, two on each 384-well plate. Array CGH is robust and scalable, turnaround time as short as 12 hours, decreased cost per sample as increased case numbers. SNP arrays and NGS-based methods for copy number analysis are likely to be the most accurate and informative but complex and high-cost equipment. Low pass NGS-based PGS is moving to be cost effective in addition to performance same as array CGH.
Conclusion: Clearly, the largest reported increases in implantation and live birth rates to date have been with TB. TB is a good choice for good-prognosis patients particularly for those wishing to have elective single-embryo transfer. However, the cleavage-stage embryos of some poor-prognosis patients may implant and develop in utero but not develop to the blastocyst stage in vitro. At the moment, array CGH seems to be the most applicable method for PGS however future would be with higher resolution methods following decrease of related costs, hence ethical concerns on designer babies will need more serious considerations.
Key words: PGS, Biopsy, Stage, Technique.
P-147
Expression of CYP19A1 in patients with endometriosis and normal endometrium during the menstrual cycle
Khateri E1, 2, Shahhoseini M2, Mahdian S3, Aflatoonian R3, Afsharian P2.
Introduction: Endometriosis is an estrogen-dependent inflammatory disease defined by the growth of endometrial tissues outside of the uterus. Epidemiological and clinical studies show that estrogen is essential for the growth of endometriosis. Molecular studies have revealed the presence of aromatase P450, the key enzyme in the biosynthesis of ovarian estradiol, inside the endometriotic tissues, indicating local synthesis of estradiol. So, it is proposed that the enzyme aromatase P450, which is coded by CYP19A1 gene, will be expressed aberrantly in endometriosis.
Materials and Methods: To evaluate the expression profile of CYP19A1 gene in women with endometriosis during menstrual cycle, ectopic endometriotic lesions and eutopic endometrium samples were collected using laparoscopy from 20 women with endometriosis and endometrial biopsies were obtained from 10 healthy fertile women as a control group. For this respect, ethical approval and informed patient consent was gained for the use of tissue samples. Quantitative expression analysis was performed using real-time PCR technique.
Results: Data showed that mRNA expression of CYP19A1 was significantly higher in ectopic endometrium of patients with endometriosis, in both proliferative and secretory phase, in comparison with eutopic and control group. CYP19A1 expression in eutopic endometrium was significantly higher in comparison to control group in proliferative phase.
Conclusion: Higher expression of CYP19A1 mRNA in the endometriotic tissues, may contribute to the etiology and progression of endometriosis and it is involved in disease pathogenesis.
Key words: Endometriosis, CYP19A1, Menstrual cycle.
P-148
A headache in prenatal diagnosis: A case of vanished twin or hermaphroditism?
Mousavi F1, Mohammad Khani N2, Bagherizadeh E1, Hadipour F3, Hadipour Z3, Shafeghati Y4, Behjati F5.
Introduction: The finding of a mixture of both 46,XX and 46,XY cells in amniotic fluid culture has been frequently described. In the great majority of cases, the finding is followed by the birth of a normal male infant, leading to a consensus that the finding is the result of contamination with maternal cells in a normal male fetus. There are, however, several other possible explanations including the presence of cells from an undiagnosed twin pregnancy, cross contamination in the laboratory, the presence of cells from a “vanished” male twin, and true fetal chimerism. An accurate obstetric history and thorough sonography can be of great value in correct assessment of such situations.
Materials and Methods: A 29 Years-old woman was referred for genetic counseling. Her gestational age was 16 weeks and maternal biochemical serum screening test indicated high risk for Down Syndrome. She underwent amniocentesis for chromosome study using standard high resolution GTG banding technique.
Results: Karyotype result was 46,XY and sonography revealed a female fetus with normal internal genital organs. A normal baby girl was borned with 46,XX karyotype. Upon reviewing of amnio GTG slides both 46,XY and 46,XX cell lines were detected. The patient admitted that she had experienced a miscarriage and heavy bleeding early in pregnancy. The sonography at the time of amniocentesis had shown a shrinking cyst next to the fetus. These findings strengthened the vanished or resorbed twin as the reason for such a finding.
Conclusion: The fetus was most likely a non-identical twin. Most of grown cells were from the vanished male fetus which led to misdiagnosis. Thorough genetic counseling with the view of obstetric history is of great value in such situations.
Key words: Amniocentesis, Resorbed twin, Hermaphroditism, Genetic counseling.
P-149
+49A/G CTLA-4 gene polymorphism in NorthWest Iranian population with recurrent pregnancy loss
Parsa S, Jabbarpour bonyadi M, Zeinal zadeh N, Taghavi S.
University of Tabriz, Tabriz, Iran.
Email: sara.parsa6928@gmail.com
Introduction: Recurrent pregnancy loss is defined as three or more pregnancy loss before 20th gestational weeks. There are several factors which involved in pregnancy loss including anatomical, hormonal, immunological and infectious factors. Immunological factors are important due to the interaction between mother and fetus. In spite of allogeneic proteins encoded by paternal genes, it is clear that a series of regulatory mechanisms should exist in maternal immune system not to reject fetus during pregnancy. T regulatory cells which are one of the important component of humeral immune response plays role in the fetu-maternal interface. One of the regulatory ways for these cells is mediated by antigen independent co-stimulatory signals. Interaction of B7/ CTLA-4 is one of these signals. Cytotoxic T Lymphocyte Antigen 4(CTLA-4) is a glycoprotein transmembrane molecule which down regulate the activation and proliferation of T cells in a competitive interaction with CD28 to bind to B7.
Materials and Methods: We have studies 120 cases; patient group consist of 60 women with the experience of two or more pregnancy loss and control group consist of 60 women with at least two live births without any previous history of pregnancy loss and autoimmune diseases. Genomic DNA was extracted from whole blood using standard protocols. The CTLA-4 +49 A/G were detected using polymerase chain reaction- restriction fragment length polymorphisms assay.
Results: In this study CTLA-4 +49 A/G polymorphisms showed no significant differences between patients and controls. Polymorphic allele G showed the frequency of 39.16% among patients whereas its frequency for controls was 35.83%. The age of patient varied from 19 years to 39 years.
Conclusion: The results of the present study suggest that the CTLA-4 do not have association with recurrent miscarriage in NorthWest Iranian population.
Key words: Recurrent pregnancy loss, CTLA-4, Immunological factors.
P-150
Investigation of mitochondrial Gln tRNA molecular alterations in idiopathic repeated pregnancy loss
Vahidi Mehrjardi MY, Seyedhasani SM
Shahid Sadoghi University of Medical Sciences, Yazd, Iran.
Email: mmvahidi@gmail.com
Introduction: Recurrent pregnancy loss (RPL) is a critical medical problem in about 0.5-2% of women. The molecular genetics background for spontaneous abortion is being increasingly understood, and some polymorphisms associated with it have been reported
Materials and Methods: The nucleotide variations of threonine and proline were investigated in 96 women with idiopathic repeated pregnancy loss. The related mitochondrial area was amplified using a polymerase chain reaction (PCR). The PCR products were demonstrated by 2% agarose gel electrophoresis, and all the positive samples were purified and verified by an automated DNA sequencing method.
Results: The sequence analysis revealed 2 mutations in tRNA Gln . These mutations were 4343 in 1 cases and 4336 in 1 cases .
Conclusion: These tRNAs mutations can alter their steady state level and affect the structure of tRNAs. It results in protein synthesis defects and, in turn, mitochondrial dysfunction. The mutations of these genes may help in the assessment of RPL. Further study of an expanded series of these tRNA mutants is recommended to describe their etiologic role in idiopathic RPL.
Key words: tRNA, Mitochondrial mutation, Repeated pregnancy lo.
P-151
A novel mutation in morquio syndrome
Yavari M1, Asadollahi S2, Hasheminasab SH2, Harazi F3.
Introduction: The Mucopolysaccharidoses (MPS) are a group of inherited metabolic disorders caused by a deficiency or malfunctioning of lysosomal enzymes which are needed to break down complex carbohydrates known as mucopolysaccharides or glycosaminoglycans (GAGs). Accumulation of GAGs causes a cascade of events leading to the progressive damage of cells, tissue and organs. Morquio disease or Mucopolysaccharidosis Type IV (MPS IV) belongs to this group and has two sub-types, A and B. Type A is also known as Morquio A, GALNS deficiency, N-acetylgalactosamine-6-sulfate sulfatase deficiency or more simply MPS IVA. This lysosomal enzyme involved in the catabolism of keratan and chondroitin sulfate. Patients who inherit two mutated GALNS gene alleles have a decreased ability to degrade the glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate, thereby causing GAG accumulation within lysosomes and consequently pleiotropic disease. The objective was to report the results of clinical characteristics, enzyme activity determination and mutation analysis of GALNS gene in an Iranian patient with mucopolysaccharidosis (MPS) type IVA (Morquio A disease).
Materials and Methods: The 10 years old Iranian boy with MPSIV was firstly diagnosed by urin GALNS determination who w:as char:actraised by multiple skeletal abnormalities and dwarfism, kyphosis, hypermobility joints, difficultly in walking to 8 years old and now he can’t walk. But his intelligence was normal. We investigated for all coding exons and adjacent intron regions of GALNS gene by PCR sequencing method.
Results: We performed urin test (MS/MS) from patient. In this test total GAGS was high and MPS was likely. So lysosomal enzymes in dried blood were checked and MPS IVA was diagnosed. Two heterozygous missens mutation as c.135 G>T (S>I) and c.510 C>A (P>H), and a homozygous mutation as c.181 A>G (y>C) in GALNS gene were detected in this patient. The mutation c.181 (y>C) is a novel variant which is not reported yet.With the method of gene analysis of new variant, the mutation c.181 (y>C) was considered to be a pathogenic mutation.
Conclusion: The MPS IVA patient showed severe multiple skeletal deformities, normal intelligence, muscle weakness, short stature, who carries homozygous mutations c.181 A>G (y>C). The bioinformatics analysis in POLYPHEN predicted this mutation as being probably pathogenic.
Key words: Mucopolysaccharidoses (MPS), Morquio disease, GALNS gene.
P-152
A newborn with ambiguous genitalia and a complex X;Y rearrangement
Dehghani MR1, Hashemian Z2, Zuffardi O3.
Introduction: In most mammals, sex is determined at the beginning of gestation by the constitution of the sex chromosomes, XY in males and XX in females.
Materials and Methods: Array Comparative Genomic Hybridization (Array-CGH) analysis was performed by using oligonucleotide aCGH platforms (180K SurePrint G3 Human Kit, Agilent Technologies, Santa Clara, CA), as reported elsewhere. Changes in DNA copy number at a specific locus were observed as the deviation of the log2 ratio value from 0 of at least three consecutive probes, by using Genomic Workbench v. 5.0.14 software (Agilent, ADM-2 algorithm with a threshold of 5). Oligomer positions refer to the Human Genome GRCh37 (hg19) assembly.
Results: Array-CGH revealed an unbalanced rearrangement resulting in the deletion of the distal Xp and the duplication of the proximal Xp contiguous region with presence of the Y chromosome from Ypter to Yq11. Fluorescent in situ hybridization (FISH) showed that this portion of the Y was translocated to the tip of the abnormal X and that the duplicated portion of chromosome X was inverted. Altogether, the abnormal chromosome was a dicentric one with the centromere of the Y chromosome apparently inactivated.
Conclusion: The presence within the translocated Y chromosome of the SRY gene explains the devolopment of testes although it is not clear the reason for the genitalia ambiguity.
Key words: Ambiguous genitalia, 46, XX testicular DSD, Inverted duplication and Xp terminal Deletion (Invdup del), Rearrangement, Array Comparative Genomic Hybridization, FISH.
P-153
Effect of genetic variation of beta defensin 126 on ICSI and IVF outcome in unexplained infertile men
Abedini M, Mohseni Meybodi A, Sabbaghian M.
Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: maria.abedini@gmail.com
Introduction: Despite improved methods for evaluation of sperm quality, infertility remains unexplained in about 20% of affected couples. During sperm maturation, a Cysteine-rich secretory glycoprotein α-defensin 126 secreted by the epididymal epithelium adsorbed to the entire sperm surface. It remains on the sperm until sperm become capacitated in the female reproductive tract. Its removal from over the head of sperm is required for sperm zona recognition. A cytosine dinucleotide deletion in the open reading frame of second exon of DEFB126 gene generates an abnormal mRNA. Men homozygous for this mutation have reduced chances of successful fertilization.
Materials and Methods: Genomic DNA from the peripheral blood of 92 male partners of unexplained couples who underwent ICSI (n=74) and IVF (n=18), were extracted. PCR was performed and molecular genotyping for the DEFB126 variant was done by single strand conformational polymorphism (SSCP), tetra PCR and DNA sequencing. ELISA and immunocytochemistry by indirect immunofluorescence antibody performed for the assessment of this protein expression on sperm cells.
Results: In our study this allele frequency in Iranian men was 0.54. Statistical analysis shows, no significant differences were found between homozygote mutation and wild type carriers in fertilization rates, implantation rates and clinical pregnancy of IVF and ICSI. Our results by ELISA and immunocytochemistry showed that the protein expression was less in men with del/del genotype in comparing to other genotypes (p<0.005).
Conclusion: Although previous studies found that DEFB126 variation would affect sperm function and male fertility rate, in the present study, no significant differences were found between homozygote mutation and wild type carriers in fertilization rates, implantation rates and clinical pregnancy. Further confirmation in a larger scale study is needed.
Key words: Unexplained male infertility, Deletion, DEFB126, IVF, ICSI.
P-154
Association of TNFR1 36 A/G polymorphism with azoospermia in Iranian infertile males
Ashrafzadeh Mehrjardi HR1, 4, Dehghan Tezerjani M2, Khademi M1, Ghasemi-Esmailabad S2, Mossadegh AM3, Ghasemi N4.
Introduction: Infertility is the failure to achieve a pregnancy after one year of regular sexual intercourse without contraception. Half of the cases of infertility are due to male factors and nearly 60-75% of reasons of male infertility are unknown, due to unknown mechanisms of molecular defect. Tumor Necrosis Factor α (TNF-α) is a multi-functional cytokine that regulates cellular processes related to spermatogenesis. Tumor Necrosis Factor Receptor 1 (TNFR1) mediate TNF-α activity and changes in its structure can affect TNF-α activity. Studies show that variation in the TNFR1 gene may be associated with male infertility.
Materials and Methods: This case-control study includes 108 azoospermic men and 119 healthy controls. We investigated the association of TNFR1 36 A/G in the population with idiopathic azoospermia who referred to Yazd Institute of Reproduction Sciences. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR- RFLP) method was used to genotype the single nucleotide polymorphism in the both groups. PCR fragments digested by enzymes Mspa1I, and were separated by gel electrophoresis and then the frequency of A→G substitution in azoospermic males and fertile men were counted.
Results: According to our study, G/G genotype in the control group has higher frequency among men with azoospermia [p=0.01; OR=2.29 (1.248-4.229)]. Our findings also show that the allele frequency of G allele in azoospermic men is significantly different in comparison with control group [p<0.001; OR=2.302 (1.580-3.55)].
Conclusion: It seems that the G/G genotype and G allele are significantly associated with increased risk of idiopathic azoospermia.
Key words: Polymorphism, Male infertility, Sperm, Cytokines, Tumor necrosis factor alpha.
P-155
Epigenetic evaluation of histone methylation on HOXA1-5 genes in cumulus cells of polycystic ovary syndrome patients
Asiabi Kohneh Shahri P1, 2, Shahhoseini M2, Favaedi R2, Hassani F3, Movaghar B3, Karimian L3, Eftekhari Yazdi P3.
Introduction: Polycystic ovary syndrome (PCOS) is the major cause of an ovulatory infertility with uncertain etiology. Epigenetics and environment play critical roles in PCOS. Of important genes correlated with human reproductive system disorders are HomeoboxA (HOXA) cluster genes. Regarding the epigenetic role of histone modifications in regulation of gene expression, lysine (K) methylation of histone3 (H3K9me), as a repressive epigenetic mark, on the promoter regions of HOXA1-5 genes were evaluated in this study. For this respect, cumulus cells (CCs) which have critical roles during folliculogenesis, oocyte maturation, ovulation and fertilization, were aimed to monitor expression profile and epigenetic alterations of HOXA1-Poster Presentations
P-1
Fertility preservation in cancer patients
Abtahi NS, Ebrahimi B, Rezazadeh Valojerdi M, Fathi R.
Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: b.ebrahimi@royaninstitute.org
Introduction: Fertility preservation before gonadotoxic treatments is necessary in cancer patients. Ovarian tissue cryopreservation is the only available option for prepubertal girls and women who cannot delay cancer treatment or when ovarian stimulation is contraindicated.
Materials and Methods: Human ovarian tissue biopsies were obtained from six women (29-40 years old) who were candidates for oophorectomy for benign gynecologic conditions. Their medulla were removed, cortical parts were thinned and then cut into 10×5×1 mm strips. Cortical strips were vitrified in two steps by using vitrification solutions including; V1 [HTCM as base medium (BM)+ 7.5% DMSO+ 7.5% EG+ 20% HAS] and V2 (BM+ 15% DMSO+ 15% EG+ 0.25M sucrose+ 20% HSA) for 15 and 10 min respectively. Morphology of ovarian tissue and enclosed follicles were compared in control and vitrification groups by histological assessment.
Results: Ovarian stromal integrity, granulosa cells distribution, follicular population and morphology were well preserved in vitrified-warmed strips as fresh one. Oocyte degeneration, vacuolization and granulation were rarely observed in vitrified-warmed ovarian tissues. Primordial follicles were more distributed in cortical region in both groups. The mean percentage of intact follicles (primordial, primary and pre antral) are higher in control groups compared to vitrification one but the difference was not significant.
Conclusion: Accordingly, used vitrification method could be suitable for human ovarian tissue preservation in cancer patients.
Key words: Human, Fertility preservation, Vitrification, Ovarian tissue.
P-2
Effects of sesame-supplemented diet on the histology of adult rat reproductive system
Amini Mahabadi J1, Hassani Bafrani H1, Nikzad H1, Taherian AA2, Seyed Hoseini ES1.
Introduction: Studies show that antioxidants are important in improving male infertility. There are several antioxidant compounds in sesame seeds.
Materials and Methods: This experimental study was carried out on 30 adults Wistar rat (200 gr). Rats were randomly divided into experimental and control groups. The control group received standard diet and experimental group received a diet containing 70% standard diet and 30% sesame seed for 12 weeks. At the end of the study, body weight, testis weight and volume were measured and histology of testis, epididymis and prostate were evaluated. Serum FSH, LH and Testosterone levels were measured as well. SPSS software was used to calculate t-test and p<0.05 was considered as significant.
Results: The measured sperm count and motility, the number of epithelial cells and lumen diameter were increased significantly in the experimental group (p<0.0001). The number of spermatogonial cells, primary spermatocytes, spermatid and spermatozoa were increased significantly in the experimental group compared to the control group (p<0.0001). The epididymal diameter, lumen diameter and epithelial thickness did not change significantly. Fibromascular and epithelium diameter of seminal vesicle in treatment group were very significant differences and volume density epithelium significantly increased in treatment group compared to control group but volume density, Fibromascular and lumen significantly decreased in treatment group compared to control group. LH concentration increased significantly in the experimental group (p<0.03).
Conclusion: This is the first study, which evaluated the histology effect of sesame seed on reproductive system of adult Wistar rat. These results highly suggest that sesame seed can improve male reproductive parameters.
Key words: Rat, Male Sex hormones, Sesame seed, Testis, Prostate, Epididymis.
P-3
The effects of sperm preparation media on motility, viability and DNA integrity of human spermatozoa
Anbari F1, Halvaei I1, Nabi A1, Ghazali Sh1, Khalili MA1, Johansson L2.
Introduction: The main goal was to compare the effects of three different sperm preparation media on sperm motility, viability and DNA integrity of semen samples from normozoospermic men.
Materials and Methods: A total of 15 normozoo spermic males were included in the study. The semen analysis was performed in accordance with the WHO guidelines (2010). After semen analysis, each sample was divided into three aliquots and swim-up performed with three different sperm preparation media (Sperm Preparation Media, Origio, Denmark, Ham’s F10, Biochrome, Berlin, Germany, and VitaSpermTM, Innovative Biotech, Iran). Sperm motility, viability and DNA fragmentation,were evaluated at 0, 1, 2, and 24 hr after swim-up.
Results: There were no significant differences, at any time intervals, in the totalsperm motility between the different sperm preparation media. However, the rate of progressive motility was significantly higher in spermatozoa prepared by the media from Origio in comparison to VitaSperm TM (68.3±11.5 vs. 58±9.8, p=0.03), whereas no significant difference was found against Ham’s F10. No significant differences in sperm viability were seen between the media products .One hour after swim-up therate of sperm DNA fragmentation was also significantly lower inthe medium from Origio versus VitaSpermTM (p=0.02).
Conclusion: The type of medium for preparation of semen samples from normozoospermic men significantly affects the performance of spermatozoain assisted conception.
Key words: Sperm preparation media, DNA fragmentation (SCD), Viability, Motility.
P-4
Investigating the underlying factors of preterm delivery prevalence preceded by preeclampsia among pregnant visitors of Shariati Hospital, Bandar-Abbas, Iran: 2011-2012
Aslavi N1, Fallahi S2, Nowroozi S1, Najafian A3, Ghanbarnejad A1.
Introduction: Along with blood loss and infection, disorders pertaining to blood pressure are the three deadly factors in preterm childbirth morbidities and disabilities. In other countries the prevalence rate of this crisis is maximally 10%, while in Iran, it is 7%.
Materials and Methods: The present descriptive/ analytic research is a cross-sectional retrospective study. It was carried out on 863 pregnant women (167 had a preterm delivery due to pre-eclampsia and 696 were diagnosed with preterm delivery due to other reasons). The data were collected via a checklist about mother’s age, blood group, pre-eclampsia history, systemic disease, preterm delivery pain, etc. SPSS 16 analyzed the data. T-test and Chi-squared tests were used too. Significance level was set at p<0.05.
Results: Participants with a preterm delivery after pre-eclampsia were found to have a higher average age than peers afflicted with preterm delivery for other reasons. Most of the women afflicted with pre-eclampsia were of the B+ blood type. Significant differences were observed in these variables: pre-eclampsia history, preterm delivery pain, preterm rupture in fetal membrane, vaginal bleeding, intrauterine growth restriction, mother’s systemic diseases, blood group, history of intrauterine death and amniotic fluid disorders.
Conclusion: There is an increase in the probability of preterm childbirth due to pre-eclampsia with intrauterine growth restriction, history of intrauterine death and mother’s background diseases. Timely medical and preventive attempts and instructions for pregnant mothers on pre-natal healthcare can help to decrease irrevocable a consequence that endanger mothers and baby’s health.
Key words: Pre-eclampsia, Premature childbirth, Disease prevalence, Bandar-Abbas.
P-5
Infertility and quality of life in women with polycystic ovary syndrome
Bahri Khomami M1, Ramezani Tehrani F1, Azizi F2.
Introduction: Polycystic ovary syndrome (PCOS), the most common endocrine disorder, may cause infertility among affected women. Infertility is a life crisis and blamed for psychosocial distress and poor quality of life (QoL). Ethnicity and socio-cultural factors may have an impact on this negative influence on QoL.
Materials and Methods: The study procedure was completed by 796 women with polycystic ovary syndrome, aged 15-49 years. A reliable validated Persian version of the health related quality of life questionnaire (HRQoL) for polycystic ovary syndrome patients (PCOSQ) was filled for each participant. They were subdivided into 2 groups according to their fertility status: fertile and infertile. Using linear regression the association between infertility and health related quality of life in women with PCOS was assessed.
Results: Out of all participants 120 (15.1%) and 482 (60.6%) women were infertile and fertile, respectively. Infertility was associated with lower HRQoL score before and after adjustment for age, body mass index and other perceived PCOS symptoms (CI 95%: -16.76, -3.81; p=0.002 and CI 95%: -15.02, -3.67; p=0.001). Women who had longer duration of infertility, had got better scores.
Conclusion: We found that infertility causes poor quality of life in PCOS women. Care providers should pay attention to the effect of each PCOS symptoms on patients emotional health along with physical health and plan to treat them accordingly.
Key words: Quality of life, Health-related quality of life, Infertility, Polycystic ovary syndrome.
P-6
The effect of hydrostatic pressure on parthenogenetic activation of mouse oocytes
Bashiri Z, Azadbakht M.
Departeman of Biology, Faculty of Sciences, Razi University, Kermanshah, Iran.
Email: zbashiri88@gmail.com
Background: Parthenogenetic activation of mammalian oocytes using artificial stimuli is commonly used in various reproductive biotechniques. Hydrostatic pressure can act as a mechanical stimulator that rearranges egg contents. In this study, we investigated the effect of hydrostatic pressure on parthenogenetic activation of MП oocytes derived from superovulation and matured oocytes in vitro.
Materials and Methods: In experiment 1, immature oocytes were dissected from ovary of female NMRI mouse (8-week-old) and transferred to α-MEM medium for in vitro maturation. After 24 hr, MП oocytes were transferred to T6 medium. In experiment 2, mice were superovulated by injections of 10 IU of PMSG and 10 IU of HCG 48 hr apart. MП oocytes were collected 12 hr after HCG injection and transferred to T6 medium. Then oocytes from two groups (experiment 1, 2) were divided into experiment and control groups. Oocytes of experiment group were subjected to 20 mmHg pressure for 10, 20, 30 min (treatments I, П, III). Oocytes without exposure to pressure were considered as control. Oocytes were cultured for 72 hr and embryo development was assessed.
Results: In experiment 1, cleavage rate in treatments I, П, III and control was 21.42%, 15.71%, 8.52%, 5.71% respectively. The best cleavage rate were associated with treatment I which were significantly different with treatment III and control group (p<0.05). In experiment 2, cleavage rate in treatments I, П, III and control was 22.91%, 53.12%, 29.15% and 9.34% respectively. Oocyte activation rate in experiment group was higher than control group. The highest cleavage rate associated with treatment П which were significantly different with treatments I, III and control (p<0.05).
Conclusion: Exposure of MП oocytes derived from superovulation to hydrostatic pressure, on account of cumulus cells presence around oocyte, could improve embryonic development and by affecting on calcium channels, probably leading to increase rate of cleavage in the mouse oocyte.
Key words: Parthenogenetic activation, Hydrostatic pressure, Oocyte, Mouse.
P-7
Evaluation the knowledge and awareness of nulliparous women from adverse outcome of delayed childbearing
Behboudi-Gandevani S.
Reproductive Endocrinology Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Email: s.behboudi@hotmail.com
Introduction: Recently the numbers of women who delay childbearing in older age were rapidly increased. The aim of this study was to evaluate the nulliparous women’s knowledge and awareness from adverse outcome of delayed childbearing.
Materials and Methods: In this cross-sectional survey study, a total of 700 healthy nulliparous women 35 years and older that had self-choice delayed pregnancy were selected by cluster sampling in Tehran. To evaluate the knowledge and awareness of these women, a standard questionnaire about adverse pregnancy outcomes in older age were used. The questionnaire consists of three parts: demographic characteristics, assessment of maternal adverse outcomes (10 questions) and to assessment of fetal-neonatal adverse outcomes in maternal older age (6 questions). Data were analysis by SPSS software and the significance level was set at p˂0.05.
Results: The psychometric properties of questionnaire including forward and backward translation, face and content validity and reliability with cronbach's-alpha: 0.078 were done. The result of the study showed that women’s awareness about the risks of delayed childbearing including gestational diabetes, congenital anomaly, Down syndrome, caesarean section, low birth weight, stillbirth and long-term health problems like learning difficulties among neonate varies between 40%-72%. In this respect, most of the women were aware about the increased risk of infertility in older age (82.9%), but a little were aware about the possibility of a twin or multiple pregnancy and gestational hypertension in older ages of mother (35.2%). Awareness of delayed childbearing risks were independently related to the women’s educational levels (OR: 1.87; 95% CI: 1.25-2.78) and family income (OR: 3.12, 95% CI: 0.01-5.12).
Conclusion: It seems that women are largely unaware about the adverse consequences of pregnancy in older age among mother and child. Women’s should be more inform about these risks by health care professional.
Key words: Awareness, Delayed childbearing, Adverse outcome.
P-8
Study the protective role of jujube extract on teratogenic effects of Carbamazepine in Balb/c mice embryos
Doostabadi MR, Hasanzadeh Taheri MM, Afshar M, Hami J, Hosseiny M.
Birjand University of Medical Sciences, Birjand, Iran.
Email: doostabadim@yahoo.com
Introduction: Carbamazepine (CBZ) is an anticonvulsant medication that is consumed during pregnancy and can produce congenital anomalies including; neural tube defects, cardiac, skeletal and craniofacial abnormalities.
Materials and Methods: In this experimental study, 100 pregnant mice of Balb/c 25±3 gr body weight randomly divided into 8 experimental groups (E) and 2 control groups (C) (n=10). Experimental groups (E1, E5 and E6) and (E2, E7 and E8) received daily intraperitoneal injection (IP) of 50 and 100 mg/kg/ of CBZ respectively from gestational day zero (GD 0) to GD 15 and also groups (E5, E7) and (E6, E8) in addition to medicine, received jujube extract (AJE) at doses of 200 or 400 mg/kg/ from ten days prior to gestation, to GD 15. E3 and E4 groups gavaged only by 200 and 400 mg/kg of AJE respectively. Two control groups (C1, C2) received normal saline or tween -20. Dams underwent cesarean section on GD 18 and fetuses harvested from uterine. First, absorbed embryos were counted. Thereafter, morphological studies were done on the offspring by stereomicroscope. All malformed fetuses were stained with Alizarin red S and alcian blue for detection of skeletal anomalies. Data were analyzed by ANOVA, Tukey and c2 tests and using SPSS software version 18 and p<0.05 were considered significant.
Results: The findings of this study showed that administration of CBZ purely in pregnant mice induced various anomalies in their fetuses such as; limb defects, deformities of vertebral column, craniofacial malformations and etc in experimental groups so that their differences were significant when compared with control groups. But these anomalies decreased significantly in those experimental groups which received CBZ and AJE synchronously when compared with experimental groups that received CBZ only.
Conclusion: According to our findings it can be concluded that although administration of CBZ can induce several malformations in fetuses of pregnant mice, but consumption of AJE synchronously with CBZ, can prevent teratogenicity of CBZ. Therefore, probably AJE can play a protective role against CBZ induced anomalies.
Key words: Teratogenic, Jujube, Carbamazepine.
P-9
Depression and clinical markers in polycystic ovary syndrome
Enjezab B1, Ghadiri anari A2, Eftekhar M3, Mohajeri M1.
Introduction: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among reproductive-age women. Previous studies have raised conflicting results of depressive disorders in PCOS compared with healthy people.
Materials and Methods: In this cross-sectional analytic study, 62 patients with PCOS diagnosed by Rotterdam criteria and 61 women without PCOS were selected after an initial survey conducted to determine demographic characteristic, systolic and diastolic blood pressure and reproductive status, the Beck depression questionnaire short form was used to assess depression in both groups. All women were introduced to determine fasting insulin and blood sugar and testosterone.
Results: The mean age, BMI, systolic and diastolic blood pressure of PCOS and Non PCOS was respectively (29.96±6.85, 29.49±7.44 years), (29.15±6.56, 25.65±5.84), (117.42±10.17, 117.18±66.57) and (75.41±9.27, 76.42±6.56) respectively that only mean of BMI was different (p˂0.05). The average Beck score in patients was 7.47±5.54 and in the control group was 7.57±5.79, (p˂0.05), which was not statistically significant. 37.1% of the patient group and 36.1% of the control group have degree of moderate to severe depressed mood (p˃0.05).
Conclusion: Despite non significant difference in prevalence of depression in both groups, due to the high prevalence of depression in both groups may be there is other reasons for depression in this population that overcomes on the psychological effects of this disease.
Key words: Depression, Polycystic ovary syndrome, BMI.
P-10
Comparison of metabolic syndrome in postmenopausal women with natural or surgical menopause: Community based cohort study
Farahmand M, Ramezani Tehrani F, Azizi F.
Introduction: The ovaries produce female sex hormones that after natural menopause gradually and following surgical menopause abruptly end.
Materials and Methods: The study subjects were selected from 5019 women, 59-35 years old, which were participated in the Tehran Lipid and Glucose Study. They include 357 natural menopauses and 63 surgical menopauses during the follow-up that was 10 years. Data were collected by questionnaires in this project.
Results: Changes in metabolic and biochemical profiles of these two groups of women during follow-up were compared with each other. The findings of our study showed that the incidences of metabolic syndrome in surgical menopause and natural menopause subjects were 14.5 and 8.9% respectively. Mean serum low density lipoprotein in surgically menopausal women was significantly lower than natural menopause subjects. Also, mean systolic blood pressure in naturally menopausal women was significantly higher than surgical menopause women.
Conclusion: It seems the metabolic disorders associated with menopause differ in surgical and natural menopause women. The metabolic complications should be considered following menopause.
Key words: Surgical menopause, Natural menopause, Metabolic syndrome.
P-11
Medical staffs’ viewpoints on “ART nursing” working at Reproductive Institutes in Iran
Faramarzi A, Khalili MA, Borzouei Z, Hosseini A, Rahiminia T.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: faramarzi_90@yahoo.com
Introduction: As, assisted reproductive techniques (ART) advances, multifaceted changes are noted in the roles, responsibilities, and commitments of each members of the infertility team. The aim was to determine the level of knowledge and attitude of medical staff working at reproductive institutes about role of nursing in ART program.
Materials and Methods: In this descriptive cross-sectional study, knowledge and attitudes of 199 Bio-medical staffs working at reproductive institutes were investigated through a questionnaire including 20 questions. All participants based on work experience, education and communication with patients were classified. Chi-square test was used for data analysis and p<0.05 was considered as statistically significant.
Results: There was no significant relationship between educational levels and viewpoints on ART nursing. But, significant relationship between work experience, kind of relation to the patients and viewpoints on ART nursing were found. 52.5% of participants were familiar with ART nursing. By increasing experience, knowledge and attitudes of Bio- medical staffs enhanced. Laboratory technician, embryologist, ART Laboratory technician and nurses were more familiar with ART nursing; but, Urologists were less familiar with ART nursing. Over 97% of participants requested offering a master degree of ART nursing in Iran (p<0.05).
Conclusion: There is a need for nursing professionals to assume an ongoing, visionary, scientific, and academic approach to advancement. It seems that appropriate continuing education and opportunities are essential to support this group of nursing professionals.
Key words: Viewpoint, Nurse, ART, Medical staffs.
P-12
The effect of microtubule stabilizer pretreatment in IVM program
Fesahat F1, Khalili MA1, Khoradmehr A1, Anbari F1, Bazrgar M2, Taheri F1.
Introduction: It has been reported that in vitro maturation (IVM) increases the risk of abnormal spindles and chromosome configurations of oocytes compared with oocytes matured in vivo. This is one possible explanation for the reduced developmental potential of IVM oocytes compared with those matured in vivo. Paclitaxel is known to stabilize the microtubules that constitute the spindle. The aim was to investigate whether pretreatment by a microtubule stabilizer, paclitaxel, would improve IVF outcomes in IVM program.
Materials and Methods: In this ongoing experimental study, 75 GV oocytes were retrieved from 5 mice primed with rec-FSH for 48 hr. The immature oocytes were cultured 24-48 hr for IVM. After identifying mature oocytes by the presence of a first polar body extrusion under a stereomicroscope, Normal morphologically MII oocytes were divided in control and 5 experiment groups. Experiment groups were incubated in presence of 1μM paclitaxel for different times (30 min/group 1,1 hr/group 2,2 hr/group 3,3 hr/group 4,4 hr/ group 5 and group 6 as control) prior to IVF. Standard IVF program was performed for the 60 MII oocytes after pretreatment with paclitaxel and embryo development was followed until blastocyst formation.
Results: Total maturation rate of GV oocytes was 80% and fertilization rates of MII oocytes were 85% in groups 6, 83% in groups 1, 2 and 3 and 74% and 65% in groups 4, 5 respectively. Blastocyst formation rates of embryos were 95% in group 1, 2 and 6, 60% in group 3 and 40% in groups 4 and 5. The final results, which include probable relationship between paclitaxel pretreatment and the aforementioned variables, will be presented.
Conclusion: The preliminary results showed by performing critical concentration with optimized timing of microtubule stabilization procedure IVM, MII oocytes could efficiently develop to the blastocyst stage. Paclitaxel may cause cell toxicity with irreversible harm on normal fertilization and embryo development in higher concentrations.
Key words: Paclitaxel, IVM, IVF outcomes.
P-13
The effect of ovarian drilling on poly cystic ovary syndrome (PCOS) women
Ghasemi-Esmailabad S, Eftekhar M, Pourmasumi S, Rajabi Firoozabadi Sh, Sabeti P, Heidarian Meimandi H.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: Qasemisaeed@yahoo.com
Introduction: One of the most common causes of female infertility factors is ovulation disorder. Poly cystic ovary syndrome (PCOS) has the highest prevalence rate about 5-10% among women. There are different diagnostic methods and treatments for PCOS syndrome, including medical and surgical treatments. Laparoscopic surgical treatment is for the patients resistant to medical treatment, and could be done with ovarian drilling at some points.
Materials and Methods: In this retrospective study we evaluated 289 patients. All patients had proven clomiphene resistant PCOS with ultrasound and hormonal tests. Laparoscopic ovarian cautery was performed for all these women. Age, duration of infertility and assisted reproductive techniques in each patient were asked.
Results: Of the 289 patients, we access to the information of 135 patients. The mean age and the duration of infertility were 26.90 and 4.83 years respectively. Among these, 63 patients (46.6%) had not pregnancy and 72 patients (53.33) were pregnant, 52 patients (72.22%) were naturally pregnant and 20 patients (27.77%) used assisted reproductive techniques.
Conclusion: PCOS patients who resistant to clomiphene could be treated with ovarian drilling.
Key words: Polycystic ovary, Clomiphene, Ovarian drilling.
P-14
Comparison of two different embryo loading techniques for embryo transfer in IVF/ET cycles
Halvaei I, Mangoli E, Omidi M, Khalili MA.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: ihalvaei@gmail.com
Introduction: Embryo loading (EL) is one of main steps in embryo transfer (ET) technique that plays an important role in IVF success. This study was aimed to compare the effect of two different techniques for EL on rates of pregnancy and delivery in IVF/ET cycles.
Materials and Methods: In total 195 fresh ET and 171 frozen-thawed ET were included in this retrospective study in which 76 and 119 cycles in fresh ET and 76 and 95 cycles in frozen-thawed ET were placed in two groups of A and B, respectively. Embryo catheter loading techniques were divided into two groups of A and B. In group A, the whole catheter was flushed with a 1-ml air-tight syringe with Ham’s F10 medium. Then the embryos were drawn into ET catheter between two air brackets. In group B, 70 µl airs was held in the syringe and the catheter was flushed by Ham’s F10 medium. Then the media, air, embryos, and air were respectively drawn into catheter. The main outcome measure of the study was delivery rate.
Results: The groups were matched for number of fertilized oocytes, etiology of infertility, source of sperm, type of stimulation protocol, percent of conventional IVF or ICSI, high quality embryos, type of embryo loading catheter, fresh ET and ease of transfer. In fresh ET cycles, delivery rate showed an increasing trend in group B compared to group A (21% vs. 11.8%, respectively, p=0.1). In frozen-thawed ET cycles, the rate of delivery rate was also higher in group B compared to group A (16.8% vs. 13.1%, respectively), but the difference was not significant (p=0.5).
Conclusion: It seems different embryo catheter loading has no effect on delivery rate.
Key words: Embryo loading technique, Embryo transfer, Pregnancy.
P-15
Application of sonography in infertility treatment cycles: instructions for midwives and nurses
Irani Sh, Ahmadi F, Javam M.
Department of Reproductive Imaging at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: Irani_shohreh@yahoo.com
Introduction: Ultrasound has a pivotal role in imaging modality in the study of the female pelvis, and provides fundamental information in detecting and characterizing pelvic masses of uterine, ovarian, or adnexal origin.
Materials and Methods: A narrative review was performed within articles published at PubMed, Elsevier, SID and original text books to reach the aim.
Results: Every patient in the process of infertility treatment needs to go through four steps of sonography to roll out disorders and malformations in ovaries, uterus and tubes then to opt for the best treatment method and to follow up the results. Infertility Workup: 1) Base sonography is applied in examination of the following organs: -For overall evaluation of the pelvis and determining any pathologic condition in uterus such as polyps, fibroms, uterus malformations. -Ovarians are examined for ovarian cysts and Poly Cystic Ovary Syndrome (PCOS). 2) Pre-treatment sonography is applied in checking the result of operative surgeries- like Hysteroscopy, Laparoscopy, Myomectomy. 3) Monitoring sonography is done at different stages of treatment cycle, especially at ovulation stimulation period, in order to investigate ovarians and in picking up the eggs as well as endometrial thickness in response to drugs. 4) Post-Assisted reproductive techniques (ART) cycles is done for confirmation of pregnancy (gestational sac, fetal heart), and the side effects of treatment cycles such as ovarian hyper stimulation syndrome (OHSS), pregnancy complications such as ectopic pregnancy (EP) and mole.
Conclusion: Ultrasound has a key role in diagnosis and proposing of different options of treatment regimes, and the post-treatment follow-ups. It is important to know the application of the aforementioned four steps to improve the accuracy and efficiency of infertility treatment.
Key words: Ultrasonography, ART cycle, Infertility.
P-16
Evaluation of anxiety, depression and risk factor associated with them in women after tubal ligation
Jahanian Sadatmahalleh Sh1, Ziaei S1, Kazemnejad A2, Mohamadi E3.
Introduction: Tubal ligation is recommendable for women completed their family. The existence of anxiety and depression following this procedure has been the subject of debate for decades.
Materials and Methods: A historical cohort study was carried out on 200 subjects with tubal ligation and on 200 subjects using condom as contraceptive method. The two groups were matched in demographic and personal characteristics. Data collection tool was a questionnaire including questions regarding demographic and obstetrical characteristics. Anxiety and depressive symptoms were evaluated by the Hospital Anxiety and Depression Scale (HADS). All statistical analyses were carried out using software package used for statistical analysis (SPSS) version 20 (SPCC Inc., Chicago, IL, USA). Student’s t-test and chi-square test were carried out to reveal the statistical differences between the groups. Multiple linear regression was done to build a prediction model in anxiety and depression.
Results: The mean (SD) duration of tubal ligation was 4.1 (1.6) years. The overall prevalence of anxiety and depression in the two groups was 81.5% and 48.5% respectively (p<0.0001). Multiple linear regression analysis revealed a significant association between low education (p<0.0001), post-sterilization regret (p=0.03) and no consultation prior tubal ligation (p=0.03) with risk for anxiety and depression in women who have undergone tubal ligation.
Conclusion: Termination of fertility with tubal ligation may be a risk factor for anxiety and depression. We found significant differences in anxiety and depression between women with and without tubal ligation. Therefore, women should be informed by the health providers regarding the advantages and disadvantages of tubal ligation before the procedures.
Key words: Historical cohort study, Tubal Ligation, Anxiety, Depression.
P-17
Polymorphism in CGA affects the function of miR-1302 and increases the risk of men infertility
Jamalvandi M, Motevali bashi M.
Department of Biology, Faculty of Sciences, Esfahan University, Esfahan, Iran.
Email: mina.jamalvandi@yahoo.com
Introduction: Infertility occurs in 10-15% of couples worldwide and close to half of it is caused by male factors. Despite decades of efforts to clarify mechanism of male infertility, most of cases are still idiopathic. Many factors such as genetics and sexual problems can affect infertility. Among these problems, genetic disorders are the most common factors. A study has shown that one of genes that can affect male infertility is CGA. This gene is involved in miotic. CGA, α subunit of glycoprotein hormones, is the main part of thyrotropin glycoprotein hormone (pituitary TSH), lutropin (LH), follitropin (FSH), and Chorionic gonadotropin (human placental gonadotropin, hCG) that has essential role in development and function of tyroid and gonads. CGA gene is located on 6q14-q21. Rs6631 in CGA has strong association with men infertility. Studies have shown that miR-1302 can negatively regulate CGA and substitution of T with A may interfere this process. This miRNA can band with rs6631-A more strongly than rs6631-T.
Materials and Methods: Tetraprimer technique is an appropriate way to study this polymorphism; because it is faster and cheaper than ordinary PCR. Also laboratories with low equipment can use this method.
Results: Primers designed by the use of Primer1 and then checked by Oligo7 software. By the use of these primers and tetraprimer technique, this polymorphism can be studied for the first time in Iran.
Conclusion: SNP in genes specially the ones that are target of miRNAs, can have important role in complex disease such as infertility. In this study, miR-1302 negatively regulates CGA, and the substitution of T by A at rs6631 within the binding site disrupts its regulation. It is not exactly clear that what is the real role of this polymorphism in idiopathic male infertility; but it has been suggested that the variant allele of rs6631 may elevate the risk of idiopathic male infertility through up-regulation of the expression of CGA. It has been showed that α-subunit, that is produced by CGA, can has growth factor activity and can induce the differentiation of lactotrope and secretion of RPL. Because CGA and PRL play an important role in the development of gonads, the abnormal hormone level may elevate the risk of male infertility.
Key words: Men infertility, miRNA, SNP, CGA.
P-18
Evaluation of testosterone levels after GnRH agonist administration in the adult male rat
Khadivi B1, Peirouvi T1, Zirak Javanmard M2, Rasmi Y3, Mohammadbeigi H1.
Introduction: Gonadotropin-releasing hormone (GnRH) is secreted from the hypothalamus in a pulsatile manner and stimulates the biosynthesis and release of gonadotropins, LH and FSH, via GnRH receptor located in gonadotropin cells. These gonadotropins regulate various gonadal functions such as gametogenesis and steroidogenesis.
Materials and Methods: 24 male adult Wistar rats were divided into three groups. In the first study group, 300 µg/kg buserelin, in the second study group, 600 µg/kg buserelin and in the control group, salin was injected subcutaneous for 5 days. Thirty day after the first injection, blood samples were collected from the heart, centrifuged and plasma was isolated from blood. Plasma testosterone level was measured by ELISA.
Results: The findings reveal no significant differences in plasma level of testosterone in the first study group (2.71±0.71) and second study group (2.12±1.74) compared with control group (3.14±1.20) (p>0.05).
Conclusion: Short-term administration of buserelin has no effect on the plasma testosterone level.
Key words: GnRH, Buserelin, Testosterone, Adult rat, ELISA.
P-19
Apoptotic cells and loss of follicle development were resulted after administration of Nano dioxide titanium on immature mouse ovary
Khoradmehr A1, Danafar AH1, 2, Hosseini M3, Halvaei I1, Golzadeh J1, Akyash F1, Anvari M1.
Introduction: Titanium dioxide (TiO2) is used as an antimicrobial and whitening agent in food and products such as chewing gums, candies, toothpastes, lip balms, shampoos, deodorants and sunscreens. Previous studies showed that ovary might be one of the target organs that TiO2 nanoparticles induced genotoxicity and cytotoxicity in ovary cells and exposure to TiO2 can effect on follicle development and fertility with changes the levels of sex hormones. The aim of the present study was to evaluate ovarian dysfunction by detect of follicle development.
Materials and Methods: 40 immature Balb/C female mice (4week ages) randomly divided into five groups. First, second and third groups were administrated by oral gavage 2.5, 5 and 10 mg/kg TiO2 for thirty days respectively. Forth group was gavaged normal saline as placebo and in control group we didn’t gavage anything. Finally female mice were dislocated and ovary was fixed in bouin's fluid. Ovaries were dehydrated with ethanol series, embedded in paraffin and then serially sectioned at 5μm thickness. Hematoxylin and Eosin used to evaluate primordial, primary, preantral and antral follicles by light microscope. TUNEL assay was used to detect cell apoptosis. Statistical significance was analyzed by Kruskal-Wallis test and the level of significance was determined to be at p≤0.05.
Results: According to data, primordial, primary, preantral and antral follicles were significantly decreased in third group in comparison with the controls and shams (p≤0.05) and also, there was increasing trend in primary and preantral follicles in first and second groups compared to control group during TiO2 exposure. Although, significant reduction of apoptotic cells was observed in third group compared to control and sham groups (p≤0.05).
Conclusion: Furthermore, there is little knowledge about physicochemical characteristics of TiO2 nano materials; our findings suggested that TiO2 exposure can defect follicle development and perhaps fertility potential in young mice.
Key words: Titanium dioxide, Ovary, Follicle development, TUNEL.
P-20
Correlation between 25-OH vitamin D in follicular fluid and implantation rate in infertile women undergo IVF/ICSI
Latifi Z1, Farzadi L2, Khayatzadeh Bidgoli H2, Fattahi A1, Nouri M2.
Introduction: Vitamin D in combination with its receptors can be involved in implantation by regulating gene expression, endometrium immune response and stimulation of endometrium decidualization. So, it seems that the amount of vitamin D in follicular fluids (FF) may has an association with ART success.
Materials and Methods: In a prospective study, 80 infertile female candidates for IVF/ ICSI were enrolled. Blood samples (on the day of human chorionic gonadotropin) and follicular fluids were taken, and then levels of serum estradiol and follicular fluids 25-OH vitamin D were measured. Also clinical characteristics of patients (duration of infertility, causes of infertility, and menstrual status), number and quality of oocytes, number of fertilized oocytes, estradiol levels, and clinical pregnancy were evaluated.
Results: Concentration of FF 25-OH vitamin D in pregnant women was significantly higher than non-pregnant women (p=0.007) but there were no significant differences in age, body mass index (BMI), duration of infertility, menstrual status, number of oocytes, oocytes quality, number of fertilized oocytes and serum estradiol levels between the two groups (p>0.05). Statistically positive correlation was found between 25-OH vitamin D levels with patient age and implantation rate (r=0.264, p=0.018 and r=0.301, p=0.007 respectively).
Conclusion: The obtained results suggest that vitamin D without affecting the number and quality of oocytes can independently improve implantation rate and IVF outcome.
Key words: Embryo implantation, Vitamin D, IVF outcome.
P-21
The protective effect of N-acetyl-L-cysteine on spermatogenesis and sperm characteristics in mice following exposure to Para-Nonylphenol
Malmir M, Soleimani Mehranjani M, Naderi Noreini S.
Faculty of Science, Arak Univercity of Medical Scinecs, Arak, Iran.
Email: mehhdi@yahoo.com
Introduction: Para-Nonylphenol (p-NP) is an environmental contaminant with wide industrial applications which causes oxidative stress in different organs such as the reproductive system. The purpose of this study was to investigate the protective effect of N-acetyl-L-cysteine (NAC), as a powerful antioxidant, on spermatogenesis indices, sperm parameters, chromatin quality and tail length following treatment with para-Nonylphenol in adult mice.
Materials and Methods: 24 adult male NMRI mice (32±4 gr) were divided randomly into 4 groups (n=6), control, NAC (150 mg/kg/day), p-NP (250 mg/kg/day) and p-NP+NAC, and they were treated orally for 35 days. By the end of the treatment, mice were weighed and sacrificed, Their right testis was also weighed then the left caudal epididymis was cut in Ham's F10. Released spermatozoa were used to analyze the motility, viability and the abnormalities of the sperm. The sperm tail length was estimated by stereological methods. Sperm chromatin quality was also assessed by nuclear staining using acridine orange and aniline blue dyes.In continue, the right testis were taken out, fixed, sectioned, processed and stained using heidenhain azan method. Spermatogenesis indices including the tubular differentiation index (TDI), Sertoli cell index (SCI) spermatogenesis index (SI), meiotic index (MI) and repopulation index (RI) were studied. Data were statistically analyzed using one way ANOVA and means were considered significantly different at p<0.05.
Results: A significant decrease in the spermatogenesis indices, sperm motility, viability, sperm tail length and the number of sperms with normal morphology was observed in the p-NP group compared with the control (p<0.002), while no significant change was found in the sperm chromatin quality. The above parameters significantly increased in the p-NP+NAC group compared to the p-NP treated ones (p<0.01).
Conclusion: N-acetylc-L-ysteine, as an antioxidant, can prevent the adverse effects of para-Nonylphenol exposure on spermatogenesis indexes and sperm parameters in mice.
Key words: Para-Nonylphenol, N-acetyl-L-cysteine, Spermatogenesis indices, Sperm parameters.
P-22
The effect of vitamin C on human vitrified sperm parameters in normozoospermic men: raw semen and washed semen
Mangoli E1, Anvari M2. Talebi AR3, Taheri F1, Rahiminia T1, Vatanparast M1.
Introduction: Vitamin C is a common component in seminal fluid. It can improve sperm parameters as an antioxidant. The objective was to evaluate the effect of Vitamin C on human vitrified sperm parameters in normozospermia men.
Materials and Methods: Semen samples were collected from 40 normozoospermic samples and divided into 4 groups. Group 1, raw semen was vitrified. Group 2, semen processed by swim up method and then vitrified. Group 3, vitamin C (600 µm) was added to raw semen and then vitrified. Group 4, vitamin C (600 µm) was added to prepared spermatozoa and then vitrified. The semen analysis was performed according to WHO criteria before and after vitrification.
Results: In this study data showed that progressive motility and immotile spermatozoa after vitrification were significantly different between groups (p=0.014, 0.005 respectively). All groups were compared, and the results revealed that progressive motility and immotile spermatozoa after vitrification were statistically significant in washed semen+vitamin C group compared with wash semen and raw semen groups (p=0.05, 0.01 for progressive motility and p=0.04, 0.007 for immotile spermatozoa respectively).
Conclusion: Adding vitamin C, as an antioxidant, to washed semen has shown beneficial effect on sperm motility after vitrification.
Key words: Sperm parameters, Vitamin C, Vitrification.
P-23
Review of fertility diet and its effect on infertility among women from the viewpoint of Iranian traditional medicine in comparison with modern medicine
Tork Zahrani Sh1, Mirahi A1, Tansaz M2, Alaeddini F3, Hekmatdoost A4.
Introduction: In the recent years the infertility diagnosis and treatment methods have undergone a radical change and development but still, the success rate of modern treatments like in vitro fertilization (IVF) even in the best centers is between 30-40%.
Materials and Methods: The available texts on the Iranian traditional medicine were reviewed. Besides, the relationship between fertility diet factors and infertility among women was investigated through reviewing databases on the world-wide web.
Results: Iranian traditional medicine investigates the relationship between nutrition and infertility from a perspective postulating that complying with fertility diet principles helps maintain healthiness and improve fertility. From this view, body members have their special temperaments; if balanced, body members function normally. Any kind of abnormal temperament happened to the semen or uterus results in infertility or hardship in fertility. These are seen as disorders in sanguine, choleric, melancholic, and phlegmatic temperaments. It should be noted that the bad temperaments are mainly caused by not following the health and fertility diet.
Conclusion: The different viewpoint of the Iranian traditional medicine towards the relationship between infertility and nutrition and its focus on the nature of food and nature of a person could be of interest to researchers in the field and if approved, help to prevent infertility or treat infertile couples.
Key words: Infertility, Iranian traditional medicine, Infertility diet.
P-24
The in vitro fertilization outcome and luteal phase GnRH antagonist administration
Eftekhar M1, Miraj S1, 2.
Introduction: Genital tissues (ovary, endometrium and placenta) express GnRH receptors. GnRH plays essential roles in embryo implantation, invasion of trophoblastic tissue and steroid synthesis in the placenta. In IVF-ICSI cycles, the use of GnRH antagonists is limited to the last days of ovulation. The aim of this study was to evaluate the effects of GnRH antagonist at pharmacological doses given in the early implantation period on pregnancy.
Materials and Methods: This retrospective study was performed in Yazd Research and Clinical Center for Infertility, 2014-2015. Women under 40 years old, with >20 follicles (>11mm) and risk of OHSS were included. Participants with history of endometriosis, hysteroscopy and history RIF were excluded. The treatment for all of participants was antagonist protocol. Twenty seven patients did not receive Cetrotide in luteal phase, and 67 patients received Cetrotide. Pregnancy outcomes were assessed based on chemical and clinical pregnancy.
Results: Totally 94 patients were included. The mean age of participants was 28.40±4.25 years old. There were no differences in ART and demographic characteristics (p>0.05). The most frequent causes of infertility were: male factor (45.2%) and polycystic ovary syndrome (19.4%). The pregnancy outcome was not significantly different between Cetrotide and non Cetrotide group (p=0.224).
Conclusion: The present study proposed that luteal phase GnRH antagonist administration does not influence the chance of successful pregnancy outcome. The incidence of chemical and clinical pregnancy in two groups was not significantly different.
Key words: GnRH antagonist, Pregnancy outcome, IVF.
P-25
Hazardous effect of acrylamide on development landmarks in rat offspring and the role of Glycyrrhiza Glabra
Mirgalili A1, Yadegari M1, Talebi AR2, Dortaj H1.
Introduction: Acrylamide (AA) is a chemical substance used mainly in certain industrial process. Monomer of Acrylamide formed in food stuffs containing carbohydrates during high temperature cooking.
Materials and Methods: 24 pregnant rats were selected for this study. The pups of these mothers divided into four groups: group A:(control group); group B: (ACR administration 10 mg/kg/day orally); group C: (GG administration 150 mg/kg/day orally); group D: (ACR+GG). At day 21 the pups were evaluated for developmental study. The results were analyzed by SPSS software (15) and p<0.05 was significant.
Results: The result demonstrate that eye opening and fur development appeared occurred slightly later in AA administration group.
Conclusion: These results showed that AA treatments induce delay in developing offspring and GG as an antioxidant reduce these changes.
Key words: Acrylamide, Glycyrrhiza glabra, Eye opening, Fur appearance, Offspring.
P-26
The effect of long-term exercise via TNF-α level on the type-1 diabetic rats in testis tissues
Moghaddami Z1, Sheikhzadeh Hesari F1, Banan khojasteh SM1, Hemmati AR2, Dastranj A3.
Introduction: Approximately 90% of diabetes patients often show sexual abnormalities and impotence infertility. Damages caused by hyperglycemia and oxidative conditions result in inflammatory mediators increasing such as cytokines in tissues of sex organs. The TNF-α is the first locally produced cytokines in chronic inflammation pathway. TNF-α level in diabetics blood is higher than normal people that probably histological studies of TNF-α could determine source of this increasing. Also exercise can reduce complications of diabetes especially in tissue by reducing of oxidative elements and establishing of Hypoglycemia.
Materials and Methods: In this study, 40 male rats weighing 200-250 gr were used and randomly were classified into four groups; each group consist 10 rats (control, diabetic, healthy with period of 60-day exercise, diabetic with period of 60-day exercise). Treadmill exercise daily was 1 hr with 22 m/min speed. Diabetes was induced by injection intraperitoneal of streptozotocin with the amount of 60 mg/kg. After expelling of the testis under general anesthesia, samples were homogenized and TNF-α protein levels were measured according to ELISA kit (special rat TNF-α).
Results: The level of TNF-α showed no significant changes among diabetic groups in testicular tissue compare to control groups. Also, exercise didn’t have any negative effects on the level of TNF-α in exercise control group compare with control group. Therefore, we didn’t show any significant change of TNF-α level between our groups.
Conclusion: In this study, the period of disease time may be is not enough to determine the level of TNF-α via exercise treatment in testis tissues. It seems that duration of diabetic developing, severity and type of exercise could be many important reasons to change inflammatory mediators.
Key words: Cytokine, Hyperglycemia, ELISA Kit, Sexual dysfunction.
P-27
Evaluation of progesterone and estradiol levels after GnRH agonist administration in the adult female rat
Mohammadbeigi H1, Peirouvi T1, Rasmi Y2.
Introduction: Gonadotropin-releasing hormone (GnRH) is secreted from the hypothalamus in a pulsatile manner and stimulates the synthesis and release of gonadotropins, LH and FSH, via GnRH receptor located in adenohypophysis. These gonadotropins, regulate various gonadal functions such as folliculogenesis and steroidogenesis. Various data are reported of GnRH agonists effects on ovary steroidogenesis.
Materials and Methods: 24 female adult Wistar rats were divided into three groups. In the first study group, 300 µg/kg buserelin, in the second study group, 600 µg/kg buserelin and in the control group, the same amount of salin was injected subcutaneous for 5 days. 30 days after the first injection, blood samples were collected from the heart, centrifuged and plasma was isolated from blood. Plasma esteradiol and progesterone levels were measured by ELISA.
Results: The findings reveal a significant incereas in plasma level of estradiol in the second study group (165±104.89 pg/ml) in comparison with control group (55.47±50.32 pg/ml) and in the second study group compared with the first study group (47.28±32.95 pg/ml) (p<0.05). But no significant differences were observed in plasma level of progesterone in the three groups (p>0.05).
Conclusion: Short-term administration of high doses buserelin increase plasma estradiol level, while has no effect on the plasma progesterone level.
Key words: Buserelin, Progesterone, Estradiol, Adult rat.
P-28
A comparative study on the effect of face-to-face or group education during the pregnancy period on sexual function of the couples under coverage of selected clinics in Isfahan in 2013
Mohammadi M1, Khani MB1, Bahadoran P2.
Introduction: Pregnancy can conflict with sexual function that can be affected by physical and psychological changes during Pregnancy.
Materials and Methods: In this quasi-experimental pre-post-test study, 64 couples with pregnant women were selected and randomized in two groups in Isfahan. The data were collected via the triangulation of FSFI, BFSI and demographic characteristics questionnaires. SPSS -18 was used to analyze the data by descriptive and inferential statistics.
Results: No significant difference was found in the demographic characteristics between two groups. Education was effective on sexual function in two groups of women (p<0.001), but no significant difference was found between two groups (p=0.61). Also education was effective on sexual function of the men in both groups (p<0.001), and there was a significant difference between the two groups (p=0.003). Meanwhile, there was no significant difference between couples regarding the education (p=0.104).
Conclusion: The results of the study showed that type of education plays a role in improvement of sexual function in pregnancy. In addition, sex education is effective on prevention of sexual disorders in pregnancy. Therefore, having a special approach toward sex education classes during pregnancy is inevitable for the health providers, particularly midwifery professionals.
Key words: Group education, Face to face education, Sexual function, Pregnancy, Iran.
P-29
Assisted reproductive technology outcomes in couples with hepatitis virus infection
Mohsenzadeh M, Omidi M, Nabi A, Khalili MA.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, iran.
Email: mohsenzade@sums.ac.ir
Introduction: Currently, many hepatitis positive infertile couples attend assisted reproductive technology (ART) program, so there is concern as this viral infection may be deleterious to human fertility. Some have suggested that hepatitis virus have an adverse effect on pregnancy outcomes.
Materials and Methods: The study was conducted in two groups of patients: A) seropositive for hepatitis B virus (HBV) and hepatitis C virus (HCV) (n=46); and B) patients lacking these viruses as a control group (n=32). Patients included couples seeking ART from January 2010 to October 2014. ART cycle characteristics and clinical outcomes were assessed between two groups.
Results: Demographic characteristics and age were similar between two groups. The mean±SD age was 30.19±5.54 and 34.31±6.86 years in female and male, respectively. Regarding to oocyte number and embryo score, there was no significant differences between groups A and B. The rate of pregnancy was, however, significantly higher in group B in comparison with A (p=0.03).
Conclusion: Although, the data on embryo formation was similar in both groups of infertiles, but the pregnancy outcomes were noticeably reduced in infertile patients with hepatitis virus infections.
Key words: Hepatitis virus infection, ART, Pregnancy outcome.
P-30
The influence of the Corpus Luteum (CL) on hormonal and biochemical metabolites composition of follicular fluid from different sized follicles and their relationship to serum concentrations in dairy cows
Moradi kor N.
Research Center of Physiology, Faculty of Medicine, Semnan University of Medical Sciences, Semnan, Iran.
Email: moradikor@semums.ac.ir
Introduction: Metabolic changes in blood serum may be rejected in the biochemical composition of follicular fuid (FF) and could indirectly influence oocyte quality. In addition, the levels of hormonal and biochemical metabolites in the FF were related to follicular size and interestingly to the presence or absence of a corpus luteum. The purpose of this study were to examine the influence of the corpus luteum on hormonal and biochemical metabolites composition of follicular fluid (FF) harvested from different sized follicles and their relationship to blood serum in dairy cows.
Materials and Methods: Ovaries were recovered from 30 female adult cows (Holstein Friesian) 4-7 years of age with clinically normal reproductive tracts after slaughtering. Blood samples were collected from the jugular vein before slaughter from each cow. The stage of the cycle in the cows slaughtered was diestrus determined post mortem. The ovaries collected per cow were classified with corpus luteum (CL+) and without corpus luteum (CL-). Visible follicles on the surface of the ovaries were classified, based on their diameter, into (I) small (3-5 mm), (II) medium (6-9 mm) and (III) large (10-20 mm) categories. Follicular fluid was aspirated from different sized follicles in CL+ and CL- ovaries. Serum and FF samples were analyzed for hormones (estradiol-17β, progesterone, testosterone, T3 and T4) and biochemical metabolites (glucose, cholesterol, triglyceride, total protein, albumin and globulin).
Results: Results showed that the FF concentration of estradiol-17β, progesterone and testosterone in different size follicles categories (small, medium and large follicles in CL+ and CL- ovaries) were significantly higher (p˃0.05) when compared with the serum. The FF concentration of estradiol-17β, testosterone, glucose and cholesterol in same follicle size categories of CL- ovaries were significantly higher (p˃0.05) when compared with CL+ ovaries. In the present study, the serum concentration of glucose, cholesterol and triglyceride were significantly higher (p˃0.05) when compared with the fuid from different sized follicles categories in CL+ and CL- ovaries. The differences between follicle size categories in CL+ ovaries were only significant for concentration of estradiol-17β and in CL- ovaries were significant for concentrations of estradiol-17β, glucose and triglyceride.
Conclusion: According to the results of the present study, the levels of the biochemical metabolites in the FF were related to follicular size and interestingly to the presence or absence of corpus luteum.
Key words: Dairy cow, Follicular fluid, Corpus luteum, Hormone, Metabolite.
P-31
Health related quality of life in women with polycystic ovary syndrome
Moridi M1, Rahebi SM1, Seyf Zadeh Z2.
Introduction: Polycystic ovary syndrome (PCOS) is associated with symptoms that affect psychological wellbeing and health-related quality of life (HRQoL).
Materials and Methods: This was a retrospective case-control study which was conducted on 116 women from April-August 2014 in Rasht, Iran. Cases were 60 newly diagnosed women with PCOS, according to the Rotterdam criteria while the controls were 60 healthy women and groups were matched on sociodemographics characteristics. Data needed to determine health-related quality of life were collected via convenience sampling using modified PCOS quality-of-life questionnaire (MPCOSQ) that contains 6 subscales: emotional disturbances, hirsutism, infertility, weight, menstrual and acne. Data collected were analyzed using statistical T-test, chi square and Kolmogorov-Smirnov.
Results: Finding demonstrated that groups were matched in age, body mass index, marital status, occupation, educational status and socioeconomic status. The mean HRQoL score was significantly lower among women with PCOS (20.96±7.4) than among controls (29.8±4.5) (p<0.001). The mean scores of all subscales in cases (32.01±13.56 for emotional disturbances, 23.34±9.35 for hirsutism, 12.02±6.6 for infertility, 21.25±10.16 for weight, 16.89±6.71 for menstrual and 20.27±7.25 for acne) were significantly lower than controls (47.53±8.28 for emotional disturbances, 31.81±6.68 for hirsutism, 25.83±4.19 for infertility, 20.23±7.70 for weight, 21.15±7.27 for menstrual and 24.26±6.17 for acne) (p<0.001).
Conclusion: According to the results of this study, the average of HRQoL and all the six subscales of MPCOSQ in the case group was significantly lower than in the control group. The psychological implications of PCOS are easily underestimated and have been largely ignored. Clinicians has a pivotal role in recognizing these concerns and implementing therapy to improve quality of life in women with PCOS.
Key words: Polycystic ovarian syndrom, Quality of life, Infertility.
P-32
Counseling during pregnancy and women with precedent infertility
Naeimi N.
Department of Biology, Faculty of Science, University of Sistan and Baluchestan, Zahedan, Iran.
Email: nnaeimi@rocketmail.com
Introduction: Delivery is physiological phenomenon and among the beautiful events in the life of every woman and yet stressful reality. In the present era considering the increasing of caesarean sections, it require using consulting services for informed, correct decisions.
Materials and Methods: In this descriptive-analytical study, 271 pregnant women, completed a researcher-made questionnaire including, demographic characteristics (age and precedent of infertility) and kind and rates of counseling. To analyze the obtained data, chi-square test was used through applying SPSS19 software.
Results: Our results showed that 7 pregnant women (2.6%) had a precedent of infertility while 259 pregnant women had not (95.6%). In total 53 (19.2%) subject did consulting during pregnancy and 213 (80.8%) did not. From these, 6 women with precedent infertility (85.7%) and 47 (14.3%) without, perform counseling during pregnancy. There was a significant relationship between counseling during pregnancy and fertility (p<0.05).
Conclusion: The results showed that women with precedent infertility because of the importance of pregnancy and because of the history of information which were obtained during the course of treatment were more acquainted with the role of consultant, and therefor used consultant services mostly during pregnancy. Ferthermore, considering to the high prevalence of elective cesarean, counseling during pregnancy can informe the pregnant women about the advantage of normal delivery, causing the reduction in cesarean section prevalance.
Key words: Counseling, Precedent infertility, Pregnancy termination.
P-33
Overweight and obesity of patients: Blind spot in assisted reproductive technology treatment
Naghibzadeh M.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: naghib330@yahoo.com
Introduction: The effects of lifestyle on health were investigated via several studies. But, the impacts of overweight and obesity on fertility and reproductive system are poorly understood. Overweight and obesity may reduce fertility rate via reducing the IVF success rate in both women and men undergoing treatment via assisted reproductive technology (ART). Improve lifestyle, including physical exercise and weight loss program, may improve fertility and ART outcome.
Materials and Methods: The results of the related epidemiological and randomised control trials (RTCs) sited on Pubmed and ISI database were selected as references.
Results: The results indicated that the patients with a BMI above 30 have about 68% less chance to have a live birth following their first ART cycle compared with women with a BMI less than 30. Also, overweight and obesity of the patients were associated with both lower implantation and more risk for cycle cancellation. A correlation between obesity and the risk of spontaneous abortion (22% increased risk) was observed in obese women compared with normal weight women. The body composition, specific fat mass, was adversely associated with the success of the IVF procedure. Finally, some studies indicate that weight loss is associated with a higher spontaneous pregnancy rate.
Conclusion: Lifestyle behaviours may have a significant impact on pregnancy rates in women with infertility problems. The results support the clinical recommendation of advising overweight and/ or obese women to lose weight prior to ART. Nevertheless, it seems that the prospective randomized controlled trials are required to prove efficient evidence-based guidelines for weight loss interventions in overweight and/ or obese women before the ART procedure.
Key words: Lifestyle, Obesity, Fertility, ART.
P-34
Role of nitric oxide in the developmental process of ovarian follicles in pregnant rats
Naseri Sh, Khanehzad M, Noori Moogehi SMH.
Department of Histology, Faculty of Medicine, Tehran University of Medical Sciences (TUMS), Tehran, Iran.
Email: naserishafighe@yahoo.com
Introduction: Nitric Oxide (NO)- as one of the smallest active products, involved in control of a number of physiological processes including cell growth, apoptosis, regulation of blood pressure, defense mechanism and especially in the reproductive process.
Materials and Methods: We used 40 Wistar pregnant rats between 200 and 250 grams in weight and aged eight weeks. Based on observation of vaginal plug, pregnant mice were divided into five groups. The first group was received 2 mg/kg normal saline and the others were received respectively 200 mg/kg L-Arginine, 20 mg/kg L-NAME and a mixture of the same doses of L-Arginine and L-NAME on 3, 4 and 5th gestational days via intraperitoneal. The control group did not received any injection. Ovaries were removed on 18th gestational days, and after fixation and tissue preparation via staining by the routine H&E method, studied by Light microscopy.
Results: Comparing the groups using ANOVA, there was a significant difference in reducing the number of primary follicles and increasing atretic follicles in the L-Arginine group in comparision with the other groups. The histological changes were also observed in the L-Arginine group.
Conclusion: The study results showed that nitric oxide during pregnancy has damaging effects on ovaries and is recommended to be used with caution during pregnancy.
Key words: Ovarian follicles, Nitric oxide, L-Arginine, L-NAME.
P-35
Comparison of ovarian stimulation protocols base on AMH level in patients undergoing intracytoplasmic sperm injection
Nikmard F1, Hosseini E1, Bakhtiyari M1, Ashrafi M2, Aflatoonian A3, Aflatoonian B4, Aflatoonian R2.
Introduction: Controlled ovarian hyperstimulation (COH) plays important role in reproductive medicine because selection of appropriate ovarian stimulation strategy can improve assisted reproductive technology outcomes. Antimullerian hormone (AMH) is a predictor of ovarian response, which can help to select the best treatment strategies in women undergoing agonist and antagonist protocols to optimize safety and clinical pregnancy rates.
Materials and Methods: This study is a retrospective study conducted in a private assisted reproductive unite and a total of 243 patients with tubal factor infertility are selected.
Results: In both of GnRH agonist and antagonist protocol, with increasing of AMH level, oocyte and good embryo number is increased. On the other hand in AMH˂1.1, ˃ 2.8 ng/ml levels, GnRH agonist leads to higher oocyte and embryo number that is significant. On the contrary, pregnancy rate with increasing of AMH level is not increased and the highest rate of pregnancy is observed in AMH 1.1-2.8 ng/ml levels that it observed with antagonist protocol.
Conclusion: Based on AMH levels we can predict assisted reproductive outcomes. In three range of AMH levels, GnRH agonist protocol can lead to better results. In the women with poor prognosis and low and high levels of AMH, it should focus on improving results with increasing of endometrial receptivity or embryo quality.
Key words: Antimullerian hormone, Ovarian response, ovarian stimulation protocols.
P-36
Prenatal exposure to a single dose of testosterone leads to appearance of polycystic ovary syndrome in female rat’s offspring in adulthood
Noroozzadeh M1, Ramezani Tehrani F1, Zahediasl S2, Piryaei A3, Azizi F2.
Introduction: Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women, affecting 8-12% of reproductive-aged women. PCOS is associated with reproductive and metabolic disorders, including infertility, hyperandrogenism, luteinizing hormone hypersecretion, polycystic ovaries, insulin resistance and dyslipidaemia, with an increased risk of cardiovascular disease and type 2 diabetes in those affected. PCOS is known as a genetic disease. Beside genetic factors, environmental factors may contribute to appearance of this syndrome.
Materials and Methods: Pregnant rats were divided into two groups, experimental and control groups (n=10 in each group). Experimental group were subcutaneously injected with 5 mg free testosterone on gestational day 20 and control rats received only solvent at the same time. The female offspring of these mothers were examined for the functioning of their reproductive system in adulthood.
Results: Levels of testosterone and luteinizing hormone and the luteinizing hormone/follicle-stimulating hormone ratio were increased in prenatally androgenized offspring compared with control rats (p<0.05). The numbers of preantral and antral follicles in the ovaries of prenatally androgenized offspring were also increased compared with control rats (p=0.07 and p<0.01, respectively). The number of corpora lutea was decreased in prenatally androgenized offspring compared with control rats. Cystic follicles were observed in the ovaries of prenatally androgenized offspring.
Conclusion: Prenatal exposure to a single dose of testosterone during the critical period of fetal development leads to appearance of PCOS phenotype in female rats with minimal morphological disorders in their reproductive system in adulthood. Production of a functional rat model that resembles many features of PCOS may contribute to a better understanding of this syndrome.
Key words: Fetal life, Testosterone, Polycystic ovary syndrome, Rat.
P-37
Advanced paternal age does not influence the outcomes of ART cycles
Omidi M, Halvaei I, Khalili MA, Ghazali Sh, Tahajjodi S, Sabeti P.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: omidi.marjan@ymail.com
Introduction: The reports in assisted reproductive technology (ART) cycles noted that the fertilization rates were significantly decreased in men over 50 years of age. However, data have shown that there was no adequate substantiation to prove the effects of paternal age on fertility outcomes.
Materials and Methods: In a retrospective study, the outcome of intracytoplasmic sperm injection (ICSI) in ART cycles in two groups of patients was studied. Patients regarding etiology of infertility were divided into two groups: A) male factor infertility (n=47), and B) female factor infertility (n=16). Sperm parameters, ART cycle characteristics and clinical outcomes were assessed between two groups.
Results: There were no significant differences for rates of sperm count and morphology between two groups, but rate of spermatozoa with progressive motility were higher in group B in comparison with A (p=0.002). The rates of high quality embryos, pregnancy and live birth showed no significant differences between the groups (p=0.4, p=0.4, p=0.2, respectively).
Conclusion: It appears that the paternal age has no detrimental effect on rates of pregnancy and live birth in ART program.
Key words: Paternal age, ART, Live birth.
P-38
Effect of non-polar extract of Phaleria macrocarpa (Mahkota dewa) on serum testosterone level and libido behavior in male rats
Aziz Dollah M1, Parhizkar S2, Ferdaus Binti N1.
Introduction: Phaleria macrocarpa (PM) has been claimed to overcome male fertility problems related to reduction of testosterone level.
Materials and Methods: Non-polar hexane extract of PM was prepared using soxhlet extraction technique, dried to powder using rotary evaporator and been kept for later use. In total 30 male adult Spraque Dawley rats weight about 250 gr were divided into five groups. For libido study, 30 female rats weight about 250 gr each were used for the purpose of mounting latency and mounting frequency study. The male rats were kept in individual cage while for female rats, there were five rats for one cage. Three groups of rats were given three different concentration of PM non-polar extract [High dose (60 mg/kg), Medium dose (12 mg/kg) and Low dose (6 mg/kg)]. One group was given tween 20 solution (negative control) and another group was given commercial testosterone hormone (positive control) orally for seven weeks. At the end of experiment period each male rat was introduced to one female rat to determine the libido behavior. Blood sample was then collected using cardiac puncture and the testosterone level was determined by radioimmunoassay kit (TESTO-CTK P3093).
Results: Administration of non-polar hexane extract of PM showed no significant changed for the serum testosterone level and mounting latency of the rats. While, there was a significant change (p<0.05) in the mounting frequency in different groups; 6, 14, 14, 4 and 7 times for the negative control, low dose, medium dose, high dose, and positive control respectively.
Conclusion: PM non-polar hexane extract did not show a potential value as an alternative way to improve the sexual strength. However it showed a potential for the improvement of the mounting frequency.
Key words: Phaleria macrocarpa (PM), Testosterone level, Libido behavior.
P-39
Effect of vitamin C on sperm parameters and serum malondialdehyde levels in mice fallowing treatment with sodium arsenite
Pirozmanesh H, Soleimani Mehranjani M, Naderi Noreini S.
Faculty of Science, Arak University of Medical ASciences, Arak, Iran.
Email: hp457@yahoo.com
Introduction: Arsenic as an environmental toxicant is able to exert malformation in male reproductive system by inducing oxidative stress. Ascorbic acid with potent antioxidant property is able to restrict oxidative stress.
Materials and Methods: In this experimental study, 24 NMRI mice were divided into four groups (n=6): control, sodium arsenite (7 mg/kg), ascorbic acid (150 mg/kg) and ascorbic acid + sodium arsenite. Oral treatment was performed for five weeks. At the end, left cauda epididymis was removed and used to analyze sperm viability, morphology, motility and sperm tail length. Serum MDA levels, as lipid peroxidation index, was measured by spectrophotometeric method. Data were analyzed using one way ANOVA and means difference was considered significant at p<0.05.
Results: The mean of sperm viability, morphology, motility and sperm tail length significantly decreased in sodium arsenite group compared to control (p<0.04). A significant increase in the serum MDA levels was found in sodium arsenite group compared to control (p<0.05). In ascorbic acid+ sodium arsenite group, a significantly reversed in adverse effect of sodium arsenite on these parameters was observed when compared with sodium arsenite group (p<0.02). In addition sperm viability, motility and sperm tail length was significantly increased in the mice treated with ascorbic acid alone in comparing to the control ones (p<0.001). Also, a significant reduction in MDA levels was found in ascorbic acid group compared with control group (p<0.001).
Conclusion: Ascorbic acid could compensate the adverse effects of sodium arsenite on viability, morphology, motility, sperm tail length and serum MDA levels in adult mice. Hence, this study suggests that ascorbic acid may improve the male fertility by improving sperm parameters.
Key words: Sodium arsenite, Ascorbic acid, Viability, Motility, Sperm tail length.
P-40
The predictors of spiritual growth and interpersonal relations in infertile couples referring to the Infertility Center, Al-zahra Hospital, Tabriz, Iran.
Rahimi M, Sehhatie Shafa'I F, Mirghaforvand M.
Tabriz University of Medical Sciences, Tabriz, Iran.
Email: marieh.rahimi@yahoo.com
Introduction: In all cultures, involuntary childlessness is recognized as a crisis that has the potential to threaten the stability of individuals, relationships, and communities. Every society has culturally approved solutions to infertility involving, either alone or together with alterations of social relationships (e.g., divorce or adoption), spiritual intercession (e.g., prayer or pilgrimage to spiritually powerful sites), or medical interventions.
Materials and Methods: This study is a descriptive cross-sectional study on 322 infertile couples referring to the Infertility Center at Al-zahra Hospital in Tabriz. Samples were selected through simple random sampling method. Questionnaires used in the study were demographic data questionnaire and Lifestyle Profile II (HPLP-II) questionnaire with 52 questions. The multivariate linear regression analysis method was used for defining the social-individual predictors of Spiritual Growth and Interpersonal relations. Data were analyzed by SPSS Win/13.
Results: The findings showed that the mean (SD) score of Spiritual Growth and Interpersonal relations in couples was 2.6 (0.5) out of 1-3 grade range. The variable of job, family member, history of contraception, reason of infertility, and education level were the predictors of Spiritual Growth and Interpersonal relations.
Conclusion: According to the results of the study, the mean score of Spiritual Growth and Interpersonal relations in infertile couples is average, therefore we should focus on predictive variables for recovering the infertile couples’ Health-promoting behaviors status.
Key words: Health-promoting behaviors, Spiritual Growth, Interpersonal relations.
P-41
Comparing in-vitro maturation of oocytes in presence of mature or immature cumulus cells in mice
Rahiminia T, Faramarzi A, Khoradmehr A, Khalili MA.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: tahere_1980@yahoo.com
Introduction: Cumulus cells (CCs) are somatic cells which are coupled with oocyte during maturation. During process of maturation, the secretions by CCs influence oocyte to maintain the functional competence.
Materials and Methods: Superovulated mice were killed and ampulla was ruptured to release cumulus oocyte complex (COCs) in IVM culture media. COCs were incubated for 30 min before removing oocytes. Then, denuded mature oocytes were removed and remaining mature CCs were collected for culturing germinal vesicles (GVs) for exp II. After dissecting ovaries, denuded GVs were considered as control group and put in IVM medium. In exp I, collected GVs with intact immature CCs were put in IVM medium and observed for maturation after 24 and 48 hr. In exp II, GVs were cultured with mature CCs and maturation was checked as mentioned for exp I group.
Results: The rate of maturation was (77.36±14.4) in control group. In exp I and exp II, the rate of maturation was (91.32±22.5) and (63.33±7.4) respectively. Difference in maturation rate was significant between two groups of exp I and exp II (p=0.04) at 24 hr. However, maturation rate did not increase after 24 hr. The average of maturation rate in metaphase I (MI) oocytes was as low as about 15% in different groups at 24 hr and did not increase by 48 hr. Degeneration rate was increased from time 0-48 hr in exp II more than this rate in the other two groups.
Conclusion: Presence of mature CC did not improve maturation of GVs comparing to exp I with immature CCs, or control group as conventional IVM.
Key words: In vitro maturation, Cumulus cells, Mice.
P-42
Investigating the effect of culture pH, on Chinese hamster ovary (CHO) cells growth and recombinant human chorionic gonadotropin (hCG), expression
Rahnama M, Sanati MH, Amiri Yekta A, Gourabi H.
Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: mhsanati@yahoo.com
Introduction: The demand for production of recombinant therapeutic proteins by mammalian cell lines, such as Chinese hamster ovary (CHO) cells, continues to grow. Significant achievements in process optimization including development of cell culture strategies for large-scale, cost effective production have been made. One of the key parameters, that has effect on mammalian cell growth, metabolism and productivity, is pH. Human chorionic gonadotropin (hCG), is a member of pituitary glycoprotein hormone family, which has an important role in regulating human reproductive functions and is widely used in assisted reproductive technologies (ART).
Materials and Methods: Equal number of rCHO cells were cultured in four DMEM media with different pH ranging from 6.8-7.7 (6.8, 7, 7.3 and 7.7). The cells were harvested at confluence and viable cell concentration was determined by trypan blue exclusion. The rhCG production was assessed using SDS-PAGE, Bradford and Western blotting techniques. ImageJ software was used for quantitating Western blotting results.
Results: Results demonstrated that maximum viable cell concentration was at pH=7. Bradford assay showed that total protein concentration reached its maximum level at pH=7 and its minimum at pH=6.8. hCG expression was confirmed by SDS-PAGE and western blotting methods. According to ImageJ analysis, maximum hCG expression level was found at pH=7, and minimun expression at pH=7.7 but no significant difference was found at pH=7.7 and pH=6.8. Overall, pH=7 was assessed as optimum pH for culturing rCHO cells.
Conclusion: Several strategies including culture condition optimization, have developed to improve productivity of a recombinant cell line. Findings suggest that optimizing simple parameters such as culture pH, had the potential to increase the viability and productivity of recombinant CHO cells.
Key words: Chinese hamster ovary (CHO) cell, Optimization, pH, Human chorionic gonadotropin (hCG), Assisted reproductive technology (ART).
P-43
Noscapine induced cell death in human endometriotic epithelial cells
Rashidi Z1, Khazaei M2, Khazaei MR2.
Introduction: Endometriosis is the presence of a functional endometrial tissue outside the uterine cavity and defines as cancer like model. Noscapine is a safe cough suppressant which has been introduced as cancer suppressor.
Materials and Methods: Human endometriotic biopsies (n=7) were digested by enzymatic method (collagenase, 2 mg/ml). Epithelial glands were collected by sequential filtration through nylon meshes (70 and 40 µm). The cells were divided to five groups: control and 10, 25, 50 and 100µM concentration of noscapine and cultured for 24, 48 and 72 hr.Viability of cells was assessed by MTT assay; cell morphological analyses with Acridine orange (AO)-ethidium bromide (EB) double staining also cell death was assessed by TUNEL assay.
Results: Viability of endometrial epithelial cells were decreased in 10, 25, 50 and 100 µM noscapine concentration (84.1%, 80.8%, 72.7%, 67.1%) compared to control group (91.0%) in 48 hr respectively. Cell death increased in high concentrations noscapine and were increased TUNEL positive cells in 10, 25, 50 and 100 µm noscapine concentration (9.04%, 13.07%, 15.15% and 17.82%) respectively compared to control group (4.96%) in 48 hr (p<0.05).
Conclusion: Noscapineas inducer cell death decreased endometriotic epithelial cells viability and decreased apoptotic index in dose dependent manner. It can suggest for endometriosis treatment.
Key words: Apoptosis, Epithelial Cell, Endometriosis, Noscapine.
P-44
The effect of chronic prenatal stress on insulin secretion from Langerhans isolated islets in male offspring rats
Rostamkhani F1, Zardooz H2, Shirvani H3.
Introduction: A large number of studies have reported associations between prenatal stress and offspring lifetime consequences. Chronic gestational stress alters maternal glucocorticids and subsequently disturbs intrauterine environment which may lead to metabolic disorders in the offspring.
Materials and Methods: In this study the effects of gestational 8 and 20 days foot-shock and psychological stress on body weight, plasma corticosterone, insulin, glucose, concentrations and also insulin secretion from isolated pancreatic islets of rats' offsprings were examined. Stress was induced by Communication Box twice a day (1 h/session) for 8 consecutive days beginning on E8 in 8-day stressed group and for 20 consecutive days beginning on E1 in 20-day stressed group.
Results: The results obtained from this investigation indicate that 8 and 20-day chronic foot-shock stress arises maternal plasma corticosterone concentration. Prenatal stress induces lower birth weight and body weight gain in offspring. Insulin secretion from isolated pancreatic islets of offspring in 8 and 20-day foot-shock stress groups in presence of 8.3 and 16.7 mM glucose increased as compared to the control and psychological groups. Furthermore, prenatal stressed offspring had significant elevation in plasma glucose concentration without marked alteration in plasma insulin and corticosterone concentrations.
Conclusion: These data suggest that prenatal stress could result in impaired glucose metabolism in the offspring which is independent of timing of the stress exposure.
Key words: Prenatal stress, Corticosterone, Insulin, Glucose, Langerhans isolated islets.
P-45
In vitro culture of vitrified mouse ovarian tissue derived preantral follicles in two and three dimensional systems
Sadr SZ1, Ebrahimi B1, Shahhoseini M2, Fatehi R1, Favaedi R2, Maroufizadeh S3.
Introduction: Cryopreservation is the best choice to preserve fertility in patients exposed to premature ovarian failure. Designing an appropriate system for in vitro follicle culture that resembles in vivo conditions is really valuable and could improve the usage of cryopreserved tissue.
Materials and Methods: Ovaries of 12-day-old female NMRI mice were exposed to EG and DMSO combination (7.5% and 15%) in two steps for 15 and 30 minutes respectively, then cryopreserved by needle immersed method (NIV). Afterwards, middle sized preantral follicles (110-130 µm) were mechanically isolated and were distributed into two different groups: two dimensional culture system (2D) and three dimensional culture system (3D; alginate encapsulation). Both groups were cultured for 12 days in α-MEM that supplemented with 10 mIU FSH, 1% ITS, 50 ng/ml activin A and 5% FBS. Finally, follicular morphology, survival, growth rate and also quantitative expression of oocyte maturation genes (Gdf9, Bmp15 and Bmp6) were studied on the first and last days of culture.
Results: At the end of culture period, although follicular morphology in 3D culture system was better preserved as compared to the 2D one, survival rate of cultured preantral follicles was significantly (p<0.05) lower than in 2D group. Expression analysis of oocyte maturation genes indicated a reduction process in both groups during 12 days of culture. In the last day of culture period, genes expression level in 3D culture system was significantly lower than 2D group.
Conclusion: Although the better survival rate was seen in 2D culture system, morphologic characteristics including antrum formation and follicle development was extremely better preserved in 3D culture system. Overall, as three dimensional culture system mimic in vivo conditions, so could be used as a more appropriate system for follicle culture.
Key words: Ovarian tissue, Preantral follicle, Two dimensional culture system, Three dimensional culture system, Vitrification.
P-46
Routine use of EmbryoGlue® as embryo transfer medium does not improve the ART outcomes
Safari S1, Razi MH1, Safari S2, Razi Y3.
Introduction: The assisted reproductive technology (ART) success rates have been significantly improved over last decades; however, embryo implantation still remains a major limiting factor. The composition of an embryo transfer (ET) medium is important for interaction between embryo and endometrium at the time of implantation. Some modifications to the embryo culture media are developed to mimic the in vivo conditions. One of the examples for that is supplementation of ET media with hyaluronan (HA, hyaluronic acid), a major glycosaminoglycan in uterine fluid, which assumed to improve the process of implantation.
Materials and Methods: A cohort of total 229 patients was retrospectively enrolled for the present study. They were subjected for ET on day 2 either in EmbryoGlue® (n=117) as study group or in conventional ET medium with low concentration of HA as control group (n=112).
Results: Patients in the both groups, in regards to the mean level of day 3 FSH, the etiology of infertility, the history of implantation failure and the rate of good quality embryos showed similar characteristics. There were no significant differences between two groups in terms of clinical and ongoing pregnancies, implantation, delivery and live birth rates. In spite of a decreased abortion and increased multiple pregnancy rates in the study group compared to the control group (15.8 vs. 19% and 20.6 vs. 15.6 respectively), the differences were not statistically significant.
Conclusion: Routine use of EmbryoGlue® as a HA enriched ET medium for cleavage stage embryos does not have advantage to the conventional one for infertile patients undergoing ART.
Key words: Hyaluronan, EmbryoGlue®, Embryo transfer, Pregnancy outcome.
P-47
The relation between vitamin C in follicular fluid with the morphology of oocyte and the quality of embryo in IVF patient
Saffari S1, Bahadori MH2, Sharami SH3, TorabZadeh P1, Goudarzvand M1.
Introduction: Oxidative stress and the inappropriate effect of reactive oxygen in the body or laboratory circumstances can decrease the quality of embryo and sexual cells. Ascorbic acid is a natural antioxidant which has a protective effect in the body.
Materials and Methods: This is a cross-sectional analytical study which was conducted on 50 women with IVF referred to Al Zahra Hospital, Rasht, Iran. 20-45 years old women with infertility caused by tubal dysfunction, ovarian factors or male factors were included. Patients underwent same protocol for stimulating; the ovulation and injection of 10,000 units of hCG. After 36 hr, follicles were suctioned and matured oocytes were separated for fertilization. Vitamin C was assessed biochemically and the morphology of oocyte and the quality of embryo were evaluated by by inverted optical microscope.
Results: Among patients 583 oocytes and 275 embryos were assessed. There was no significant relation between, age, BMI and duration of infertility with vitamin C (p>0.05). Also there was no significant relation between vitamin C, the maturity of oocyte and the quality of embryo (p>0.05). But MII oocytes were more in patients with vitamin C less than 1 in comparison with ≥1 (p=0.038). Also the mean 2PN embryos in 0-0.05 level of vitamin C was higher than other groups but no significant relation was noted (p=0.719).
Conclusion: According to our results vitamin C at special level can improve the morphology of oocytes and the quality of embryos.
Key words: Infertility, Follicular fluid, IVF, Vitamin C, Oocyte, Embryo, Antioxidants.
P-48
Polscope analysis of meiotic spindle and zona pellucida birefringence of metaphase ΙΙ oocytes in polycystic ovarian syndrome patients
Safian F1, Khalili MA1, 2, Ashourzadeh S2, 3, Omidi M2.
Introduction: Polycystic ovarian syndrome (PCOS) is one of the most common causes of infertility in women of reproductive age. Currently, one of the best therapeutic options for PCOS patients is ICSI. Moreover, IVM can be a useful technique for women with PCOS who are at the risk of ovarian hyperstimulation syndrome (OHSS). On the other hand, the oocyte quality can be a determining factor for outcome of ICSI cycles.
Materials and Methods: This prospective study included immature oocytes (30 GV and 5 MI) undergoing IVM, and MII oocytes obtained from PCOS patients (29.64±5.31 years) in ICSI program. Using a PolScope, the presence of MS and ZP birefringence was assessed in both in vivo-matured (n=32) and matured oocytes after IVM (n=24). Oocytes were classified as high birefringent (HB) ZP and low birefringent (LB) ZP. Furthermore, the rates of fertilization and embryo development were evaluated.
Results: The maturation rate was 68.5% after IVM. Analysis revealed that the percentage of a HB ZP was significantly higher in the IVM oocytes than in vivo-matured ones (58.3% vs. 31.2%, p=0.04). There was insignificant relationship between spindle detection and either in vivo-maturation or IVM oocytes (p=0.53). Likewise, there were similar outcomes for the rates of fertilization and embryo development after ICSI between two groups (p=0.80 and p=0.13, respectively).
Conclusion: Clinical IVM is a safe technology for the maturation and maintenance of oocytes integrity. Furthermore, the use of non-invasive PolScope is recommended for the detection of healthy oocytes in ICSI.
Key words: PCOS, IVM, PolScope, ZP birefringence, Meiotic spindle.
P-49
History and application of public bank of umbilical cord blood
Seyfipoor Naghne E1, Bolorytabar F1, Khoshravash S2.
Introduction: Umbilical cord blood is rich of hematopoietic stem cells. Today, umbilical cord blood constitutes about 20% of stem cells transplant. 75% of umbilical cord blood banks in the world are public and the remaining are private (trade). 27 years after the first successful umbilical cord blood transplant, more than 555,000 umbilical cord blood units have been made available around the world after performing qualitative study in public bank of umbilical cord blood in order to treat patients who need hematopoietic stem cells transplant. The first program of public umbilical cord blood bank was implemented by the New York in 1991 and in Asia in 1999 in Japan. In Iran in late 2008, the Stem Cells Association has started its activity by receiving financial supports. In this type of storing, umbilical cord blood does not only belong only to the family and public organizations are responsible for paying related costs. Cells in this banking are kept safe and number of their alive cells remains unchanged during the freezing process. Samples saved in bank in terms of health indices such as viral and microbial contaminations are evaluated by colony measurement method. With regard to high prevalence of blood malignancies and thalassemia in Iran, it has been very important to establish public bank of umbilical cord blood and support it spiritually and materially.
Materials and Methods: Subjects of this review study are derived from articles and sites related to collecting, maintain and transplanting umbilical cord blood.
Results: Development of umbilical cord blood banks to develop transplantation centers and treat patients is of unavoidable necessities in the society health system. In societies that the government has ability to establish public bank and supports this bank, development of public banks has preference to development of private banks.
Conclusion: In year 2011, research and development department established public bank of umbilical cord blood in order to scientific and practical promoting and proceeded to amniotic membrane, standardization of tissue adhesive, production and standardization of amniotic extract and platelet extract by establishing bank. Collecting this bio garbage and transforming it to biologic bands, eye drop to improve cornea wounds and mixing hydrogen with amniotic membrane extract to treat wounds for therapeutic consumptions is economical and is effective to decrease therapeutic costs. Also fibrin glue is produced only via umbilical cord blood. Fibrin glue as cell carrier and tissue adhesive is widely used in surgery and cell therapy. Transplant of stem cells of umbilical cord has opened bright and promising horizons for medical society to treat patients with different chronic and acute disorders. So, saving umbilical cord blood finds high importance and its necessity feels further.
Key words: Umbilical cord, Blood bank
P-50
Protective effect of alcoholic extract of Garden cress (Lepidium sativum) seeds on the histopathological changes of epididymis and fertility in diabetic rats
Seyed Hoseini ES1, Nikzad H1, Amini Mahabadi J1, Haddad Kashani H2.
Introduction: Diabetes mellitus (DM) is a frequent and serious metabolic illness all over the world and plants have been a desirable source of medicine recently. Diabetes has unpleasant effect on male reproductive system and it may lead to male infertility. It causes erectile dysfunction and reduces ejaculate volume by affecting the health of small blood vessels and the small nerves that control ejaculation and also decrease libido by decreasing testosterone levels. Current study evaluated the possible protective efficiency of Garden cress (Lepidium sativum) seed extract on fasting blood sugar (FBS) and then assessed epididymal histopathologic changes in streptozotocine (STZ)-induced diabetic rats.
Materials and Methods: 50 male adult Wistar rats were randomly categorized into five groups (each 10 rats). Groups 1 were control placebo group receiving only 0.1 ml normal saline via gastric gavages, Group 2 as control diabetic rats received an intraperitoneally injection of STZ 60 mg/kg body weight. Rats with FBS >250 mg/dl were considered as diabetic. Group 3 were diabetic rats receiving insulin in dose 3U/100 g body weight and Groups 4 and 5 were diabetic rats that received 0.1 cc of 200 and 400 mg/kg‚ ethanol extract of Lepidium sativum seed by gavages daily. One day after the last gavages, rats were anesthetized by chloroform. Epididymis duct was removed from abdomen and weighed with a digital scale. Afterwards, samples were putted in Bouin's solution for histological measurement.
Results: Administration of 200 and 400 mg/ml doses of Lepidium sativum seed extract increased epithelium height and decreased interstitial volume density and fibro muscular thickness significantly. Also, volume density of epithelium, fibro muscular, lumen and interstitial were decreased significantly. Tubular and lumen diameter did not change significantly in different groups.
Conclusion: It seems Lepidium sativum seed extract has benefit as a supplementary protective agent against bad effects of diabetes on reproductive system in diabetic male.
Key words: Diabetes‚ Lepidium sativum seed extract, Epididymis‚ Streptozotocine‚ Insulin.
P-51
New insights into the effect of aflatoxin on infertility of animal models
Sheikhpour R, Jabbari S.
Yazd Medical Science Branch, Islamic Azad University, Yazd, Iran.
Email: R.sheikhpour@yahoo.com
Introduction: Aflatoxins are produced via Aspergillus flavus and Aspergillus parasiticus, species of fungi. Toxic and carcinogenic properties of Aflatoxin are determined. After entering the body, Aflatoxins may be metabolized by liver to a reactive epoxide intermediate or hydroxylated to become the less harmful. Nowadays, infertility properties of Aflotoxine have been known in studies.
Materials and Methods: Literature review was conducted using the following databases: SciELO, Lilacs, Medline, PubMed, Scopus and science direct from 2000-2014. The key-words used were Aflatoxin, infertility, fertility, animal, human.
Results: When Aflatoxin level increased in diet of male goats, glucose and total protein concentration were decreased in the testes and testosterone level was significantly reduced. The study suggests that exposure of male goats to dietary Aflatoxin will reduced testicular biochemical and testosterone with resultant depression in sperm storage capability and daily sperm production in the animals. Chronic Aflatoxin exposure in animals can result in impaired reproductive efficiency. One study showed that Aflatoxin decreased fertility, abortion, lowered birth weights and caused disturbances in hormonal metabolism in sheep. Another study showed that the fertility of treated mice with Aflatoxin at a daily dose of 50 µg/kg body weight was reduced drastically. Sperm concentration in the epididymis and sperm motility decreased whereas sperm abnormalities increased.
Conclusion: The results of studies showed that Aflatoxin can cause infertility in animal models, but these results need to be evaluated by more clinical trials.
Key words: Aflatoxin, Infertility, Animal.
P-52
ART cycles outcome in donor oocyte recipient couples: A case-control study
Tahajjodi S, Khalili MA, Nabi A, Omidi M.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Science, Yazd, Iran.
Email: s.tahajjodi@yahoo.com
Introduction: Oocyte donation is an acceptable alteration in women infertility treatment for different indications. Several factors including the age of the oocyte donor and recipient, oocyte and embryo quality and characteristics of endometrium can influence on ART outcome.
Materials and Methods: Outcome of 202 cycles, consist of 113 DO cycles and 89 AO cycles, were compared. Both DO and AO groups were divided to two subgroups according to fresh and frozen embryo transfer. First DO and AO groups included the fresh embryo transfer (fDO, fAO) and another DO and AO groups included the frozen embryo transfer (fzDO, fzAO). Comparison was performed in regard to chemical pregnancy, clinical pregnancy, implantation rate, live birth and abortion rate.
Results: DO (mean=28.29 years) and AO (mean=29 years) groups were significantly matched by age. No significant differences were observed between fDO and fAO groups and also between fzDO and fzAO groups with regard to chemical pregnancy, clinical pregnancy, implantation rate, live birth and abortion rate. But, there was a significant increase in oocyte number, MII oocyte number and embryo number in DO compared with AO groups (p<0.001).
Conclusion: Despite of increasing in total number of oocyte, MII oocyte and embryo, our findings showed that oocyte donation program has no impact on ART pregnancy outcome.
Key words: Oocyte donation, ART outcome, Women infertility.
P-53
Dose stress always has a negative effect on sperm cell?
Vaseghi-Dodaran H1, Zhandi M2, Daghighkia H1, Sobhani A3.
Introduction: Cryopreservation of semen requires optimized condition to minimize the harmful effects of various stresses. Oxidative stressors are known to be major mediators of cell damages during cryopreservation. An important perspective is that free radicals are not exclusively beneficial or exclusively detrimental. Rather, they need to be maintained at appropriate levels to ensure physiological function, while preventing pathological damage. Recent studies showed that placing the cells in a controlled stress conditions could affect cell survival in later stages.
Materials and Methods: This article discusses some of the new approaches employed for improve spermatozoa before and after freezing-thawing. A review of recent bibliography was carried out in PubMed, Google scholar and SID by the use of relevant keywords.
Results: Osmotic, oxidative, hydrostatic and mechanical stressors before freezing induce expression a series of heat shock proteins (HSPs), decrease apoptotic sperm amount and increase live sperm during treatment and high mitochondrial activity observed in treatments. Cell's reaction to environmental stresses is depended on domain, concentration and acts time of this reactions.
Conclusion: Although cellular and sub-cellular mechanisms, supposedly contributing to these processes, require further research, the new principle may outline a completely new strategy in mammalian embryology, as well as cryopreservation of other cells and tissues with remarkable theoretical and practical consequences.
Key words: Cryopreservation, Sperm, Stress, Sublethal.
P-54
The effect of KIT ligand (KITL) and leukemia inhibitory factor (LIF) on maturation and apoptosis of follicles after vitrification of mouse ovary and three dimensional culture
Abdi Sh1, Salehnia M1, Hosseinkhani S2.
Introduction: In vitro ovarian follicle culture techniques not only provide a model for research into the mechanism of folliculogenesis but also, in combination with cryopreservation of ovarian tissue, it may have clinical applications in preserving of fertility.
Materials and Methods: This experimental study is carrying out on 7-days-old female mice (NMRI). In the first step, the ovaries were vitrified with a solution containing ethylene glycol and then their morphology, ultrastructure and apoptosis were compared with non-vitrified ovaries. In the second step, the non-vitrified and vitrified ovaries were cultured in base medium α-MEM supplemented with KL and leukemia inhibitory factor (LIF) for 7 days then their morphology, area, apoptosis, hormone assay were analyzed. In third step, mechanically isolated preantral follicles were cultured in two dimensional and three dimensional systems in α-MEM supplemented with 5% FBS, 100 mlU/ml, 1% ITS, 10 ng/ml rEGF and KL for 12 days then the ovulation were induced. At the end of culture survival and maturation rate, apoptosis and hormones were assayed. In the fourth step, the level of reactive oxygen species (ROS) and ATP, distribution of mitochondria and fertilization rate in MII oocyte were assessed.
Results: The results of first step showed that the morphology, ultrastructure and apoptosis of vitrified ovaries were similar to non-vitrified ovaries. The results of second step showed that the percentage of preantral follicles, ovarian area, production of hormone in the non-vitrified ovaries cultured in medium supplemented with KL were significantly higher and the level of caspase 3/7 was lower in comparision to other groups (p<0.001). The results of next step showed that the survival, maturation rates and production of hormone of follicles in KL supplemented in three dimensional system groups were higher than other groups. The level of caspase-3/7 activity was lower in three dimensional culture systems (p<0.05). The results of fourth step showed that the level of ROS and ATP in MII from in vitro culture of follicle had significant different with control in vivo group and the rate of embryo development were significantly higher in three dimensional culture system.
Conclusion: Our results demonstrated that the culture of ovarian tissue and three dimensional culture of ovarian follicle in combination of KL improved the growth and development of ovarian primordial follicle.
Key words: Vitrification, In vitro maturation, Apoptosis, Mitochondria, KIT Ligand (KITL).
P-55
Evaluating the levels of expression of TLR 9 in women with endometriosis and its comparison with normal endometria
Aflatoonian Ali1, Janan A2, Ramazanali F2, Moieni A1, 2, Aflatoonian R2.
Introduction: Endometriosis is a benign condition which endometrial glands and stroma appear outside the uterine cavity which presents by pelvic pain and infertility. Endometriosis is associated with changes in cellular and humeral immunity and impaired immune response, leading to inefficient removal of debris after a menstrual cycle. Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system. Based on recent studies TLRs are increased in endometriosis and initiate immune responses.
Materials and Methods: This case-control study assessed and compared expression of Toll like Receptor 9 in the endometrium of three type tissues: 1) Eutopic endometria of women with endometriosis 2) Ectopic endometria women with endometriosis 3) Endometrium of women without endometriosis. Eutopic and ectopic endometrial samples were taken from 10 patients with endometriosis (Case Group). Also endometrial samples were taken from 10 patients without endometriosis or infertility history whom operated for other gynecological cause. The level of TLR9 gene expression in ectopic samples, eutopic and controls were evaluated by Real Time-PCR. Patients were chosen randomly from Arash Hospital. Data was analyzed by SPSS.
Results: Real Time-PCR showed that TLR9 expressed in all three groups of eutopic, ectopic and control. According to statistical analysis, level of TLR9 was higher in ectopic group, but not significantly (p=0.13). There was no significant difference between eutopic and control groups.
Conclusion: Considering the role of TLR9 in the innate immune system, such as pathogen detection and set up a cascade of inflammatory response associated with cell proliferation and inhibition of apoptosis, this study approves the association between TLR9 and endometriosis. Because of lack of information about this new issue, we suggest further researches and more studies.
Key words: TLR9, Endometriosis, Inniate immune system.
P-56
Congenital uterine malformations: A widespread phenomenon among infertile women with recurrent miscarriages and IVF failure
Javam M, Ahmadi F.
Department of Reproductive Imaging at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: f_ahmadi@royaninstitute.org
Introduction: Uterine malformations are a various group of congenital uterine disorders found in women with recurrent abortions or IVF failure.
Materials and Methods: A narrative review was performed within articles published at “PubMed”, “Elsevier”, "EBSCO", original text books and etc. to reach the aim. Several unique high-quality images were provided in this article, using the archive of infertile patients referred to imaging department of Royan Institute.
Results: Congenital uterine anomalies are originated from development defects of mullerian ducts during fetal growth. They are also associated with higher incidences of infertility, preterm birth, intra uterine fetal death and etc. However, manifestations and severity of the obstetric/ gynecologic complications and treatment procedures vary depending on the type of anomaly. Thus, accurate diagnosis of uterine malformations and differentiation between various types of them, have a vital role in decision about treatment procedures and management of these patients. Several imaging modalities are used to investigate women suspected to have uterine anomalies. In this article, we described about various types of uterine malformations and imaging evaluation of them prior to IVF treatment cycles.
Conclusion: Congenital anomalies of the uterus are a major cause of recurrent miscarriage and pregnancy failure. Several imaging methods help midwives and obstetricians evaluate this group of infertile women to detect suspected uterine anomalies.
Key words: Congenital uterine malformation, Infertility, Recurrent miscarriages.
P-57
Study on the impacts of anti-histamine dimenhydrinate on testicular tissues and sperm production of male mature rat
Ahmadifar M1, 2, Vahidi-eyrisofla N3, Taghvaei F4, Jafarzade Rastin S4, Yousef Zadeh AR6, Daliri L5, Gharanfoli M1.
Introduction: In this study we did a research about the effects of AHDH on testicular tissues and sperm production of rats. Dimenhydrinate (DH) is used as anti-nausea as well as an anti-gastric reflux in cases of nausea caused during travelling. It is easily taken orally and it will be excreted by the kidneys within 24 hr.
Materials and Methods: This survey is conducted in laboratory on experiment basis among 30 mature male rats categorized in 3 different groups; two experimental groups and one control group. The control group was watered normally for 60 days and the first experimental group was watered with 200 mg DH blended in their water equal to one kg of their body weight for 60 days. In the end, after the rats were anesthetized, testicles were removed, sectioned and painted with Hematoxilin and Eozine and were investigated.
Results: The obtained results have revealed that in the experimental groups, testicles weight decreased substantially in comparison to those of the control group (p<0.001). The reduction of the number of sperm cells within the experimental group, in comparison to those of the control group was significant (p<0.001). The reduction of the Leydig cell was very substantial in the second experimental group (p<0.001). The difference of the Sertoli cells was not noticeable in neither of the experimental groups.
Conclusion: DH has impact on testicular tissues and weakens their mechanism.
Key words: Dimenhidranate, Testis.
P-58
Investigating association of G103T polymorphism of Coagulation factor XIII and recurrent pregnancy loss
Ahmadpour Nazm S1, Bonyadi M2, Taghavi S3, Ajamian F1.
Introduction: Recurrent miscarriage (RM) occurs in 1-3% of couples that attempting to bear children. Thrombophilia is one of the suspected cause of recurrent miscarriage. At the end of coagulation cascade there is Factor XIII that makes blot stable. The polymorphism G103T of factor XIII gene is the most common polymorphism that affects FXIII’s activity.
Materials and Methods: The study groups consisted 50 patients with two or more consecutive miscarriage. The control group included 50 women with at least two successful delivery and no history of pregnancy loss. By using PCR-RFLP, DNA from both groups analyzed for carrying mutation of FXIII.
Results: 4% in the case group were homozygote (TT) for 34 Leu mutation whereas no homozygote (TT) was found in control group (p<0.05). 28% patients in the case group and 26% women in the control group were found to be heterozygote for G103T polymorphism (p>0.05). No significantly differs was observed between patients with RPL and healthy women for G103T mutation.
Conclusion: No statistically differs between case group and control group was observed.
Key words: Recurrent miscarriage, Thrombophilia, F XIII.
P-59
Effect of pumpkin seed and ginger extracts on adult rat’s sperm characteristics, biochemical parameters and epididymal histology treated with cyclophosphamide
Amini Mahabadi J1, Nikzad H1, Aghaie S2, Taghizadeh M3, Azami-Tameh A2, Taherian SS2.
Introduction: Infertility is one of the problems of human society and 10-15% of couples have experienced some forms of infertility problems. Reproductive toxicity is one of cyclophosphamide (CP) side effects in cancer treatment.
Materials and Methods: Male adult Wistar rats were randomly divided into six groups. Group 1 as control received an isotonic saline solution injection intraperitoneally (IP). Group 2 was injected intraperitoneally with a single dose of CP (100 mg/kg) once. Group 3 and 4 received CP plus 300 and 600 mg/kg combined pumpkin seed and Zingiber officinale extract (50:50). Group 5 and 6 received only 300 and 600 mg/kg combined pumpkin seed and Zingiber officinale extract. Six weeks after treatment, sperm characteristics, histopathological changes and biochemical parameters were assessed.
Results: Results showed that in CP treated rat’s sperm characteristics were diminished significantly. Biochemical analysis showed that the administration of combined extracts could increase the Total Antioxidant Capacity (TAC) level significantly in groups 3, 4, 5 and 6. Also, in these groups, the sperm viability, motility, count, normal sperm morphology, epididymal epithelium and fibromascular thickness were improved compared to control and CP groups. Interestingly, the mixed extract could improve histopathological changes such as vacuolization, disorganization and separation of epididymal tissue in CP treated rats as well.
Conclusion: Overall our findings indicated that the combined extracts might be used as a protective agent against CP-induced reproductive toxicity.
Key words: Pumpkin seed extract, Zingiber officinale extract, Cyclophosphamide, Total antioxidant capacity (TAC), Sperm parameters, Rat epididymis.
P-60
Effect of vitamin D insufficiency treatment on fertility outcomes in frozen-thawed embryo transfer cycles: A randomized clinical trial
Aflatoonian A, Arabjahvani F, Eftekhar M, Sayadi M.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: F_arabjahvani@yahoo.com
Introduction: Frozen- thawed embryo transfer is an essential part of ART treatment and outcomes of this procedure are associated with several clinical factors. Several studies have showed an increase level of IVF outcomes in women with sufficient vitamin D. whether treatment of vitamin D insufficiency can improve pregnancy rates in frozen-thawed embryo transfer cycles.
Materials and Methods: This is an interventional, randomized clinical trial. Serum 25-(OH) vitamin D level of 128 women who had undergone IVF/ ICSI with cryopreservation of embryos was checked. One hundred fourteen infertile women with insufficient serum vitamin D (less than 30 ng/ml) were included in the study. Fifty seven women were treated with supplementary vitamin D, 50000 IU weekly, for 6-8 weeks and 57 women were received no supplementation. 106 women completed frozen thawed embryo transfer cycles and included in the final analysis. Primary and secondary outcomes were chemical and clinical pregnancy respectively.
Results: Our study did not show any significant difference between vitamin D insufficient and treated women in term of chemical (29.40% vs. 29.10% respectively, p=1.00) or clinical (25.50% vs. 21.80% respectively, p=0.81) pregnancy rates.
Conclusion: Vitamin D insufficiency treatment is not associated with higher pregnancy rate in frozen-thawed embryo transfer cycles.
Key words: Vitamin D, Embryo transfer, Pregnancy rate.
P-61
Association of rs10954213 polymorphism of IRF-5 gene with idiopathic recurrent miscarriage
Arjmand F1, Ashrafzadeh Mehrjardi HR2, 3, Ghasemi N3.
Introduction: Interferon regulatory factor (IRF) 5 is a transcription factor that can induce transcription of IFN-α mRNA. Recent studies have shown that IRF-5 promotes the proliferation of T cells and the activation of TH1 and TH17 cells but does not induce TH2 or Treg differentiation. TH17 cells produce inflammatory cytokine IL-17 which plays an important role in the induction of inflammation via neutrophil infiltration and stimulation of inflammatory cytokines. There are many reports that inflammatory processes play a major role in recurrent miscarriage in a manner that an uncontrolled and persistent inflammatory response during pregnancy can harm placental growth. We analyzed the rs10954213 polymorphism of IRF-5 gene in normal pregnancy and recurrent miscarriage patients in order to discover a possible mechanism for the genetic control of immune regulation in patients with recurrent miscarriage.
Materials and Methods: Genomic DNA from 100 recurrent miscarriage (RM) patients and 100 normal fertile control individuals using the routine salting out method were isolated. DNA fragments were then analyzed by real time PCR with SYBR Premix Ex Taq II master mix. Statistical analysis used SPSS 19 software. IRF-5 allele frequencies and genotypes in RM women and the fertile control group were compared using a Chi-square test.
Results: Our results so far indicate that there is a detectable relationship between rs10954213 polymorphism of IRF-5 and susceptibility to recurrent miscarriage.
Conclusion: IRF-5 gene plays a possible role in the induction of inflammation and therefore leads to recurrent miscarriage. However, additional studies are needed in this regard.
Key words: Rs10954213, IRF-5, Recurrent miscarriage.
P-62
Role of 45,X mosaicism in couples with fertility problems
Bahman I1, Bagherizadeh I1, Abdi A1, Shajarehpour L1, Hadipour F1, Hadipour Z1, Saremi A2, Shafaghati Y1, 3, Behjati F1, 3.
Introduction: Infertility is typically defined as the biological inability of a person to contribute to conception after one year of attempting to have a child. Genetic factors such as chromosomal abnormalities are one of the major causes of infertility and spontaneous abortions. The objective of this study was to establish chromosome X abnormality rate among women referred for infertility problems to Sarem Women's Hospital in Tehran between October 2007 and January 2015.
Material and methods: A total number of 1764 women with the age range of 12-60 years were referred for chromosomal investigation. Referral reasons included recurrent abortions, primary infertility, secondary infertility, Turner syndrome, premature ovarian failure, amenorrhea and ART failures. Cytogenetic analysis was performed on cultured peripheral blood lymphocytes stimulated with phytohaemagglutinin M, using standard techniques. Karyotype was done in all patients using high resolution GTG banding technique. For each patient, at least 15 chromosome spreads were examined by light microscopy, and extended to 100 in the case of mosaicism.
Results: Cytogenetic investigation was performed on 1764 patients. The overall chromosome X abnormality rate in infertile and subfertile women was 4% (74 out of 1764). The cytogenetic result for these patients with fertility problems is as follows: 74 patients had a numerical chromosome abnormality and 65 (85%) of them had mosaicism of 45,X with other different cell lines. The age range of women with 45,X mosaicism was 12-53 years old.
Conclusion: Accurate genetic diagnosis is the most important prerequisite for genetic counseling in patients with fertility problems. Finding of chromosome X numerical abnormality are of great value in better management of the patients. However, the attribution of chromosome X instability due to age should be considered in Genetic counseling of these couples.
Key words: Fertility problems, Chromosome X abnormality, 45,X cell line.
P-63
Effects of mitochondrial gene deletions on human sperm motility
Bahrehmand Namaghi I, Vaziri H.
Department of Biology, Faculty of Science, Guilan University of Medical sciences, Rasht, Iran.
Email: i_man_bahrehmand@yahoo.com
Introduction: Asthenozoospermia is an important cause of male infertility. It has been primarily characterized by reduced sperm motility owing to a variety of factors, including ultra-structural abnormalities, abnormal semen liquefaction, anti-sperm antibodies, varicocele and endocrine abnormality, etc. The mitochondrion is the major energy provider for sperm motility. Mitochondrial DNA contains several genes encoding for proteins that play an important role in oxidative phosphorylation and in ATP production. Mutations in sperm mtDNA result in either functionless or malfunctioning proteins, subsequently affecting sperm motility leading to asthenozoospermia.
Materials and Methods: To detect 4977 bp deletion in spermatozoa mtDNA, semen samples of 42 asthenozoospermic infertiles and 50 controls from northern Iran were collected. After extraction of spermatozoa total DNA, Gap-PCR was performed.
Results: 4977 bp deletion was observed in 73.80% of patients with asthenozoospermia, compared with 36% in controls (OR=5.0101, 95% CI: 2.0408-12.2998, p=0.0004).
Conclusion: Large-scale mtDNA deletions in spermatozoa may induce bioenergetic disorders and cause subfertility or infertility in men. It is concluded that there is a strong association between sperm mtDNA 4977 bp deletion and asthenozoospermia-induced infertility in the population examined. Nevertheless, to validate our results broader research may be needed.
Key words: Asthenozoospermia, mtDNA deletion, Male infertility, Motility.
P-64
Effects of Nano TiO2 on chromatin, apoptosis and parameters of sperm in mice
Danafar AH1, 2, Khoradmehr A1, Hosseini M3, Halvaei I1, Golzadeh J1, Anvari M1.
Introduction: Titanium dioxide nanoparticles (TiO2 NPs) are manufactured worldwide and used in a broad range of applications. In mammals, there are only limited reports regarding the effect of TiO2 NPs on male reproductive system. It has been demonstrated that TiO2 NPs taken up by mouse Leydig cells reduced the viability and proliferation of these cells. Also it is improved that titanium compounds can disrupt the mouse blood-testis barrier. In this study we decide to evaluate the effects of nano-TiO2 on chromatin, apoptosis and sperm parameters of male mice.
Materials and Methods: 35 NMRI male mice were classed in five groups: control, sham and three treated groups. Low, medium and high treated groups were gavaged 2.5, 5 and 10 mg/kg doses of TiO2 respectively for thirty five days. Sham group only received saline instead. The mice were weighted before and after administration. Sperm count, motility, morphology and viability were analyzed accordingly. Sperm DNA integrity and apoptosis were assessed using acridine orange, aniline blue, toluidine blue, CMA3 and TUNEL assay.
Results: Non-significant decrease in body weight gain was observed. Sperm analysis showed no count, motility and viability changes but morphological changes were significant. Most of the morphological abnormalities were observed in sperm neck and tail. Tail morphological changes in treated groups were significant in comparison to non-treated ones (p<0.001). Compared to non-treated groups, sperm neck changes in medium and high treated groups were significant but not in low treated group (p<0.001). Results of acridine orange, aniline blue, toluidine blue and CMA3 were non-significant in all groups. Also increasing apoptotic cells in treated groups were discovered by TUNEL assay but there were non-significant statistically.
Conclusion: TiO2 significantly may increase sperms with abnormal morphology especially in tail and neck part. Also apoptosis boosting occurred but the exact mechanism is still controversial.
Key words: Nano-TiO2, Sperm parameters, Apoptosis.
P-65
Protective role of vitamin C on hazardous effects of acrylamide in rat offspring
Dortaj H, Anvari M, Yadegari M.
Shahid Sadoughi University of Medical Scienses, Yazd, Iran.
Email: hengameh.dourtaj@gmail.com
Introduction: Acrylamide (ACR) is a substance chemical used in industrial and laboratory procedures. Acrylamide according to the method of cooking foods are increasingly used and its adverse effects on multiple organ systems have been described sporadically in the literature. The purpose of this study was to evaluate the effects of ACR during pregnancy and lactation on the development and changes in cortical layer of the cerebellum and cerebellar Purkinje cells in fetal and neonatal rat by using histological and morphometric and stereological technique.
Materials and Methods: In this study 20 adult female Wistar rats weighing 180 gr and aged two months were used. Animals were randomly divided into four groups. Female pregnant rats were orally administered 10 mg/kg ACR and/or 200 mg/kg vitamin C (vit C). Pregnant rats were sacrificed on the 15th day of gestation and mother’s weight was measured. After that, their fetuses were taken out and were evaluated for fetus number, weight, crown-rump length (CRL) and cerebellar development. To study the neonatal period, 6 infants at day 21 were randomly selected and placed under deep anesthesia and transcardial perfusion. The cerebellum was taken out and fixed and cerebellum changes were evaluated by crysel violet and Hematoxylin-Eosin staining method. The cerebellar cortex layers volume were investigated by Cavalieri's principle method. Data were analyzed by SPSS software and by ANOVA and LSD Test. P<0.05 considered as statistically significant.
Results: The results showed that ACR decreased fetal weight and CRL, but this reduction in weight and volume of the cerebellum p˂0.001 and the number of embryos with p<0.05 was significant. Histological and stereological examinations revealed that the cerebellar volume was decreased in ACR and ACR+vitC group vs control (p˃0.001). While in vitamin C group the cerebellar volume was inversed (p˃0.05). ACR in newborn decreases body weight, brain weight, thickness of cerebellum with p˃0.001. The extent of this reduction in the weight of the cerebellum was significant with p˂0.05.
Conclusion: ACR exhibits a harmful effect on the development of the cerebellar cortical layers, which may be prevented by administration of vit C as an anti oxidant.
Key words: Acrylamide, Vitamin C, Cerebellum, Development, Rat.
P-66
Robust stem cell isolation from human dental pulp
Ebrahimi B, Banaei M, Namiranian G, Tabatabaei SA.
Yazd Cardiovascular Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: bhnmebrahimi@yahoo.com
Introduction: Dental pulp stem cells (DPSCs) are a population of clonogenic and highly proliferative cells derived from enzymatically digested Dental pulp tissue. Providing scaling-up of stem cells at early passages is of importance for regenerative medicine purposes. Typically, two protocols are employed to isolate stem cells from human dental pulp: tissue explants culture and enzyme digestion of pulp tissue and culture of released cells.
Materials and Methods: Dental pulp was extracted from third molars of 60 healthy subjects. In the first method pulp was digested with 1 mg/mL collagenase/ dispase (Roche) for 30 min and released cells obtained using a 70-µm cell strainer for culture, in the second method intact pieces of pulp were cultured and in the third method digested pulp pieces were immobilized and cultured. The cells and tissues maintained in alpha-MEM supplemented with 20% fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, and 25 ng/mL amphotericin B and incubated in humidified incubator with 5% CO2 at 37oC. In each group cells and colonies were counted and compared.
Results: Results showed that treating pulp segments with enzymes and culturing them (combinatory method) increased the efficiency of cell isolation up to 60% significantly in 3-4 days of culture compared with other methods which this value was <20% in 10-15 days.
Conclusion: According to the small size of pulp tissue and its low stem cell contents, acquiring substantial quantities of cells in primary culture will facilitate the in vitro expansion and providing adequate production of the stem cells at early passages with minimum risk of losing their ‘stemness’ and aberrant genetic changes for use in research, tissue engineering and regenerative medicine. Optimized method increase efficiency of cell isolation and provides significant quantities of stem cells in primary culture more than other methods.
Key words: DPSCs, Cell isolation, Cell culture.
P-67
Evaluation of exon 8 of DPY19L2 gene in total globozoospermic patients referred to Royan Institute
Ebrahimi Nasab M1, Totonchi M2, Sabbaghian M3.
Introduction: Lack of acrosome decreases the capacity of the sperm for penetrating in the oocyte which consequently results in infertility. Globozoospermia is a rare and severe teratozoospermia characterized by round-headed spermatozoa lacking acrosomes. DPY19L2 is one of the genes which are dominantly expressed in the testis and it has been shown that is involved in the cause of this phenotype. Recent studies have shown that in large majority of globozoospermia patients a 200 kb deletion including DPY19L2 gene occurs. Different mutations in exon 8 of this gene have been also observed.
Materials and Methods: In total 24 men with total globozospermia and 24 men with normal spermogram referring to Royan Institute were selected. Then we sequenced exon 8 and intron boundaries in the non-deleted patients using specific primers and PCR technique.
Results: In our results no mutations were detected in exon 8 of the patients without the gene deletion, which were 29.17% of the total globozospermia patients.
Conclusion: According to our data, exon 8 mutations or polymorphisms has no effect on globozospermia but whereas 70.83% of globozospermia patients had whole DPY19L2 deletion and on the critical role of DPY19L2 protein in acroplaxome attachment to the nucleus, it can be concluded that the absence of this protein is one of the major causes of globozoospermia in Iranian infertile men. In addition, exploring other coding exons of this gene is our next aim.
Key words: DPY19L2 gene, Globozoospermia, Male infertility.
P-68
Anti-oxidant effects of herbal supplements on seminal parametmeters
Eghbal Eftekhaari T, Fallahi S, Malekzadeh K.
Molecular Medicine Research Center, Hormozgan University of Medical Sciences, BandarAbbas, Iran.
Email: tasnim.eftekhaari@gmail.com
Introduction: Varicocele is one of the leading causes of male infertility, causing a decrease in sperm parameters such as motility, count, concentration, and abnormal sperms such as azoospermia and asthenospermia.
Materials and Methods: A herbal compound capsule containing 4 herbs was prescribed 5 times perday (3 times during morning and evening and 2 times at night), and because of the warm nature of these herbs, 3 herbal extracts including Cichorium intybus L, Salix Aegyptiaca and Fumaria Parviflora 3 cups/day were added to this medication.
Results: After 4 weeks of consuming these medications, the body warmness decreased , ejaculated sperm volume and other sperm parameters increased including motility, concentration and morphology of sperm changed form astenia and oligospermia to normal morphology.
Conclusion: The herbal compound containing antioxidant supplements and enriched in minerals and fat soluble vitamins showed promising effects on improving sperm parameters and increase the chance of male fertility.
Key words: Varicocele, Increased sperm motility, Increased sperm count, Sperm paramteters, Antioxidants.
P-69
The effect of brown algae Sargassum extract on freezing of sperm
Fardmanesh H1, Fallahi S2, Shakerizadeh S3, Masoudi M3, Fadaei- Arsanjan M4.
Introduction: Oxidative stress process resulting from freezing and thawing of sperm, influences parameters of sperm and decreases its fertility. Antioxidants play a conservative role against oxidative damages during freezing of sperm. Using an appropriate freezing bank effects on fertility and motility of sperm, so we decided to evaluate effect of antioxidant extract of brown algae Sargassum on reactive oxygen species (ROS) and parameters of frozen sperm.
Materials and Methods: 11 normal semen samples were divided in to 3 groups, including A treated with 250 µg/ml and B with 500 µg/ml of extract of algae Sargassum and C group without any treatment (as a control group) then froze throughout rapid freezing method. Using CASA software and oxisperm kit, the motility/morphology and ROS levels of sperm were measured, respectively. Finally data were analyzed with SPSS software assumed significant level of p<0.05.
Results: The analysis of sperm parameters demonstrated that general motility (p=0.006) and advanced motility (p=0.007) significantly increased in both A and B groups, after treatment compared with C group. Moreover, the level of ROS notably declined in both treated group. However this treatment revealed any changes in sperm morphology.
Conclusion: The brown algae Sargassum extract is rich in antioxidant component that leads to decrease oxidative damage via neutralizing of ROS and improve sperm motility. Our results suggested that the extract can be used as a potential antioxidant factor in sperm freezing bank.
Key words: Sperm motility, Antioxidants, Reactive oxygen Species, Brown algae sargassum.
P-70
Immunohistochemical evaluation of a testis-specific histone demethylase, JMJD1A, in tissue samples of infertile men referred to Royan Institute
Favaedi R1, Shahhoseini M1, Kordestani shargh E2, Sodeifi N3, Sadighi Gilani MA3.
Introduction: Post-meiotic stages of mammalian spermatogenesis require unique and dynamic epigenetic events leading to histone removal followed by chromatin condensation. Through these events, histone demethylases such as JMJD1A play an important role in compaction of sperm chromatin due to regulation of histone methylation dynamics and alteration of chromatin structure. As “histone methylation” is one of the best-characterized modifications in the study of germ cell development, evaluation of presence/ absence of JMJD1A protein as in impaired spermiogenesis were aimed in this study.
Materials and Methods: For this respect, consent was obtained from azoospermic infertile men referred to Royan Institute according to local ethical approval, and then testis tissue samples were collected from three groups including complete maturation arrest, Sertoli cell only syndrome, and hypo spermatogenesis as positive control. Immunohistochemical analysis of paraffin embedded tissue samples was performed qualitatively using anti-JMJD1A antibody to elucidate presence/ absence of this protein in nucleus of germ cells.
Results: Immunohistochemical analysis data showed absence of JMJD1A protein in nucleus of germ cells in groups with spermatogenesis impairment (complete maturation arrest and Sertoli cell only syndrome groups) compared to control group.
Conclusion: It can be concluded that there is an obvious association between absence of histone demethylation as a chromatin condensing state with impairment of spermatogenesis and male infertility.
Key words: Spermatogenesis, Male infertility, Epigenetic, JMJD1A, Demethylation.
P-71
Pregnancy outcome of intracytoplasmic injection with epididymal and testicular sperm retrieval in azoospermic patients
Moein MR, Ghasemi-EsmailabadS, Ghasemzadeh J, Fesahat F, Tabibnejad N.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: qasemisaeed@yahoo.com
Introduction: Intra cytoplasmic sperm injection (ICSI) is an assisted reproductive technique for treatment of infertility in azoospermic men. It seems that pregnancy outcome in ICSI following percutaneous epididymal sperm aspiration (PESA) is better than using testicular sperm retrieving from testis due to greater motility and better morphology of epididymal spermatozoa. To determine clinical pregnancy outcome following ICSI with epididymal sperm comparing to that of testicular sperm in men with azoospermia.
Materials and Methods: 60 men with azoospermia who were candidate for ICSI have been selected. Sperm retrieval was performed using PESA (n=30) or testicular sperm extraction (TESE) (n=30). The number of embryos and live births were analyzed and evaluated between two mentioned groups and also thawed sperms after freezing in TESE group (n=30).
Results: No difference was seen in age and duration of infertility between groups. The number of embryos were not different significantly between TESE and PESA groups (p>0.05), but the different was significant between PESA and TESE freeze groups (3.67±2.89 vs. 2.10±1.53 respectively; p<0.05). The live birth rate was higher in the PESA group compared with the TESE group p<0.05).
Conclusion: Intracytoplasmic sperm injection using sperm from epididymis is more effective than testicular sperm injection and can successfully be performed to treat men with azoospermia.
Key words: ICSI, TESE, PESA, Freeze, Live birth rate.
P-72
Prevalence and risk factor of congenital malformation in Kashan
Haddad Kashani H1, Seyed Hoseini ES2, Dadkhah M3, Nikzad H2.
Introduction: Congenital malformation (CM) is a major childhood health difficulty. Treatment and rehabilitation of children with congenital malformations is costly and complete recovery is usually impossible. The prevention and treatment of congenital malformations are basic concerns for child health. The purpose of this study was to define rate of CM in Beheshti Hospital in Kashan, Iran to find out if there has been any variation in the rate and types of CM in this area.
Materials and Methods: This descriptive-observational study carried on 2700 births delivered from Beheshti Hospital in Kashan during 2012 and determines the prevalence of CM and type of it in this city.
Results: Prevalence of CM was 1.851% (2% in male and 1.61 % in female). Out of the 50 cases, 28 (56%) were males and 21 (42%) were females and 1 with uncertain genitalia. 13 members of the family were CM positive in musculoskeletal system (26%). Overall, anomalies of the cardiovascular system were second in frequency which involved 10 out of 50 patients (20%), also Down syndrome and skin malformation were the lowest rate anomaly in this study in Kashan (4% and 2% respectively). There wasn't statistical difference between prevalence of CM and neonatal gender and mother’s age, father’s age, mother’s number of abortion, and mother’s number of live children. But there was statistical difference between prevalence of CM with gestational age and Apgar number.
Conclusion: In this study the overall prevalence of congenital malformation among the newborn was less than those previous reported in Tehran and Yazd and higher than the rate of malformation in Gorgan and Arak. This difference determining the needs of more extensive studies.
Key words: Congenital malformation, Newborn, Beheshti Hospital, Kashan.
P-73
Relevance between testicular tissue vitrification and short term culture with degeneration and apoptosis genes expression
Hajiaghalou S1, 2, Ebrahimi B2, Shahverdi AH2, Sharbatoghli M2.
Introduction: Cryopreservation of testicular tissue has been recommended as a promising technique for fertility preservation in pre-pubertal boys who scheduled to undergo gonadotoxic treatment. Our aim was finding the quota of apoptosis genes involved in extrinsic pathway in testicular cell death after vitrification and during short term culture.
Materials and Methods: Testes were obtained from 7 days old NMRI male mice, divided and randomly distributed into control and vitrification groups. Vitrification was performed in 3 step by increasing concentration of vitrification solution (DMSO, EG). Both fresh and vitrified-warmed testes was cultured in RPMI and 10% KOSR for 20 hr. Real-time PCR, flow cytometery and light microscopy were used respectively for evaluation of gene expression, cell death and tissue integrity at 0, 3 and 20 hr of culture.
Results: Decreasing of tissue integrity was obvious in vitrification group as compared to the control one at all times of culture. Mean percentage of cell death was significantly (p<0.05) higher in vitrification group in comparison with control during culture period. Although expression of Fas was significantly (p<0.05) higher in vitrification group at 0 and 3 hr of culture, it was significantly lower at 20 hr of culture as compared to the control group. Significant (p<0.05) increase of Fas ligand was found in vitrification group at 3 and 20 hr of culture. Mean percentage of Caspase 3 was significantly (p<0.05) higher in vitrification group than control group.
Conclusion: Concurrent increment of cell death and apoptosis genes expression in vitrification group during culture period, could be a reason for extrinsic pathway involvement in degeneration of testis tissue after vitrification and also during culture period.
Key words: Extrinsic pathway of apoptosis, Tissue degeneration, Vitrification, Short term culture.
P-74
Magnetic activated cell sorting and its application for selection of human non apoptotic spermatozoa in ART
Hassani Bafrani H1, Catt S2, Pangestu M2, Smith Peter T2.
Introduction: Hydrogen peroxide has been implemented in literature to induce a significant increase in caspase. Activation of caspase 9 triggers a cascade of caspase activation, including caspase 3, which promotes cellular apoptosis. Magnetic cell sorting (MACS) using annexin V-conjugated microbeads eliminates apoptotic spermatozoa with annexin V-positive.
Materials and Methods: A total of 20 semen samples were obtained from male partners of couples for analysis. One aliquot (0.5ml) of the sperm suspension was subjected to MACS. Motility and concentration was checked, and a sample taken for tunnel before and after MACS. The remaining sperm suspension was divided into 6 tubes (2x control, 2x peroxide, 2x peroxide/ melatonin). DNA fragmentation was evaluated using the TUNEL assay (Roche, Indianapolis, IN, USA) (14), with some modifications. Data were analyzed using Graph Pad Instat Ver. 3.10.
Results: Results of the TUNEL assay in pretreatments of human spermatozoa with 100uM peroxidase for 24 hrs revealed that the percentage of sperm with fragmented DNA was significantly lower on the sored sperm after sorting MACS (p˂0.001 vs. control). Following the pretreated human spermatozoa with peroxidase for 24 hr, percentage of sperm motility and progressive motility were significantly reduced (p˂0.001 vs. control). Pretreatments of human spermatozoa for 24 hr revealed that the percentage of sperm motility and progressive motility were significantly reduced by 100 µM peroxidase and peroxidase with MACS, versus zero hour in control group (p<0.001).
Conclusion: The sperm was treated with 100 µM peroxidase sorting using MACS retain appropriate spermatozoa and select sperm good quality. The use of MACS will select only sperm with intact this result in high percentage motility and progressive motility.
Key words: Apoptosis, Human spermatozoa, MACS.
P-75
Bibliometric mapping and clustering analysis of Iranian-based research on reproductive medicine
Bazm S1, Ramezani A2.
Introduction: Nowadays infertility is a major problem in the world. Increasing the rate of infertility in the world and Iran led our study to assess the trends in Iranian research output related to reproductive medicine through the year of 2010-2014.
Materials and Methods: We used bibliometric mapping and clustering analysis method to visualize representing bibliographic data of Iranian production. All publication data from Scopus was retrieved for 2010-2014.
Results: Analysis of data showed that a total number of 3035 papers had been indexed in Scopus through the period of study.
Conclusion: The study indicated that scientific production in the field of reproductive medicine had increased in the past 5 years.
Key words: Reproductive medicine, Bibliometric analysis.
P-76
Menstrual pattern following tubal ligation: A historical cohorts
Jahanian Sadatmahalleh Sh1, Ziaei S1, Kazemnejad A2, Mohamadi E3.
Introduction: Tubal ligation (TL) is recommendable for women completed their family. The existence of the menstrual disorders following this procedure has been the subject of debate for decades.
Materials and Methods: A historical cohort study was carried out on 140 women undergoing tubal ligation and on 140 women who used condom as the main contraceptive method. They aged between 20 and 40 years and were selected from a health care center, during 2013-2014. The two groups were comparable in demographic and personal characteristics. Data collection tool was a questionnaire including questions regarding demographic, menstrual and obstetrical characteristics. A validated Pictorial Blood Loss Assessment Chart (PBLAC) was also used to measure the menstrual blood loss. All statistical analyses were carried out using software package used for statistical analysis (SPSS) version 20 (SPCC Inc., Chicago, IL, USA). Student’s t-test and chi-square test were carried out to reveal the statistical differences between the groups. Logistic regression was done to build a prediction model in menorrhagia.
Results: Women with TL had more menstrual irregularity than those without TL (24.3% vs. 10% respectively, p=0.002). Women with TL had more polymenorrhea (9.3% vs. 1.4%; p=0.006), hypermenorrhea (12.1% vs. 2.1%; p=0.002), menorrhagia (62.9% vs. 22.1%; p<0.0001) and menometrorrhagia (15.7% vs. 3.6%; p=0.001) than those without TL. There is a significant difference in the PBLAC score between women with and without TL (p<0.0001). According to logistic regression, age (OR=1.08, CI:1.07-1.17; p=0.03), TL (OR=5.95, CI:3.45-10.26; p<0.0001), and cesarean section (OR=2.72, CI:1.49-4.97; p=0.001) were significantly associated with menorrhagia.
Conclusion: We found significant differences in menstrual disorders between women with and without TL. Therefore, women should be informed by the health providers regarding the advantages and disadvantages of TL before the procedures.
Key words: Historical cohort study, Tubal ligation, Menstrual disorders, PBLAC.
P-77
Effect of nano silver on morphological and chromosomal abnormality of NMRI mouse fetus
Mozafari M1, Kalantar SM2, Khoradmehr A2, Danafar AH2, 3, Miresmaeili M4.
Introduction: Development of nanotechnology caused the use of the materials in nano sizes. Nowadays, special biological properties of nano silver are playing an important role in our life. Some studies suggest a potential for adverse effects of nano silver on fetal development of mammals, but additional research is needed. In this study we decide to evaluate the effects of nano silver on fetus chromosomal structure and its development.
Materials and Methods: 24 pregnant mice were divided into four groups. Nano silver (1 mg/kg, 70 nanometer) were gavaged to the first, second and third group, from the 1th-7th, 8th-14th and 1th-14th gestational days respectively. Nothing was gavaged to the control group. On 14th day the pregnant mice were dislocated and the liver of fetus was used for karyotyping analysis. The fetuses were weighed and their crown-rump length and head circumference were measured by caliper.
Results: According to the effects of nano silver, the results indicated that body weight of embryos were significant decreased in third group in comparison with the others (p<0.001). Also there were significant reduction of crown-rump length of fetus in third and second group compared to other groups (p<0.05). There was lower fetus head circumference among nano silver treated and controls (p<0.05). Karyotyping analysis of fetal liver was normal in all groups.
Conclusion: It seems that abuse of nano silver during pregnancy can reduce weight, crown-rump length and head circumference in mice fetus, but its mechanism is not completely clear.
Key words: Pregnant mice, Nano silver, Karyotype.
P-78
Neuronal markers expression of induced human adipose-derived stem cells in alginate hydrogel
Khosravizadeh Z1, Khosravizadeh Z1, Razavi Sh2.
Introduction: Hydrogels provide appropriate three-dimensional environment for cell cultures and cell encapsulation in hydrogels is a promise plan for tissue engineering applications. Alginate is a biocompatible hydrogel that provides a supportive system for the encapsulated cells. Moreover human adipose derived stem cells (hADSCs) might be a suitable source of cells for use in autologous cell therapy; also these cells can differentiate into neuron-like cells. Therefore, in this study we evaluated effect of alginate hydrogel on the neural potential of induced hADSCs.
Materials and Methods: Isolated hADSCs were induced in neural medium and encapsulated in alginate hydrogel. Using Immunocytochemical and real-time RT-PCR analysis, some neural markers were evaluated in differentiated hADSCs.
Results: The expression of Nestin, GFAP and MAP2 markers significantly increased in alginate cell cultures relative to monolayer induced cells (p<0.001).
Conclusion: These findings showed that alginate hydrogel can provide a suitable environment for neural differentiation of hADSCS.
Key words: Human adipose-derived stem cells, Alginate hydrogel, Neural differentiation, Tissue engineering.
P-79
Protective effect of N-acetyl-L-cysteine on testicular tissue in mice treated with Para-Nonylphenol
Malmir M, Soleimani Mehranjani M, Naderi Noreini S.
Faculty of Science, Arak Univercity of Medical Scinecs, Arak, Iran.
Email: mehhdi66@yahoo.com
Introduction: Para-Nonylphenol (p-NP) is an alkylphenol considered as an environmental pollutant with estrogenic and toxic effects. It can also cause morphological and functional alterations in the male reproductive system inducing infertility. The purpose of this study was to investigate the protective effects of N-acetyl-L-cysteine (NAC), as a powerful antioxidant, on p-NP-induced testicular toxicity in mice.
Materials and Methods: Adult male NMRI mice (32±4 gr) were divided randomly into 4 groups (n=6), control, NAC (150 mg/kg/day), p-NP (250 mg/kg/day) and p-NP+NAC, and they were treated orally for 35 days. Finally, mice were killed, their right testis were removed and fixed followed by sectioning, tissue processing and Heidenhain azan staining. Testicular tissue sections were then evaluated using stereological method. The level of Malondialdehyde (MDA), a lipid peroxidation index, was also measured in the testis of mice. Data were statistically analyzed using one way ANOVA and means were considered significantly different at p<0.05.
Results: A significant decrease in the mean total volume of testis, diameter, length and the volume of the seminiferous tubules, height of the germinal epithelium and the thickness of the basement membrane along with a significant increase in the MDA level was observed in the p-NP group compared to the control (p<0.01) while the above parameters were compensated to the control level in the p-NP+NAC group (p<0.01).
Conclusion: Our findings indicate that N-acetyl-L-cysteine can protect the testicular tissue against the tissue damage and oxidative stress induced by para-Nonylphenol in mice.
Key words: Para-nonylphenol, N-acetyl-L-cysteine, Stereology, Testis.
P-80
Effects of dietary canola oil on sperm quality parameters at Afshari Ram Breed
Mansouri S, Pirestani A.
Islamic Azad University, Khorasgan Branch, Isfahan, Iran.
Email: nmansorrin@yahoo.com
Introduction: In mammalian and non-mammalian spermatozoa, there are natural fatty acids, cholesterol, phospholipids (mainly lecithin, cephalin and sphingomielin) and glycolipids. Phospholipids of mammalian sperm cell membranes characteristically contain very high proportions of long-chain polyunsaturated fatty acids, particularly n-3 series
Materials and Methods: 18 Kurdish rams were selected with weight average 54.47±2.58 kg and with the age of 3-4 years approximately. They were divided to two experimental groups randomly. Experimental groups were control and canola oil (2.5% of DMI). Before of study, 10 day was considered as adaptation period and then Sperm was collected by electro ejaculation at 6 week and 11 week after begging of adaptation period and sperm motility was analyzed by using CASA software at zero time and 24 hours post ejaculation. Also, percentage of live/dead sperm and morphology was evaluated by staining of Eosin-Nigrosin and Papanicolaou, respectively.
Results: The results showed that motility parameter wasn’t significantly different between whole experimental groups at first time (week 6) but PM% and TM% was significantly different in canola oil group compare to control group at second time (week 11), separately. Percentage of live/dead sperm and morphology was higher significantly in canola oil group compare to control group.
Conclusion: It was concluded that canola oil can improve sperm motility, morphology and viability in Afshari ram.
Key words: Canola oil, Motility, Afshari Ram, Sperm.
P-81
Mutation analysis of bone morphogenetic protein-15 gene in Iranian patients with polycystic ovarian syndrome
Mehdizadeh A, Kalantar SM, Sheikhha MH.
Department of Genetics, International Campus, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: anahita.mz@gmail.com
Introduction: With the prevalence on the order of 6-10%, polycystic ovarian syndrome (PCOS) is considered the most common endocrinologic disorder affecting women in their reproductive age. Major criteria that have been proposed for the diagnostic characteristics of PCOS are clinical and/ or biochemical hyperandrogenism, ovulatory dysfunction and polycystic ovary in ultrasound. It has been suggested that genetic factors participate in the development of PCOS. Follicular development has been considered as one of the impaired processes in PCOS. BMP-15 gene is a candidate gene in follicular development and its variants may play role in pathogenesis of PCOS. Previous investigations have revealed controversial results on BMP-15 mutations in PCOS women among various racial groups.
Materials and Methods: A cross-sectional study was carried out on 70 PCOS patients. Following taking the informed consent, 5ml venous blood was taken from each participant. Genomic DNA was extracted from the blood sample by salting out Method. Then a set of PCR reactions for BMP15 gene was performed using specific primers followed by genotyping with direct sequencing.
Results: As for exon1 in BMP-15, 20 heterozygote (G/C) and 2 homozygote (G/G) cases were found in 70 PCOS patients. Also, one -9 C>G polymorphism in 5’UTR and 3 cases of A308G mutation (A/G) were discovered in our patients.
Conclusion: Result of this study indicates that variants of BMP-15 gene could be related to susceptibility of development of PCOS and may be used as genetic markers for detecting PCOS in the future.
Key words: Polycystic ovarian syndrome, Bone morphogenetic protein 15.
P-82
Effect of adding human chorionic gonadotropin to frozen thawed embryo transfer cycles with history of thin endometrium
Davar R1, Farid Mojtahedi M1, Miraj S1, 2.
1. Department of Obstetrics and Gynecology, Research and Clinical Center for Infertility, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Department of Obstetrics and Gynecology, School of Medicine, Shahrekord University of Medical Sciences, Shahrekord, Iran.
Email: miraj.sepideh@gmail.com; dr.s.miraj@gmail.com
Introduction: Embryo implantation process is a complex phenomenon and depends on fetal and maternal factors interaction. Endometrial thickness is needed for successful implantation. Increasing endometrial thickness, raise the chance of clinical pregnancy. The triple line pattern, with thickening more than 7mm, is indicating the greater chance of successful implantation. We designed this study to assess adding HCG to the conventional protocol in endometrial preparation in women with thin endometrium and history of IVF-ET failure.
Materials and Methods: This non-randomized clinical trial study (quasi experimental design) was performed in Yazd Research and Clinical Center for Infertility on 28 patients. Participants were women who candidate for frozen thawed embryo transfer and had 2 previous failed ET cycle because of thin endometrium. All patients received 8 mg estradiol valterate on second day of menstrual cycle and continued during the study. HCG was administrated (150 IU, IM) from 8th days of cycle. In 12th-13th day Trans-vaginal sonography was done, when endometrial thickness reached at least 7mm, HCG was discontinued and frozen thawed embryo transfer was done.
Results: Totally 28 patients were included. The mean age of participants was 30.39±4.7 years. The mean of endometrial thickness before and after HCG were 5.07±0.43 and 7.85±0.52 mm which were significantly different (p=0.00). After HCG administration 100% patient's endometrial thickness reached more than 7mm. The frequency of 20% improvement after HCG was 89.3% (25 patients). Also there were 5 (17.8%) clinical and chemical pregnant women after HCG.
Conclusion: Our study findings suggest that adding HCG to the conventional preparation method is an effective protocol and significantly improved endometrial thickness and pregnancy outcomes in women with previous embryo transfer failure because of thin endometrium.
Key words: Human chorionic gonadotropin, Frozen thawed embryo transfer, Thin endometrium.
P-83
Glycyrrhiza glabra and vitamin C can reduce toxic effects of acrylamide on sperm parameters in rat
Mirjalili A1, Talebi AR2, Mangoli E2, Ghasemzadeh J2.
Introduction: Acrylamide (AA) is a chemically reactive substance used in various industries. Recently, acrylamide was discovered in a variety of human foods including heat-processed starchy foods such as potato chips and bread. AA is able to induce sperm damage in male mice.
Materials and Methods: In this experimental study, 30 male Wistar rats of 28 days of age were divided in to five groups: Acrylamide, Acrylamide+ Vitamin C, Acrylamide+ Glycyrrhiza glabra, Acrylamide+ Vitamin C+ Glycyrrhiza glabra and Control. All treatments were administered (oral Acrylamide 10 mg/kg, Vitamin C 200 mg/kg and Glycyrrhiza glabra 150 mg/kg) daily for two months. Thereafter, the cauda epididymis of each rat was dissected and placed in 1 mL of pre-warm Ham’s F10 culture medium for 30 min. The swim-out spermatozoa were analyzed for count, motility, morphology and viability.
Results: The results showed that almost all of the sperm parameters except non progressive motility were significantly different between groups (p=0.001). Also, the mean of sperm parameters in Acrylamide+ Vitamin C+ Glycyrrhiza glabra group was higher than other groups.
Conclusion: This study showed that the co-administration of vitamin C and Glycyrrhiza glabra as antioxidant can reduce the detrimental effects of acrylamide on sperm parameters in rats.
Key words: Glycyrrhiza glabra, Vitamin C, Acrylamide, Sperm, Rat.
P-84
Infertility in male by neurological disorders
Mollaei AR, Seihei E.
Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
Email: ptalireza@yahoo.com
Introduction: Normal sexual and reproductive functions depend largely on neurological mechanisms. Neurological defects in men can cause infertility through erectile dysfunction, ejaculatory dysfunction and semen abnormalities.
Materials and Methods: Science Direct, PubMed, Cochrane, CINAHL, Embase, ProQuest Dissertations, Scopus, (2000-2015) were searched for English-language studies using a list of keywords. The books about physical therapy and medical and neurological were studied too.
Results: Among the major conditions contributing to these symptoms are pelvic and retroperitoneal surgery, diabetes, congenital spinal abnormalities, multiple sclerosis and spinal cord injury. Erectile dysfunction can be managed by an increasingly invasive range of treatments including medications, injection therapy and the surgical insertion of a penile implant. Retrograde ejaculation is managed by medications to reverse the condition in mild cases and in bladder harvest of semen after ejaculation in more severe cases.
Conclusion: An ejaculation might also be managed by medication in mild cases while assisted ejaculatory techniques including penile vibratory stimulation and electro ejaculation are used in more severe cases. If these measures fail, surgical sperm retrieval can be attempted. Ejaculation with penile vibratory stimulation can be done by some spinal cord injured men and their partners at home, followed by in-home insemination if circumstances and sperm quality are adequate. The other options always require assisted reproductive techniques including intrauterine insemination or in vitro fertilization with or without intracytoplasmic sperm injection. The method of choice depends largely on the number of motile sperm in the ejaculate.
Key words: Ejaculation, Electro ejaculation, Infertility, Nervous system diseases.
P-85
Adenosine deaminase G22A polymorphism and recurrent spontaneous abortions
Mohammadi A1, Bonyadi M1, Khalaj kondori M1, Taghavi S2.
Introduction: Adenosine deaminase (ADA) is an enzyme of purine salvage pathway and has two important isoenzymes ADA1 and ADA2. The adenosine deaminase G22A polymorphism (ADA*2) increases the level of adenosine. Adenosine may play a protective role against recurrent spontaneous abortions (RSA), since it regulates blood flow into the uterus and placenta.
Materials and Methods: A total of 100 women were classified in two groups: First group, with a history of recurrent spontaneous abortions (n=50), and second one, without a history of abortions (n=50). Genomic DNA was extracted from peripheral blood with a commercial kit and PCR-RFLP analysis was used to identify the G22A genetic polymorphism.
Results: The frequency of homozygotes (AA) was 2% in control group, whereas no homozygote (AA) was found in the case group (p>0.05). The frequency of heterozygotes (AG) was 20% in the control group and 8% in the case group (p<0.05). The frequency of homozygotes (GG) was 78% in the control group and 92% in the case group (p<0.05). A significant increase in the frequency of AG genotype in controls (p<0.05, OR=0.348) relative to women with the history of RSA demonstrates the protective effect of AG genotype in controls.
Conclusion: The data suggest that women carrying the ADA*2 allele which is associated with the lower enzymatic activity are better protected against recurrent spontaneous abortions. Because decreased enzymatic activity increases adenosine levels. High adenosine levels may play a protective role against recurrent spontaneous abortions, since it regulates blood flow into the uterus and placenta.
Key words: PCR-RFLP, Recurrent spontaneous abortion, Adenosine deaminase.
P-86
Seminal bacterial contaminations: Probable factor in unexplained recurrent pregnancy loss
Nabi A, Khalili MA, Halvaei I, Ghasemzadeh J, Zare E.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: ali.nabi67@yahoo.com
Introduction: It is estimated that about 50% of causes of recurrent pregnancy loss (RPL) cases remain unknown. Sperm factors are suggested to have probable role in cases with RPL. The goal was to determine the possible relationship between semen bacterial contaminations with unexplained RPL. Also, the correlation between number of bacterial colony and sperm chromatin condensation was examined.
Materials and Methods: This study consisted of 30 fertile men (group A) and 30 infertile men (group B) with unknown RPL in their wives. Semen collection and analysis were done according to WHO manuals. Sperm count and motility were evaluated by Makler chamber. Eosin-Nigrosin and Papanicolaou staining methods were applied for viability and morphology assessment, respectively. The semen samples from both groups were cultured for aerobic bacteria. Aniline blue (AB) and toluidine blue (TB) staining methods were applied for evaluating sperm chromatin condensation.
Results: The numbers of colonies were significantly higher in group B when compared to group A. Also, S. aureus and E. coli contaminations showed significant differences between two groups. Both AB+ and TB+ sperm cells showed significant increase in group B compared to group A. There was a significant negative correlation between colony number and progressive motility (p=0.01), and sperm viability (p=0.007). In addition, positive correlations were found between colony number and AB+ (p=0.001) and TB+ (p=0.004) as well.
Conclusion: Bacterial contaminations in semen of men from RPL couples had significantly higher levels when compared to fertile controls. Presence of microorganisms in semen may be correlated with irregular sperm parameters and quality.
Key words: Recurrent pregnancy loss, Bacteria, Semen.
P-87
Precedent infertility and attitudes towards preferred Cesarean
Naeimi N.
Department of Biology, Faculty of Science, University of Sistan and Baluchestan, Zahedan, Iran.
Email: nnaeimi@rocketmail.com
Introduction: Prevalence of natural delivery and caesarean section in pregnant women of a country is one of the indicators of health system performance. Currently, we are exposure to increasing elective Cesarean with different reasons and attitudes.
Materials and Methods: In this descriptive-analytical study, 271 pregnant women completed a researcher-made questionnaire including, demographic characteristics (age, precedent of infertility) and 10 attitude questions about the influencing factors on choosing Cesarean, based on five Likert scale which was a score of 10-30 negative attitude (opposite preference for Cesarean) and 31-50 positive attitude (positive preference for Cesarean). Therefore, to analyze the obtained data, Chi-square test was used through applying SPSS19 software.
Results: Overall 74 subjects (28%) had negative attitudes, while 192 subjects (72%) had positive attitudes. In total 7 subject of pregnant women group (2.6%) had a precedent of infertility and 259 pregnant women had not (95.6%). During the study, 5 subjects did not answer questions. In addition, 5 subjects with precedent infertility (73.4%) vs. 69 subjects (26.6%) with no precedent, showed negative attitudes. 2 subjects (26.6%) vs. 190 pregnant women (73.4%) had positive attitude toward preference of Caesarean section. There was a significant relationship between precedent infertility and attitude.
Conclusion: According to the results, women with precedent infertility who are using assisted reproductive techniques, are more aware of advantages and disadvantages of different methods of termination of pregnancy and most of them opposed natural delivery. Therefor giving them information through related groups of women's health, can change their attitudes.
Key words: Precedent infertility, Attitude, Preference cesarean.
P-88
Protective effects of BDNF against oxidative damage and apoptosis in human spermatozoa
Najafi A1, Sobhani AGH1, Amidi F1, Sadighi Gilani MA2, Moawad AR3, Mikaeili S1, Fallah P2, Rezaiian Z2.
Introduction: Oxidative Stress (OS) is a condition when there is an improper balance between the amount of pro-oxidant substances and the amount of seminal plasma antioxidant factors that can be induced by repeated cycles of centrifugation and seminal plasma antioxidants, removal during the sperm preparation in Assisted Reproductive Technology (ART) Reactive Oxygen Species (ROS) can cause decrease of motility, increase of apoptosis and impaired sperm function, and ultimately, influence on fertilization and pregnancy rate. Therefore, currently there is a great interest to the use of antioxidants to prevent ROS generation and ROS-induced apoptosis during the sperm preparation processes. Brain-derived neurotrophic factor (BDNF) is member of neurotrophin family that has anti- oxidant and anti-apoptotic effects on nervous system. Recent researches show that it also plays key role in male and female reproductive system such as spermatogenesis, nuclear and cytoplasmic maturation and embryo development.
Materials and Methods: Liquefied semen samples obtained from normozoospermic men (n=25) after preparation by double wash (400× gr, 5 min) swim-up technique were divided in to two groups of control and treated group (with BDNF). Then motility was evaluated by means of CASA. Reactive oxygen species (ROS) and apoptosis were assessed by flowcytometry.
Results: Addition of BDNF to the sperm media significantly increased mean total motility and progressive motility (75.54±2.63% vs. 66.81±2.48%, p<0.001, and 46.18±2.2 vs. 39.631±1.97%, p<0.002, respectively). Also BDNF treatment caused decrease of H2O2 (50.01±6.08 vs. 38.31±5.250, p<0.02), necrosis (23.26±2.71 vs. 27.52±2.27, p<0.001) and apoptosis (3.26±0.4 vs. 3.76±0.55, p<0.49).
Conclusion: Considering this result, BDNF treatment can be a potential tool against oxidative damage and apoptosis in human spermatozoa.
Key words: Brain-derived neurotrophic factor (BDNF), ROS, Apoptosis, Flowcytometry.
P-89
Iranian couple’s sexual compatibility during the time: A qualitative study
Nekoolaltak M1, Keshavarz Z1, Simbar M2, Nazari AM3, Baghestani AR4.
Introduction: Since sexual compatibility is facilitators for marital compatibility and the root of many divorces is the sexual incompatibility, and due to the limited data available in this topic, this qualitative study was designed to exploring the changes of Iranian couple’s sexual compatibility during marriage years.
Materials and Methods: In-depth interview and written narrative methods were used. Purposive sampling with maximum variation (age, education, years of marriage, number of children, etc.) was conducted. Ale Yasin Clinic in Tehran and Fatemieh Clinic in Varamin were the setting of research. Times of interviews were almost 40 min. Data reached to saturation after 14 semi-structured depth interviews and 54 written narratives with the 68 married men and women. Conventional content analysis was done in MAXQDA10 software. Lincoln and Guba criteria were used to validate qualitative data.
Results: Emerged categories included: “duration to reach sexual compatibility" and “mainly increasing sexual compatibility during the time" and "rarely decreasing sexual compatibility during the time". Couple’s sexual compatibility increases over time due to “Getting to know each other more", “increasing sexual awareness and experience”, “talking about sex with spouse”, “skill acquisition for mutual pleasure and satisfaction”. Couple’s sexual compatibility may decrease over the time because "children growing and conflict parental role with spousal role" and “financial difficulties”.
Conclusion: With knowing the impact of passing the time on couple’s sexual compatibility many divorces can be prevented by suitable planning and counseling.
Key words: Sexual compatibility, Iranian couples, Qualitative study.
P-90
Effect of non-polar extract of Phaleria macrocarpa (Mahkota Dewa) on spermatogenesis in rats
Parhizkar S1, Aziz Dollah M2, Ferdaus Binti N2.
Introduction: Fertility is the natural capability of giving live. In male, fertility requires the production of large numbers of normal and mature spermatozoa by testes through a complex process call spermatogenesis. Evidence shows increase male age is associated with the decline of mature sperm in seminiferous tubule. Aqueous extract of Phaleria macrocarpa (PM) has been shown to improve fertility in rats by increasing the spermatogenesis.
Materials and Methods: Hexane extract of Phaleria Macrocarpa was prepared by extracting grinded dried slices of fruit followed with drying of the extract using rotary evaporator. A total of 30 male rats were divided into 5 different groups and subjected to daily treatments with PM extract at one of the following concentration: 0, 6, 12, 60 mg/kg of hexane extract, or commercial testosterone hormone for seven consecutive weeks. They will be fed with pellet once a day in the morning and drinking water will be given ad libitum. Body weight was measured once a week. On the last day of supplementation period, the rats were sacrificed and the testis was isolated for histological evaluation.
Results: The result showed that no significant effect of non-polar extract of PM on testes size and volume, thickness of seminiferous tubules and spermatogonia cell number. The mean thickness of seminiferous tubules were 62, 68, 62, 60, 60 µm for 60, 12, 6, 0 mg/kg of PM extract and commercial testosterone respectively. The mean for spermatogonia cell count in rats treated with 60, 12, 6 and 0 mg/kg and commercial testosterone were 52.8, 43.8, 47.0, 52.6, 52.0 cells respectively.
Conclusion: The study concludes that the non-polar extract of PM has no effect on the fertility of male.
Key words: Phaleria macrocarpa (PM), Spermatogenesis, Rat.
P-91
The functional SNP analysis of CYP2D6 gene in patients with Polycystic ovary syndrome (PCOS)
Peirosabet M1, 2,Khosravifar M2, Chekini Z2, Rahimi A1, 2, Shiva M3, Afsharian P2.
Introduction: PCOS is a common ovulatory disturbance, which influents about 5-10% women of reproductive age. Clomiphene citrate is used for ovulatory induction in PCOS, that is metabolized by Cytochrome P450 2D6 (CYP2D6). More than 120 variable alleles have been reported for CYP2D6 that some of them are poor metabolizer and cause resistance to drugs that are substrates of CYP2D6.
Materials and Methods: Whole blood was collected from drug control, PCOS and fertile women (60 in each group). RFLP method was used to determine CYD2D6*34 (2850C˃T). Patients should have the Rotterdam criteria. Inclusion criteria’s were age under 35 years old, candidates for IUI, not using other drugs and not have ovary cautery.
Results: Genotype distribution of CC, CT and TT in PCOS group was 47.4%, 40.4% and 12.3% respectively (p=0.479), and frequency of these genotypes in drug control group was 36.7%, 50% and 13.3% respectively (p=0.479), which was very close to results in fertile group: 33.3%, 56.7% and 10% respectively. The number of antral follicles (follicle diameter≥15mm) was measured as drug response which was 1.21 (p=0.118) in clomiphene citrate administered PCOS patients (64%) while this was 1.64 (p=0.09) in 36% of patients treated with letrozole (follicle diameter≥15mm). Same results were observed in drug control group: antral follicles were 1.66 in 80% of clomiphene citrate treated whereas it was 1.77 when they received letrozole (20% of patients).
Conclusion: Although genotype frequencies of 2850C˃T polymorphisms were not significant between three groups, we observed cases bearing this polymorphism, showed resistance to clomiphene citrate, in both groups, which is the first time to report.
Key words: CYP2D6, Polycystic ovary syndrome, Clomiphene citrate.
P-92
Does L-carnitine therapy add any extra benefit to standard inguinal varicocelectomy in terms of deoxyribonucleic acid damage or sperm quality factor indices: A randomized study
Pourmand GH1, Movahedin M2, Dehghan S1, Mehrsai A1, Ahmadi A1, Pourhosein M1.
Introduction: Varicocelectomy and anti-oxidant therapy are both documented to have positive effect on spermatogenesis and improving semen quality.
Materials and Methods: 100 patients enrolled in this study and were randomly divided into 2 groups (50 patients in each group). In group 1, standard inguinal varicocelectomy and, in group 2, standard inguinal varicocelectomy plus oral antioxidant therapy (oral L-carnitine, 250 mg 3 times a day) were performed for 6 months. For all patients, routine semen analysis and DNA damage test of spermatozoa (by 2 methods of terminal deoxynucleotidyl transferase dUTP nick end labeling and protamine damage assay) were performed at baseline and at 3 and 6 months postoperatively.
Results: In both groups, the improvement in semen analysis parameters and DNA damage was observed, but there was not any statistically significant difference between the 2 groups in these parameters, although the slope of improvement in DNA damage was slightly better in group 2 (that was not statistically significant).
Conclusion: We observed that addition of 750 mg of L-carnitine orally daily to standard inguinal varicocelectomy does not add any extra benefit in terms of improvement in semen analysis parameters or DNA damage.
Key words: DNA damage, Varicocelectomy, L-carnitine therapy.
P-93
Different haplotype frequency in AKAP3 gene in Iranian patients with short tail sperm
Poursafari E1, Mohseni Meybody A2, Sabbaghian M2, Modarresi T2, Hosseini H1, Gourabi H2.
Introduction: One kind of sperm abnormalities that leads to men infertility is short flagella of sperms. In this defect, fibrous sheath and axoneme are disorganized, the sperms tail is short, the numbers of sperms in the semen fluid reduce and the sperms are immotile. A Kinas anchoring protein 3 (AKAP3) gene encodes a protein that is involved in the fibrous sheath structure, regulation of sperm motility and head-associated functions such as capacitation and the acrosome reaction. AKAP3 interacts with the regulatory subunit of Protein Kinas A via its dimerization/ docking domain. In the present study, 30 patients with short tail sperm defect and 40 males with normal spermogram referred to Royan Institute were enrolled as case and control groups respectively. The genetic variation in exon 5 of AKAP3 gene which encodes the functional domain of this protein was studied.
Materials and Methods: PCR- sequencing was done on extracted DNA from blood samples of control and patient groups.
Results: According to the results, four haplotype polymorphisms 1378 T>C, 1391 C>G, 1437 T>C and 1573 G>A were observed in all samples studied. These polymorphisms were all observed as the mutant alleles. 92% C allele and 8% G allele of 1391 C>G polymorphism has been reported in East Asia. In the present study, 100% G allele was observed in this polymorphism.
Conclusion: This difference in frequency of mutant allele can be due to different ethnic of Asian population and ours. 1391 C>G alternation is located on the outside of the AKAP3 binding domain to AKAP4 and seems that it cannot have a role in the interaction between these two proteins.
Key words: Short tail sperm defect, Fibrous sheath, AKAP3 gene, PKA, Polymorphism.
P-94
Study of genetic alterations of STK11 in patients with polycystic ovary syndrome (PCOS) and their response to ovarian stimulation
Rahimi A1, 2, Khosravifar M2, Peirosabet M1, 2, Chekini Z2, Shiva M3, Afsharian P2.
Introduction: PCOS is the main reason of anovulatory caused infertility. Metformin use for PCOS patients to reduces hyperinsulinemia and androgens production through activation of STK11. We aimed to study the associations between c.842C>T and c.996G>A polymorphisms and risk of PCOS susceptibility in women plus the relations of two polymorphisms and response to ovarian stimulation in PCOS patients.
Materials and Methods: Blood samples were collected from patients and drug control group (male infertility) and fertile group (60 in each group). For genotype analysis, we used RFLP method. We used Rotterdam criteria for patient selection. Inclusion criteria's were age below 35 years old, candidates for IUI, not using other drugs and not have ovary cautery. Our data shown that frequency distribution of CT, TT,CC genotypes in exon6, in PCO Patients was 1.7%, 0%, 98.3% respectively (p=0.236), these frequencies in fertile group was 5%, 0%, 95% respectively and in drug control group was 0%, 1.7%, 98.3% respectively (p=0.236). We have not detected c.996 G>A polymorphism in any groups.
Results: The mean of age and BMI were 26.31±3.68 years and 27 kg/m2 in PCOS and control group. Most of PCOS patients (64%) were received clomiphene citrate and 36% used letrozole whereas 80% of drug control was treated by clomiphene citrate and 20% used letrozole. In both groups their responses to drugs was measured by the number of antral follicles (follicle diameter ≥15mm) by ultrasonography that was 1.64 in PCOS, p=0.09 and 1.77 in drug control treated with letrozole that were better than clomiphene citrate (1.21 in PCOS, p=0.118 and1.66 in control).
Conclusion: Our data did not find any significant differences between these polymorphisms and response to ART medicines in PCOS women. According to our study, PCOS patients had higher response to letrozole rather than clomiphene citrate.
Key words: STK11, Polycystic ovary syndrome, Polymorphism.
P-95
Effects of electromagnetic fields of magnetic resonance imaging on male mice fertilization
Rostamzadeh A1, 2, Anjamrooz SH1, Fathi F1, Rezaie MJ1,3.
Introduction: Currently, the use of electromagnetic waves in medicine, especially in diagnostic devices such as magnetic resonance imaging (MRI) has increased and many of its biological effects have been reported. The aim of this study was to investigate the adverse effects of 1.5T MRI on fertility and reproductive parameters of male mice.
Materials and Methods: 40 NMRI adult male mice were randomly divided into two groups of control and experimental. The mice in the experimental group were exposed to MRI at 1.5T for 36 min once a week for a period of 3 weeks. Then, in the 1st day and 35th day after the final exposure, 10 mice were used for IVF and 10 mice for In vivo studies. MRI effects on testis weight, the duration of pregnancy, the number of newborns, sperm count, and fertility were evaluated. The obtained data were analyzed using ANOVA and Tukey’s tests.
Results: According to the present study, one day after MRI exposure, testis weight, sperm count, and the number of born children were significantly decreased in the experimental group (p<0.05). Moreover, a significant number of the embryos failed to develop to the blastocyst stage. In contrast, 35 days after exposure, no statistically significant difference was found (p>0.05).
Conclusion: Based on the results of the present study, it seems that although the MRI at 1.5T has adverse effects on fertility and reproductive parameters of the adult male mice, these side-effects are reversible.
Key words: Electromagnetic fields, MRI, Sperm, Fertility.
P-96
Interference of histone modification with aberrant expression of HOXA10 gene in eutopic and ectopic endometrial tissues of women with endometriosis during menstrual cycle
Samadieh Y1, Shahhoseini M1, Favaedi R1, Mahdian S3, Ramezanali F2, Ashrafi M2, Afsharian P1, Aflatoonian R2.
Introduction: Disruption of the balance of epigenetic networks, which involves DNA and histone modifications, can cause several pathologies, including reproductive disorders such as endometriosis. HOXA10 gene expressed in endometrium plays an important role in uterine receptivity at the time of implantation, uterine organogenesis and functional endometrial differentiation.
Materials and Methods: Epigenetic analysis were assayed by Chromatin Immunoprecipitation (ChIP), using anti- H3K9ac, H3K9Me2, H3K27Me3, H3K4Me3 antibodies and quantitative expression analysis was performed by real-time PCR technique. For this respect, eutopic and ectopic endometrial samples were collected using laparoscopy from 36 women with documented endometriosis, and also endometrial biopsies were obtained from 22 healthy women with male factor problem as a control group. Ethical approval and informed patient consent was gained for the use of tissue samples.
Results: Data showed a harmonious pattern between mRNA expression of HOXA10 and epigenetic state of its promoter region, in the way that, in secretory phase the activating epigenetic marks, H3k9ac and H3k4me3 were higher in ectopic endometrial tissues and H3K9ac itself were lower in eutopic endometrium. In contrast, H3K9me2 and H3k27me3, the epigenetic marks, known to be associated with gene repression, showed a different pattern in which that, H3K9me2 were higher in eutopic endometrium and H3K27me3 were lower in ectopic endometrial tissues.
Conclusion: Our findings suggest epigenetic might be greatly responsible for aberrant expression of HOXA10 gene in eutopic and ectopic endometrial lesions of patients with endometriosis.
Key words: HOXA10 gene, Endometriosis, Menstrual cycle, Histone modifications, Epigenetics.
P-97
Evaluation of action and mechanism of L-Carnitine in improved sperm quality in male infertility
Sheikhpour R.
Yazd Medical Science Branch, Islamic Azad University, Yazd, Iran.
Email: R.sheikhpour@yahoo.com
Introduction: About 15% of married couples are infertile and that approximately 50% of this is due to male factor infertility. A number of drugs have been proposed as being possible causes of male factor infertility. Estudies showed that a diet supplemented with L-carnitine can improve sperm quality in some mammalian species. In humans, 75% of carnitine derives from diet, while 25% is synthesized from lysine and methionine, although the enzyme that catalyses the hydroxylation of the 4-butirrobetain in L-carnitine is present in few tissues.
Materials and Methods: This review was conducted using the following databases: Medline, Pubmed, Scopus and Science direct from 1990-2014 for better understanding of the mechanism and action of L-Carnitine in male infertility.
Results: L-Carnitine can increase the concentration of this component in the epididymal tubules and spermatozoa. This molecule increases their motility and fertilizing abilities. Both free L-carnitine and acetylated L-carnitine can be accumulated in spermatozoa. In sperm cells, L-carnitine transports fatty acid to mitochondria for oxidation and production of energy for epididymal spermatozoa. Also L-Carnitine has antioxidant property; it via deleting excess acyl-CoA (formyl-CoA), due to toxic effect have important role in cellular detoxification. L-carnitine as antiaging protects cellular membranes against oxidative damage. It prevents protein oxidation, pyruvate and lactate oxidative damage. During sperm maturation, a reduction in total lipid content has been seen due to the changing composition of fatty acids in sperm cells. This leads to an increase in the fluidity of spermatozoa’s plasmalemma. Moreover, L-carnitine reduces lipid availability for peroxidation which guards against potential peroxidative damage.
Conclusion: The result of studies showed that addition of L-carnitine to the diet of male infertility is effective in increasing semen quality, but these results need to be confirmed by more clinical trials.
Key words: L-Carnitine, Sperm quality, Male infertility.
P-98
Effects of cell phone radiofrequency electromagnetic waves (RF-EMW) on human spermatozoa
Soltani S1, Khalili MA1, Omidi M1, Nabi A1, Shamsi F2.
Introduction: Cellular phones emit radiofrequency electromagnetic waves (RF-EMW) in the low frequency microwave. The detrimental effects of RF-EMW on the reproductive system and human fertility have been debated in recent years. The goal was to assess the influence of cell phones RF-EMW on different parameters and DNA integrity of human spermatozoa.
Materials and Methods: 50 semen samples were categorized in two groups: a) normospermia (n=24); and b) asthenozoospermia (n=24). After liquefaction, each sample was divided into two aliquots. One aliquot (experimental) was exposed to mobile radiation for 1 hr, and the second aliquot (unexposed) served as control. The sperm parameters and chromatin/ DNA integrity were examined and compared between groups.
Results: Normal samples exposed to RF-EMW showed insignificant differences in sperm motility, viability, and chromatin/DNA integrity. Regarding sperm morphology, normal morphology was significantly decreased in experimental group (p=0.04). Moreover, sperm parameters as well as DNA structure showed no significant differences in astenozoospermic samples.
Conclusion: Data did not show any noticeable impact of RF-EMW on human sperm quality. Further studies should be considered for confirmation of the results.
Key words: Cell phone, Electromagnetic radiation, Sperm parameters, DNA integrity.
P-99
Assessment of genetic variations in intron4 and exon5 of RABL2B gene in Iranian infertile men with immotile short tail sperm defect
Hosseini H1, Sabbaghian M2, Mohseni Meybodi A2, Hosseinifar H2, Poursafari E1, Sadighi Gilani MA2.
Introduction: The immotile short tail sperm (ISTS) defect, is a syndrome which causes male infertility. Patients with ISTS disorder have immotile short-tailed sperm with disorganized axonem, and a significant decrease in sperm counts too. Numerous proteins are involved in sperm tail formation. One of these proteins is RAB Like 2B (RABL2B), which recently its essential role in sperm intra-flagellar transport and fertility in male mouse has been demonstrated. So its gene, which called RAB Like 2B (RABL2B), is an appropriate candidate gene in human studies. RABL2B protein has 4 GTP binding domains which have important roles in protein function. Exon 5 of RABL2B gene, codes one of these main domains and intron 4 is the location for binding to some important transcription factors. The purpose of this study was to evaluate the genetic variations of exon 5 and intron 4 of RABL2B gene in infertile men with ISTS defect and controls
Materials and Methods: In this study, 30 infertile men with ISTS defect and 30 normozospermic men as controls were recruited. Remarkably it took 2 years to collect patients samples. To study the genetic variations, DNA was extracted from peripheral blood, then PCR sequencing was done.
Results: Sequence analysis results did not identify any mutations or single-nucleotide polymorphisms (SNPs) in exon 5, but an intronic variant (rs:144944885), was found in heterozygote form in one patient. No mutations or SNPs was identified in controls.
Conclusion: Although our data just revealed an intronic variations and no mutations or SNPs was detected in exon 5, due to the high expression of RABL2B gene in testis, and considering the fact that RABL2B is evolutionarily conserved and not many studies have been conducted about the exact role of this gene in human male fertility, evaluation of other exons and regulatory areas of this gene is strongly recommended.
Key words: RABL2B gene, ISTS, Male infertility.
P-100
Localization of septin 14 protein in sperm
Vahabi Barzi N, Hosseinifar H, Nickhah Kalashami Z, Sadighi Gilani MA, Sabbaghian M.
Department of Andrology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: nastaranbarzi@yahoo.com
Introduction: In mammals, 14 septin genes have been identified so far. Disruption of septin functions has been implicated in the pathology of many diseases, including male infertility. Here, we study about one of the new members of the septin family called septin14 that is specially expressed in testis. This gene has two transcripts but only one of them transcript into proteins.
Materials and Methods: In this study, semen obtained from subjects attending Royan Institute that had normal spermogram. After that, immunocytochemistry, which is a common laboratory technique, was used to anatomically localize presence of a specific protein in cells by a specific primary antibody. The primary antibody allowed visualization of the protein under a fluorescence microscope when it was bound by a secondary antibody with a conjugated fluorescence.
Results: The protein expression was detected in the sperm from head to tail and highly localized in the front of the acrosome and the neck.
Conclusion: This is the first report on the localization of septin 14 in sperm. Septin 14 plays an important role in sperm morphology. Regarding the presence of this protein in sperm acrosome and neck, it can be concluded that the probable decrease of septin 14 protein expression in sperm may be associated with the pathogenesis of male infertility.
Key words: Septin 14, Immunocytochemistry, Sperm, Male infertility.
P-101
Molecular studying of sperm surface proteins and their cellular functions
Vaseghi-Dodaran H1, Daghighkia H1, Atshan M2, Jafari S3, Sobhani AGH4.
Introduction: Fertilization in mammalian is a complicated process with some connections among wide range of glycolipid, glycoprotein and antigenic indexes on sperm and egg surface of every species. We can improve our understanding in fertilization mechanisms and also get some information on molecular or immunological defects responsible for infertility by recognizing the antigens on sperm surface and investigating their role on cell physiological performance. The key challenge is to move from lists of identified proteins to informed understanding of biological function. Studying the sperm surface molecules and investigating their biochemical, biophysical and physiological properties to understand fertilization process.
Materials and Methods: A review of recent bibliography collected from internet database as PubMed, Google scholar, SID by the use of relevant keywords on different markers of cell surface and Sperm cell proteomic published in 2000-2014.
Results: Sperm membrane proteins contain an important part of membrane and are considered as special antigenic index to each species. Every change, increase or decrease in these proteins alters sperm performances and abilities. The important events which occurs during the interaction of sperm and egg and the reactions between them for fertilization relates to identification of these two cells by connecting receptors to ligands which exist on their cell membrane. Discovering the proteins on sperm surface which act as fertility biomarkers and participate in fertilization leads to more apprehension of this cell physiology and performance, so understanding molecular events like sperm acrosome reaction, capacitation and motility.
Conclusion: Sperm surface proteins can use as fertility biomarkers, therefore identification and supplementation these proteins to cryopreservation medium can improve post-thaw parameters of sperm and prevent sperm quality during cryopreservation.
Key words: Proteomics, Sperm, Marker, Fertilization.
P-102
Frequency of chromosome inversion (pericentric and paracentric) in recurrent abortions in patients referred to cytogenetics laboratory of Sarem Hospital
Abdi A1, Bagherizadeh I2, Bahman I2, Shajarehpour L1, Hadipour F3, Hadipour Z3, Shafagh Y4, Behjati F5.
Introduction: Recurrent abortion affects almost 15% of diagnosed pregnansis. More than 50% of miscarriages in the 1st trimester are due to chromosome abnormality. The aim of this study is to present the role of chromosomal inversions in recurrent miscarriages in patients referred to the Cytogenetics Laboratory of Sarem Hospital in Tehran. One group of inversions is pericentric inversion of chromosome 9, which is usually regarded as a normal population variant.
Materials and Methods: The samples were studied using high resolution GTG banding technique. For each patient, a minimum of 15 metaphases was examined by light microscopy.
Results: Pericentric inversion around centromere of chromosome 9 was observed in 29 patients (1.37%) and pericentric inversion in hetrochromatin region was found in chromosome 1 in one patient and chromosome Y in another patient, and one patient had pericentric inversion of chromosome 2. Chromosomal inversion involving other autosomal chromosomes included pericentric inversion of chromosomes 1, 5, 11 and 12 (1.9%), and paracentric inversion of chromosomes 3, 6, 7, 8, and 12 (0.23%).
Conclusion: The chromosomal imbalance of gametes may produce spontaneous abortions and malformed offsprings. This suggests that such inversions should not be ignored and they can play an important role in reproduction failure. However, we have shown that the rate of pericentric inversion of chromosome 9 is similar in different referral groups (1-2%) and similar to normal population and thus of no clinical significance.
Key words: Pericentric and paracentric chromosome inversions, Recurrent abortions, inv (9).
P-103
Kit Ligand (KL) promotes the primordial follicle growth in mouse vitrified ovaries
Abdi Sh1, Salehnia M1, Hosseinkhani S2.
Introduction: Following cryopreservation of ovarian tissue, the primordial follicles are well preserved with minimal damage. An alternative technique to improve the development of follicles within ovarian tissue is ovarian organ culture following cryopreservation. Kit ligand (KL) known as stem cell factor, steel factor or mast cell growth factor is involved in the activation of primordial follicles, oocyte growth and proliferation of granulosa and theca cells.
Materials and Methods: One week old mouse ovaries were collected and divided into vitrified and non-vitrified groups. Then they were cultured in the presence or absence of KL for 7 days. The development of ovarian follicles was evaluated by histology and also the mean area and hormonal level was analyzed during culture period. Apoptosis assessment was done using DNA laddering, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) method and caspase -3/7 activity assay.
Results: The proportion of preantral follicles and the level of hormones were increased in all cultured groups and it was significantly higher in KL treated groups than their control (p<0.001). DNA fragmentation and TUNEL positive signals were seen in vitrified cultured ovaries. The level of caspase -3/7 activity was higher in vitrified cultured ovaries.
Conclusion: KL could improve the development of follicles in vitrified cultured ovaries also it could act as anti-apoptotic factor during culture of vitrified samples. The development potential of follicles in vitrified groups was lower than fresh ovaries.
Key words: In vitro culture, Kit Ligand, Vitrification, Caspase -3/7.
P-104
Association between rs1264457 A/G polymorphism of HLA-E gene and recurrent pregnancy loss
Zarezade Z1, 4, Ashrafzadeh Mehrjardi HR2, 4, Zaimy MA3, Ghasemi N4.
Introduction: Recurrent pregnancy loss (RPL) is defined as two or more consecutive miscarriages before twentieth week of pregnancy. The cause of 50% recurrent miscarriages is unexplained. The goal of this research was to investigate the association between RPL and rs1264457 A/G polymorphism of HLA-E gene.
Materials and Methods: In this case-control study we used Polymerase Chain Reaction Restriction Fragment Length Polymorphism (RFLP-PCR) to determine frequency of this polymorphism in 105 women with RPL in comparison with 109 healthy controls. The collected data were analyzed by the statistical package for the social sciences software (SPSS) and the chi-square test.
Results: The finding showed clear correlation between this polymorphism and RPL. The results will be presented in the congress.
Conclusion: If distinctive data increase frequency of this polymorphism in RPL, it can exponent influence of this polymorphism in generating RPL. We hope result of this investigation to be useful for finding some causes of unexplained recurrent pregnancy loss.
Key words: Recurrent pregnancy loss, HLA-E, Polymorphism.
P-105
Evaluation of the meiotic spindle and zona pellucida after vitrification of mouse MII oocytes
Akyash F, Khalili MA, Safari S, Ashourzadeh S, Agha-Rahimi A, Khoradmehr A.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: fakyash@gmail.com
Introduction: Oocyte vitrification is used as fertility preservation method for young aged females with cancer. The oocyte is one of the biggest mammalian cells with large volume of cytoplasm, compares with other cells. Also, oocyte has less permeability to cryoprotectants (CPAs), since high concentration of CPAs used in vitrification procedure with possible toxic effects. Evaluation of the effect of vitrificaiton on meiotic spindle (MS) and zona pellucida (ZP) of mouse MII oocytes using Polyscope technology was the subject of this study.
Materials and Methods: Ovulation induction performed for 6-8 weeks old NMRI mice by injection of 10 IU PMSG (IP) and 48 hr later with 10 IU HCG. After oviduct removal, COCs were denuded and MII oocytes were retrieved aseptically. The equilibration (7.5% EG, 7.5% DMSO) and vitrification (15% EG, 15% DMSO) solutions were prepared in Hams F10 supplemented with 20% HSA as base medium. For 3 steps of thawing, base medium plus 1, 0.5 and 0.25 M sucrose were used respectively. Assessment of ZP and MS performed at different time intervals after thawing.
Results: After warming, vitrified oocytes showed a significantly fast ZP digestion timing compared to the control group (p<0.05). Presence of MS was detected in 69.23% of vitrified oocytes 1 hr after warming, and presence of MS was showed in 11.11% of vitrified oocytes 2 hr after warming and 26.92% of vitrified- warmed oocytes showed sign of degeneration.
Conclusion: ZP digestion was faster in vitrified group rather than control and recovery of MS structure after vitrification/ warming at 37oC. Vitrification technique still needs more modification before its application in fertility preservation program.
Key words: Oocyte, Meiotic spindle, Zona pellucida, Vitrification.
P-106
Protective effect of vitamin E on testicular germ cells and serum Malondialdehyde concentration in rats fallowing exposure to bisphenol A
Amjadi M, Soleimani Mehranjani M, Naderi Noreini S.
Department of Biology, Faculty of Sciences, Arak University, Arak, Iran.
Email: m.amjadi1369@gmail.com
Introduction: Bisphenol-A (BPA), used in plastic industries, has toxic effects on the reproductive system. It can cause testis injury through the production of Reactive oxygen species (ROS).
Materials and Methods: 24 adult male rats (220±15 gr) were divided into 4 groups (n=6): control, BPA (250 mg/kg/day), Vit E (150 mg/kg/day) and BPA+ Vit E. All groups were orally treated for 56 days. By the end of the treatment, animals were killed, their right testis were taken out, fixed, sectioned, processed and stained with heidenhain azan method. The total number of germ and Sertoli cells was estimated using the optical dissector technique. Serum MDA levels were also measured with the spectrophotometric method. Data were statistically analyzed using one way ANOVA and Tukey's test and means were considered significantly different at p<0.05.
Results: A significant decrease in the total volume of testis, number of long and round spermatids, spermatocytes and Sertoli cells (p<0.05) and a significant increase (p<0.03) in the serum MDA level were found in rats treated by BPA compared to the control group. Histopathology observations revealed morphological changes in BPA-exposed rats including atrophy and vacuolation in the germinal epithelium. The above parameters were compensated to the control level in the BPA+ Vit E group.
Conclusion: Due to the antioxidant activity of vitamin E, it may ameliorate the damaging effects of bisphenol A on spermatogenesis.
Key words: Bisphenol A, Vitamin E, Sertoli and germ cells number, Optical disector.
P-107
Evaluation of follicular genes pattern and growth of preantral follicles after culture in alginate hydrogel following vitrification of the mouse ovarian tissue
Asgari F1, Valojerdi MR1, 2, Ebrahimi B 2.
Introduction: This study was set up to evaluate the effect of ovarian tissue vitrification on the in vitro growth and pattern of follicular genes expression in mouse preantral follicles encapsulated within alginate hydrogel.
Materials and Methods: Ovaries of 12-14 days old female NMRI mice allocated into fresh control and vitrification groups. For cryopreservation, ovaries equilibrated with a solution (ES) that composed of 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 15 min and vitrification was performed by a solution (VS) that composed of 15% EG and 15% DMSO for 30 min then ovaries loaded to nitrogen with needle. Descending concentrations of sucrose (1, 0.5, 0.25 M) used for warming step. After histologic assessment of ovaries, in the next stage, pre-antral follicles was mechanically isolated from control and vitrified ovaries and was cultured for 12 days in 0.7% sodium alginate. Preantral follicles survival rate, growth, antrum formation and relative expression of oocyte- specific genes (Bmp15, Gdf9،Fgf8, Igf1, Kit, Kit ligand) was assessed after 1, 8 and 12 days of culture and finally maturation rate of oocytes was studied.
Results: Preantral follicles in vitrified group showed a lower survival rate on 8 and 12 days of culture (p<0.05) but could retain a comparable morphological appearance, growth and antral formation with the control group. Reduction of Bmp15, Gdf9, Fgf8, Kit, Kit-l showed during 12 days of culture (p<0.05). Although the expression of Gdf9, Kit, Kit-l in vitrification group was more than control group at 1st day of culture but all genes in both groups showed same expression after 12 days of culture (p<0.05).
Conclusion: Although vitrification of ovarian tissue reduces the survival rate, it is a safe method for preservation of preantral follicles and could not modify the relative expression of follicullar genes and oocytes maturation capacity.
Key words: Follicular genes, Preantral follicle, Vitrification of ovarian tissue, Alginate.
P-108
Effects of morphine on sperm parameters, protamine deficiency and DNA integrity in mice
Bahari H1, Talebi AR1, 2, Nahangi H1, Anvari M1, 2, Abbasi Sarcheshmeh A1, Khoradmehr A2, Danafar AH2.
Introduction: Morphine as a natural alkaloid (opiate) is the most effective pain-relieving drugs and can be abused because of its high addictive potential. Opiate abuse is considered as one of the problems associated with poor semen production and sperm quality. Therefore, for the first time, this experimental study was carried out to evaluate the effect of intraperitoneal injection of morphine on sperm parameters, protamine deficiency and DNA integrity of spermatozoa aspirated from cauda epididymis of mice.
Materials and Methods: Totally 24 adult male Balb/c mice (8 weeks old. 30g) were equally divided into 3 groups each containing 8 mice. Mice of group 1 served as control fed on basal diet, group 2 received basal diet and normal saline and group 3 received basal diet and morphine (15 mg/kg/daily, intraperitoneal) for 35 days. Finally right tail of epididymis of each mouse was cut and placed in Ham’s F10 for 30 min. Released sperm were used to analyze count, motility, viability (eosin-nigrosin staining), morphology (Papanicolaou), protamine deficiency with chromomycin A3 (CMA3) and apoptosis via TUNEL assay.
Results: In morphine-treated mice a significant decrease was found in sperm viability, normal morphology, count and motility compared to other groups (p<0.05). In relation with protamine deficiency the rates of CMA3-reacted spermatozoa were similar in groups (p>0.05). In addition, in morphine-treated mice there was a significant increase in apoptosis compared to other groups (p<0.05).
Conclusion: The results showed that morphine abuse disturbs sperm parameters and DNA integrity but not protamine content of the sperm nucleus.
Key words: Mice, Morphine, Sperm parameters, Apoptosis, Protamine deficiency.
P-109
Study the protective role of jujube extract on growth index of mice fetuses exposed to Carbamazepine
Doostabadi MR1, Afshar M1, Nahangi H2, Hasanzadeh Taheri MM1, Hassanpour M1.
Introduction: Carbamazepine (CBZ) is an anticonvulsant drug that is widely used, primarily to treat seizures. One of the side effects of this medication during pregnancy is the reduced fetal growth. The jujube has been widely used in traditional medicine because of its special compound. The purpose of this study was to evaluate the preventive effects of aqueous extract of jujube on growth index of mice fetuses exposed to Carbamazepine.
Materials and Methods: In this study, 100 Balb/C mice (28-30 gr; 8-9 weeks-old) were divided into eight experimental and two control groups. Experimental groups (I, III, V) and (II, IV, VI) received daily intraperitoneal injections of 50 and 100 mg/kg of CBZ, respectively. Experimental groups (III, IV) and groups (V, VI) were gavaged doses of 200 and 400 mg/kg of aqueous extract of jujube respectively. Groups VII and VIII received only doses of 200 and 400 mg/kg of aqueous extract of jujube respectively. Control groups I and II were gavaged to normal saline and tween respectively. Injections were done on 0-15 gestational days (GD), and aqueous extract was gavaged along with CBZ starting 10 days prior to gestation. On GD 18, fetuses were removed for weighing and crown-rump measuring. Data were analyzed by ANOVA and Tukey tests using SPSS (version 18) at 0.05%.
Results: Average weight and body length of fetuses in experimental groups I and II, that received CBZ, significantly reduced compared with control groups. In the fetuses of experimental groups V (1.20±0.38) and VI (1.21±0.21 gr) that recieved jujube extract (400 mg/day) with CBZ, the mean weight of these fetuses increased meaningfully in comparison with the experimental groups I (0.93±0.19 gr) and II (0.92±0.26 gr) respectively.
Conclusion: This study showed that aqueous extracts of jujube can have preventive effects on reducing effect of CBZ on birth fetal weight.
Key words: Growth index, Jujube, Carbamazepine, Mice.
P-110
Effectiveness of mindfulness-based cognitive therapy on improvement of perceived stress and infertility in infertile women undergoing IVF irrational cognitions
Dortaj A1, Mehdizadeh A1, Dortaj H2.
Introduction: Effectiveness of mindfulness-based cognitive therapy on improvement of perceived stress and infertility in infertile women undergoing IVF irrational cognitions.
Materials and Methods: This clinical trial with pre-test post-test was done in 1331 infertile women referred to the Reproductive Health Research Center, Crescent Hospital, UAE who were treated with IVF. In total, 24 infertile women filled an irrational cognitions questionnaire related to childbearing and infertility. They were divided into experimental and control groups. The experimental group received mindfulness-based cognitive therapy for 8 sessions of 2 hr each. The control group did not receive any mental health services. Those two questionnaires were completed before and after the intervention. Data were analyzed by SPSS and p<0.05 was considered significant.
Results: There were no significant differences between the experimental and control groups in terms of reduction of perceived stress of infertility. But, the improvements in the recognition of irrational for childbearing between two groups was significantly different.
Conclusion: Teaching mindfulness-based cognitive therapy on improvement of perceived stress and irrational cognitions related to fertility in infertile women undergoing IVF treatment is effective.
Key words: Perceived stress, Infertility, IVF treatment, Mindfulness-based cognitive therapy, Irrational cognitions of parents.
P-111
Genotype and phenotype frequencies of paraoxonase 1 in fertile and infertile men
Fattahi A1, Tavilani H1, Latifi Z1, Esfahani M2, Khodadadi I2, Karimi J2,Bahrayni E2, Vatannejad A3, Vaisi-Raygani A4, Ghorbani M2.
Introduction: Paraoxonase1 (PON1) is a glycoprotein associated with high density lipoprotein and has antioxidant activity. The impact of PON1 in various stages of spermatogenesis has also been suggested.
Materials and Methods: Q192R variants of PON1 were determined in 150 fertile and 150 infertile men using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Plasma arylesterase and paraoxonase activities were detected by spectrophotometry and malondialdehyde (MDA) level was measured using thiobarbituric acid.
Results: Our results showed no significant difference in the distribution of PON1 genotypes and alleles between fertile and infertile groups. However morphology and motility of sperm were associated with various genotypes of PON1. The number of fertile males with the BB phenotype (high activity) was significantly higher than that of infertile males, whereas the number of individuals with the AB phenotype (moderate activity) was statistically higher in infertile men compared with the fertile group. Additionally, MDA and arylesterase activity levels were significantly higher in infertile subjects compared with fertile men.
Conclusion: We speculate that the low activity of PON1 can be a risk factor for male infertility probably due to a decrease in antioxidant activity of PON1 and increase in lipid peroxidation.
Key words: Genetic polymorphism, Male infertility, Paraoxonase 1, Reproduction.
P-112
Saccharin consumption increases sperm DNA fragmentation and apoptosis in mice
Rahimipour M, Talebi AR.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: marziehrahimipour@yahoo.com
Introduction: Saccharin is an artificial non-caloric sweetener that used to sweeten products such as drinks, candies, medicines, and toothpaste, but our bodies cannot metabolize it. Sodium saccharin is considered as an important factor in tumor promotion in male rats but not in humans.
Materials and Methods: Totally 14 adult male mice were divided into 2 groups. Group 1 served as control fed on basal diet and group 2 or experimental animals received distilled water containing saccharin (0.2% w/v) for 35 days. After that, the left cauda epididymis of each mouse was cut and placed in Ham’s F10. Swimmed-out spermatozoa were used to analyze count, motility, morphology (Pap-staining) and viability (eosin-Y staining). Sperm DNA integrity, as an indicator of apoptosis, was assessed by sperm chromatin dispersion (SCD) and terminal deoxynucleotidyl transferase (TUNEL) assay.
Results: Following saccharin consumption, we had a reduction in sperm motility with respect to control animals (p=0.000). In addition, the sperm count diminished (17.70±1.11 in controls vs. 12.80±2.79 in case group, p=0.003) and the rate of sperm normal morphology decreased from 77.00±6.40 in control animals into 63.85±6.81 in saccharin-treated mice (p=0.001). Also, we saw a statistically significant increase in rates of sperm DNA damage and apoptosis in experimental group when compared to control one (p=0.001, p=0.002 respectively).
Conclusion: Saccharin consumption may have negative effects on sperm parameters, and increases the rate of sperm DNA fragmentation and apoptosis in mice.
Key words: Sperm, Saccharin, Apoptosis, Mice.
P-113
Increased telomeric repeat containing RNA (TERRA) levels in cumulus cells of infertile polycystic ovary syndrome patients
Ghobadi N1, 2, Shahhoseini M2, Favaedi R2, Hassani F3, Movaghar M3, Karimian L3, Eftekhari Yazdi P3.
Introduction: One of the important reasons of anovulation based infertility is polycystic ovary syndrome (PCOS) which occurs in 5-10% of women in reproductive age. It seems that the length of telomere, TTAGGG tandem repeats, is related to proliferation and differentiation events during follicular development including the mechanisms which regulate successful reproduction. It is widely recognized that bi-directional communications exist between oocytes and the surrounding cumulus cells which are essential for the production of competent oocytes. During the past few years, study on cumulus cells showed that the length of telomere is longer in cumulus cells of mature oocytes than immature ones. Until now, telomeres have been considered to be transcriptionally silent but recent studies have clearly shown that transcription of subtelomere regions produces telomeric repeat containing RNA, named TERRA. In this study, we tried to identify the correlation of TERRA levels with oocytes maturation by evaluating of TERRA transcripts in cumulus cells of PCOS patients.
Materials and Methods: For this respect, cumulus cells were collected from 6 PCOS patients and 9 healthy women with male factor infertility through ICSI/ IVF procedure. TERRA transcripts were measured by using quantitative real- time PCR.
Results: Our data showed that the level of TERRA transcripts was increased significantly in PCOS group vs. control group (p˂0.05).
Conclusion: This finding implies a considerable association between TERRA transcript levels and PCOS, so it can be concluded that any changes in levels of TERRA transcripts can be judged as a potential marker for the quality of oocytes in ART procedure.
Key words: TERRA, Telomere, PCOS, Cumulus cells.
P-114
Epigenetic modification profile of endometrium in endometriosis patients
Hosseini E1, Afsharian P2, Ashrafi M3, 4, Ramazanali F2, Mehraein F1, Shahhoseini M2, Aflatoonian R3.
Introduction: Endometriosis defined as the presence of endometrium-like tissue outside of the uterine cavity, is a common gynecologic disorder. Although endometriosis is a multifactorial disease and the exact etiology is not clearly understood, recently, some evidence suggests that epigenetic is associated with the molecular features of endometriosis. Two main epigenetic regulatory mechanisms, DNA methylation and histone modifications regulate expression of genes and recognize the states of diseases. The aim of this study was to investigate alterations of DNA methylation and histone acetylation and methylation levels in eutopic and ectopic endometrium of endometriosis patients.
Materials and Methods: Informed consents were gained from all patients then eutopic and ectopic endometrium samples (n=5) were collected from endometriosis patients undergoing surgery and biopsy, as well as endometrial tissues from healthy fertile women (n=5) during the proliferative phase of menstrual cycle. Chromatin extracts from samples were prepared following fixation and then shearing into fragments by sonication. Nucleosome ELISA was performed on chromatin extracts, in order to identify global histone H3K9 acetylation/ methylation and DNA methylation, using antibodies against H3K9ac, H3K9me and MeCP2, respectively.
Results: We have identified global histone H3K9 hypermethylation in ectopic and eutopic endometrium, compared with controls. A significant hyperacetylation at histone H3K9 was observed in eutopic samples compared to ectopic and control groups. Furthermore, eutopic endometrial samples were globally DNA hypermethylated in comparison with controls.
Conclusion: These results clearly show an epigenetic switch in endometrial and endometriotic tissue of patients with endometriosis, in the way that aberrant DNA methylation and histone acetylation/ methylation status may play a dynamic role in occurrence of endometriosis and support the opinion that epigenetic abnormalities have causative functions in endometriosis.
Key words: Endometriosis, Endometrium, Epigenetic, Methylation, Acetylation.
P-115
The effects of chronic and acute ethanol administration on sperm chromatin parameters in mice
Hosseini A, Borzouei Z, Faramarzi A, Ghasemi-Esmailabad S, Talebi AR.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: hosseinia30@yahoo.com
Introduction: Sperm chromatin can be damaged by effects of some toxic materials. Among this, ethanol is one of the reproductive toxins that consumes abuse in men and may be associated with poor sperm quality. The purpose of this investigation was to examine the acute and chronic effects of ethanol consumption on sperm parameters and chromatin assay through testing the effects in mice.
Materials and Methods: Animals were randomly divided into 3 groups consisting of 10 mice each. Group 1 and 2 served as alcohol group and were received a daily dose of (3 g/kg body weight as 25%, v/v) ethanol by I.P. for four and eight weeks respectively. Group 3 (control group) was given normal access of food and water. The subdivisions and dosages were based on past works. At the end of treatments, laparotomy was conducted to expose the reproductive system, cauda of epididymis were immediately dissected out for evaluations of sperm parameters and various tests were used to analysis of the chromatin integrity of sperm.
Results: Various tests such as aniline blue (AB), chromomycin A3 (CMA3), toluidine blue (TB), and acridine orange (AO) were used to analysis the sperm quality. We did not find any statistically significant differences in acute and control groups. Although sperm count and motility were increased but viability, normal morphology, DNA and chromatin integrity were unaffected by ethanol in acute group and were identical with control group. Our results demonstrated that count, motility and viability of sperms were dramatically decreased in chronic treated animals. Besides of that, DNA and chromatin integrity were significantly decreased.
Conclusion: Therefore, ethanol abuse can induce abnormality in sperm structure and function, and this may be one possible cause of male infertility.
Key words: Sperm, Ethanol, Chromatin integrity.
P-116
Comparison of oral dydrogesterone with vaginal progesteronefor luteal support in IUI cycles: A randomized clinical trial (RCT)
Khosravi D1, Taheripanah R1, Khosravi R1, Taheripanah A2, Tarighat Monfared V1, Defaee S1, Mehdighalb S1, Hosseini Zijoud SM3.
Introduction: Progesterone supplementation is the first line of treatment in ovarian stimulation. This study was conduct to compare the effect of oral dydrogestrone with vaginal cyclogest on luteal phase support in intrauterine insemination (IUI) cycles.
Material and methods: This prospective, randomized, double blind study was performed in a local infertility center from May 2013-2014. It consisted of 150 infertile women younger than 35 years old undergoing ovarian stimulation for IUI cycles. They underwent ovarian stimulation with oral dydrogesterone (20 mg) as group A and vaginal cyclogest (400 mg) as group B in preparation for the IUI cycle. Clinical pregnancy and abortion rates, mid luteal progesterone (7 days after IUI) and patients' satisfaction were compared between the two groups.
Results: The mean serum progesterone levels was significantly higher in group A compared to group B (p=0.001). Pregnancy rate in group A was not statistically different from group B (p=0.58). Abortion rate in group B was higher than this rate in group A, but the difference was not statistically significant (p=0.056). Satisfaction rates were significantly higher in group A compare to group B (p<0.001).
Conclusion: We concluded that oral dydrogestrone is as effective as vaginal progesterone for luteal-phase support in woman undergoing IUI cycles. Moreover, the mean serum progesterone levels and satisfaction rates in dydrogestrone group were higher than cyclogest group.
Key words: Dydrogesterone, Cyclogest, Luteal-phase, Infertility.
P-117
The detrimental effects of alcohol on sperm parameters and DNA integrity in diabetic mice
Mangoli E1, Pourentezari M2, Talebi AR1, Rahimipour M3, Taheri F1.
Introduction: Diabetes mellitus (DM) is a chronic disease that can affect male reproductive function at multiple levels. Ethanol can suppress reproductive function and sexual behavior in laboratory animals and humans. Alcohol also is considered as one of the problems associated with poor semen production and sperm quality. The main goal of this study is to examine the effect of alcohol on sperm parameters and DNA integrity in diabetic mice.
Materials and Methods: Totally 32 adult male Syrian mice (10 weeks old, 35 gr) were divided into 4 groups, mice of group 1 served as control fed on basal diet, group 2 received streptozotocin (STZ) (200 mg/kg, single dose, intra peritoneal) and basal diet, group 3 received alcohol (10 mg/kg, water-soluble) and basal diet and group 4 received streptozotocin and alcohol. After 35 days, the cauda epididymis of each mouse was dissected and placed in 1 mL of pre-warm Ham’s F10 culture medium for 30 min. The swim-out spermatozoa were analyzed for count, motility, morphology and viability. Also the sperm chromatin quality and DNA integrity, was evaluated with Aniline blue (AB), Toluidine blue (TB), Acridine orange (AO) and Chromomycin A3 (CMA3) staining.
Results: In this study all of the sperm parameters were significantly diffrent between groups (p=0.001). Also, regarding the sperm DNA integrity tests, the results from four tests showed significant differences between groups. Also, in diabetes+ alcohol mice, a significant increase was found in mean tests compare to other groups.
Conclusion: Although the DM may have bad effects on sperm fertility potential and DNA integrity, but, on the other hands, consumption of alcohol in diabetic mice, may have detrimental effects on sperm parameters, sperm function and also sperm chromatin condensation in experimentally-induced diabetic mice.
Key words: Mice, Sperm parameters, DNA integrity, Diabetes, Alcohol.
P-118
Protective effect of Silymarin on plasma membrane integrity, acrosome integrity and mitochondrial membrane potential in cadmium-treated mice
Ali Asgarnejad P, Momeni HR.
Department of Biology, Faculty of Sciences, Arak University, Arak, Iran.
Email: asgarnejad3@gmail.com
Introduction: Cadmium as a heavy metal and environmental pollutant is able to exert numerous undesirable effects on human reproduction by inducing oxidative stress. Silymarin, a polyphenolic flavonoid extracted from the seeds of Silybum marianum, has effective antioxidant properties.
Materials and Methods: Adult NMRI mice were divided into four groups: 1) control 2) cadmium chloride (5 mg/kg, sc) 3) Silymarin (100 mg/kg, ip) and 4) Silymarin + cadmium. Treatment period was 24 hours. After treatment mice were dissected and their epididymis was cut into small pieces in HTF medium in order to swim out spermatozoa. The spermatozoa from different groups were used to evaluate sperm parameters. Sperm plasma membrane integrity, acrosome integrity and mitochondrial membrane potential assessed by Hoechst and propidium iodide, coomassie blue and rhodamin staining respectively. Data were analyzed with one way ANOVA and p<0.05 was considered significant.
Results: In cadmium-treated mice, the percentage of intact membrane and acrosome were significantly decreased compared to the control. In addition, mitochondrial membrane potential was significantly decreased in cadmium-treated mice in comparison with the control. In Silymarin + cadmium group, Silymarin could significantly compensate the adverse effects of cadmium compared to cadmium group.
Conclusion: The present study showed that cadmium induces toxic effects on mice sperm plasma membrane integrity, acrosome integrity and mitochondrial membrane potential and Silymarin can compensate the toxic effects of cadmium on these sperm parameters.
Key words: Cadmium, Silymarin, Sperm parameters.
P-119
Evaluating quality of well-being, marital adjustment and sexual dysfunction between infertile women
Mousavi Nik M1, Saberi Zafarghandi MB2, Birashk B3, Assari A4, Eshaghi Farahman SR1.
Introduction: Infertility is a stressful experience and has a high impact on the infertile women’s psychological status. Infertility and it is treatment create a major and prolonged crisis for the women and create a heavy psychological trauma for the women. The effectiveness of infertility treatment depends on success rate of the treatment facility, the emotional well-being and sexual health of the women seeking treatment.
Materials and Methods: For the propose of the study, 50 fertile and 50 infertile women were selected randomly (n=100) from Midwife IVF and Fertility Research Hospital in Mysore-India. All subjects were assessed using Quality of Well-Being Scale-Self Administered (QWB-SA), Marital Adjustment Test (MAT) and Massachusetts General Hospital Sexual Functioning Questionnaire (MGH) questionnaire were administered.
Results: The results showed significant difference between fertile and infertile women assessment on Quality of Well-Being and Marital Adjustment score. Compare with the fertile women, the patients with infertility had significantly lower scores in the sexual interest and sexual arousal domains and lower frequency of intercourse. The patients with infertility retrospectively reported an orgasm, lubrication and sexual satisfaction score that was similar to that of the fertile women.
Conclusion: Women with a diagnosis of infertility were found to be at higher risk for sexual dysfunction when compare with fertile women. Further infertile women reported poor marital adjustment and quality of well being compare with controls. The results suggest to apply counseling and psychotherapy services in the infertility centers to reduce the psychological problems, increase Quality of Well-Being, Marital Adjustment and identify the factors that can contribute to the development of sexual dysfunction on women facing infertility to help them to enhance fertility chances.
Key words: Quality of well-being, Marital adjustment, Sexual dysfunction, Infertile women.
P-120
Assessment of aerobic bacterial and fungal contaminations in liquid nitrogen tanks
Nabi A, Khalili MA, Torki AR, Fesahat F, Anbari F.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: ali.nabi67@yahoo.com
Introduction: Cryopreservation technologies play an important role in assisted reproduction for both medical and research purposes. But, there are some concerns about the technical and biological safety of this technology because of pathogen transmission during cryopreservation and germplasm banking. The aim of this study was to evaluate the presence of microbial contaminations in liquid nitrogen (LN2) and carrier tanks used in different laboratories of our reproductive science institute.
Materials and Methods: In this descriptive study, different parts of 20 carrier and storage LN2 tanks, such as LN2, vapors, cryocans, and inner surface were qualitatively evaluated for microorganism contaminations through the assessment of bacterial and fungal growth in minimal and selective Petri dishes.
Results: Two fungal (Candida albicans and aspergillus flavus) and two aerobic bacterial microorganisms (Ecoli, Pseudomonas aeruginosa) were the most common findings in bottoms, vapors, and inner surfaces of 2 carriers and 11 storage LN2 tanks. There were Pseudomonas aeruginosa growths in 8 cryocans from contaminated tanks. Also, there were staphylococcus aureus and acinetobacter baumannii contaminations in one storage tank.
Conclusion: Theoretically, LN2 tanks can be considered as a possible source of pathogen transmission. Therefore, performing quality control assays, like microorganism detection tests for LN2 and supplementary equipments should be considered as a routine practice in IVF clinics. Sterilization of tanks should be performed on regular basis.
Key words: Bacterial growth, Contamination, Liquid nitrogen.
P-121
Effect of non-polar extract of Phaleria macrocarpa on sperm quality in adult rats
Aziz Dollah M1, Parhizkar S2, Baharudin N1.
Introduction: Diminish production of testosterone hormone could affect sperm quality; hence contribute to the infertility in man.
Materials and Methods: 25 Sprague Dawley adult male rats were randomly divided into five groups. Rats were supplemented orally once a day with 0.25 ml Tween 20 solution containing non-polar hexane extract of PM at one of the following concentrations: 0, 6, 12, and 60 mg/kg for seven weeks. Commercial testosterone hormone was used as positive control. On the last day of supplementation period, all rats were sacrificed and orchidectomy technique was performed to collect semen sample from cauda epididymis of the testis. The sperm count, motility, viability and morphology were determined.
Results: The results showed that the sperm motility was significantly improved by non-polar PM extract (p<0.05) with the value of 66.98% (6 mg/kg), 63.96% (12 mg/kg) and 65.21% (60 mg/kg) compared to 44.08% for 0 mg/kg group. The supplementation of PM extract however did not improved the sperm count, sperm viability and sperm morphology. The sperm count was 852 (0 mg/kg), 918 (6 mg/kg), 906 (12 mg/kg), 1030 (60 mg/kg) and 1027 million cells/ml (commercial testosterone). The sperm viability was 85.90 (0 mg/kg), 85.26 (6 mg/kg), 85.45 (12 mg/kg), 87.88 (60 mg/kg), and 89.32% (commercial testosterone). The sperm morphology was 62.5% (0 mg/kg), 63.7% (6 mg/kg), 72.2% (12 mg/kg), 67.0% (60 mg/kg) and 64.5% (commercial testosterone). The results showed that non-polar PM extract only improved the sperm motility and did not have any effect on the sperm count, morphology and viability.
Conclusion: The study concludes that non-polar extract of PM has no compound that can improve sperm quality except on the motility of the sperm.
Key words: Phaleria macrocarpa, Sperm count, Sperm motility, Sperm viability, Sperm morphology.
P-122
Protective effect of ascorbic acid on testicular tissue in mice exposed with sodium arsenite
Pirozmanesh H, Soleimani Mehranjani M, Naderi Noreini S.
Department of Biology, Faculty of Sciences, Arak University, Arak, Iran.
Email: hp457@yahoo.com
Introduction: Arsenic is one of the major environmental contaminants with carcinogenic and toxic effects. Ascorbic acid as an antioxidant is able to restrict oxidative stress. The aim of this study was to investigate the adverse effect of sodium arsenite on the mice testicular tissue as well as to examine whether vitamin c is able to ameliorate this effect.
Materials and Methods: 24 adult male NMRI mice were randomly allocated into four groups (n=6) including control, sodium arsenite (7 mg/kg/day, orally), ascorbic acid (150 mg/kg/day, orally) and finally sodium arsenite+ ascorbic acid. Mice were treated for 35 days. At the end, mice were sacrificed and their right testis were taken out, fixed, processed and stained with Heidenhain azan method. The total volume of testis, volume of interstitial tissue, volume of seminiferous tubules, diameter and of length of seminiferous tubules, germinal epithelium height and basement membrane thickness were estimated using stereological methods. Data were analyzed using one way ANOVA and Tukey's test and means difference were considered significant at p<0.05.
Results: A significant reduction in total volume of testis, volume of seminiferous tubules and its diameter and germinal epithelium height, was found in sodium arsenite group compared to control group (p<0.04). In ascorbic acid + sodium arsenite group, the above parameters were significantly increased compared with sodium arsenite group (p<0.02). In addition, germinal epithelium height and diameter of seminiferous tubules were increased in mice treated with ascorbic acid alone compared to the control group (p<0.002).
Conclusion: The results indicate that ascorbic acid may be useful in reducing the sodium arsenite- induced toxic effects on testicular tissue.
Key words: Sodium arsenite, Ascorbic acid, Stereology, Testis.
P-123
The comparison of anxiety and depression rate between medical staff of Infertility Centers and Obstetrics and Gynecology Centers of Yazd, Shiraz, Isfahan and Kerman Hospitals
Pourmovahed Z1, Yassini SM1, Khalili MA2, Halvaei I2, Nabi A2, Ghasemi M3, Fesahat F2.
Introduction: Mental health is an important issue in personal, social and occupational function of anyone in the life; moreover, mental problems have many negative consequences on the quality of professional work. Regarding the close and continuous interaction of infertility staff with hopeless infertile couples and in the contrary the atmosphere of happiness especially in Obstetric Wards it make sense that anxiety and depression rates would be different between them.
Materials and Methods: This study was a descriptive-correlation study based on cross-sectional method. 199 individuals who were the staff of Infertility Centers and Obstetrics and Gynecology Wards in four provinces enrolled in this study through stratified sampling. Data collection was done by demographic questionnaire, Spiel Berger and Beck depression inventory tests. Data were analyzed by SPSS software and ANOVA test.
Results: The results showed that there is statistically significant higher rate of anxiety in staff of Obstetrics and Gynecology Wards in Isfahan (54.69±13.58) while there was a higher rate of depression in staff of Infertility Center in Shiraz (14.94±10.87). Overall, correlation between anxiety, depression and work place was statistically significant (p=0.047 and 0.008 respectively). According to ANOVA test, the mean value of anxiety level was higher in the staff of Obstetrics and Gynecology Centers of Isfahan, Shiraz, Shahid Sadoughi and Madar Hospitals in Yazd (p=0.03, 0.006, 0.008, 0.012 respectively) and Infertility Center of Yazd (p=0.00).
Conclusion: Comparison of the mean value of anxiety in staff of Obstetrics and Gynecology Wards and Infertility Centers was not significant in these four centers except for Isfahan Center. As long as we know that infertile couples have little chance for success rate and Obstetrics and Gynecology patients have little risk of failure in treatment it could be mentioned that the anxiety and depression in the staff are not correlated with the client illness.
Key words: Anxiety, Depression, Infertility centers, Obstetrics and gynecology centers.
P-124
Developmental competence of immature oocytes aspirated from ovarian antral follicles as a method for fertility preservation
Safian F1, Khalili MA2, Karimi Zarchi M3, Mohsenzadeh M1, Ashourzadeh S1, 4.
Introduction: Advances in the treatment of cancerous women in reproductive age have markedly been increased recently. Also, in vitro maturation (IVM) of immature oocytes collected from ovary has been proposed for fertility preservation. In addition, the morphology of oocytes post IVM is one of the factors determining its developmental competence. By using the non-invasive PolScope system, the meiotic spindle and zona pellucida (ZP) can be assessed in living oocytes.
Materials and Methods: The ovarian cortex from 26 cancer patients (21-45 years old), were obtained directly from collaborating hospitals, and transported to the IVF center on ice. 61 immature oocytes were aspirated, of which 18 (29.5%) were degenerated and discarded. The remaining 43 (70.5%) healthy oocytes were cultured for 48 hr IVM culture media. The rate of maturity was assessed and the ZP birefringence and meiosis spindle (MS) were imaged with Polscope technology.
Results: 43 immature oocytes underwent IVM technology, of which 30.2% reached viable metaphase II (MII) oocytes. The ovarian tissues of 9 (34.6%) women were lacking oocytes at any stage. There was a positive correlation between the recovered number of oocytes and the ovarian volume. During polarized light microscopy examination, MS could be visualized only in one of the MII oocytes, but high ZP birefringences were observed in most of the oocytes post IVM (61.5%).
Conclusion: Oocytes maturation post IVM from unstimulated ovaries showed a good developmental competence in cancerous patients. Further studies should be performed to advance the oocyte maturation program, such as co-culture system, for fertility preservation.
Key words: Fertility preservation, Ovarian tissue, IVM, Polscope.
P-125
Effect of Heracleum persicumm oil and alcoholic extracts on sperm parameters and chromatin quality in mice
Taghizabet N1, Mangoli E2, Anbari F2, Talebi AR2, Masoodi A1.
Introduction: Seminal plasma from infertile men has lower antioxidant levels than that of fertile men, particularly of patients whose semen have poor sperm motility. Evaluation of the importance and the effects of plant derived drugs on fertility of laboratory animals have long been recognized. Antioxidant activity has been reported from Heracleum persicum (Golpar).
Materials and Methods: 80 adult male mice (10 weeks old, 35 gr) were divided to 3 groups: group1 received hydroalcoholic extract (1000 mg/kg, ip), group 2 received oil extract (200 ml/kg, ip) and group 3 serving as the sham control group that received water. After 35 days, the cauda epididymis of each mouse was dissected and placed in 1 mL of pre-warm Ham’s F10 culture medium for 30 min. The swim-out spermatozoa were analyzed for count, motility, morphology and viability. The sperm chromatin quality and DNA integrity, was evaluated with Aniline blue (AB), Toluidine blue (TB), Acridine orange (AO) and Chromomycin A3 (CMA3) staining.
Results: In sperm analysis, progressive and non-progressive motility were significantly differences between groups. Regard to sperm chromatin quality, the results of TB and AO tests showed statically significant differences between groups, but in AB and CMA3 staining, we didn’t see any differences between them. So we can say that the Heracleum persicum extracts doesn’t have any detrimental effects on histone-protamines replacement during the testicular phase of sperm chromatin packaging.
Conclusion: According to our results, Heracleum persicum extracts as an antioxidant although improved sperm motility but may influence the male fertility potential via affecting DNA/ chromatin quality in mice.
Key words: Heracleum persicum extracts, Sperm, Mice, Chromatin.
P-126
Effect of vitrification on high magnification morphology, chromatin condensation, and fertility potential of human spermatozoa
Taherzadeh S2, Khalili MA1, 2, Anvari M1, 2, Ghazali Sh1, Agha-Rahimi A1, Anbari F1, Ghasemi-Esmailabad S1.
Introduction: Sperm vitrification is a technique of ice- and CPA- free cryopreservation by direct plunging of a sperm suspension into liquid nitrogen (LN). Sperm characteristics of motility and morphology are important for normal spermatozoa-oocyte interaction. However, defects on chromatin condensation can cause vacuolization on sperm morphology. Motile sperm organelle morphology examination (MSOME) is an unstained real time high magnification that analysis viable sperm morphology. This study investigated the influence of vitrification on human sperm structure by MSOME technique, and fertility potential by zona binding assay (ZBA) and chromatin condensation by toluidine blue (TB) and aniline blue (AB) assessment.
Materials and Methods: 30 normozoospermic ejaculates were prepared by swim up technique, and supernatants were divided into two parts: fresh and vitrified groups. For vitrification, sperm suspension was mixed with equal volume (1:1) of Hams F10 +5% HSA +0.5 M sucrose. Then, 30 µl sperm suspension was dropped into LN. Warming was performed by quick submerging spheres into pre-warmed 5ml Hams F10 with 5% HSA at 37˚C. Sperm motility, stained morphology, MSOME and ZBA were evaluated for each sample. Three classes were considered for MSOME analysis: high quality sperm with a score of 4-6 (Class 1); medium-quality sperm with a score of 1-3 (Class 2); low-quality sperm with a score of 0 (Class 3). 2×10⁶ spermatozoa in each 25µl droplet containing 4 Oocytes were performed for ZBA. In addition, samples were fixed for TB and AB assessments.
Results: Cryopreservation significantly reduced both progressive motility and normal morphology of spermatozoa. There was no significant differences between the rates of MSOME in class 1 (14.93±14.66 vs. 13.56±11.34), class 2 (53.53±13.99 vs. 55.63±12.16), class 3 (31±20.81 vs. 30.80±16.03) pre and post vitrification, respectively. However, vitrification reduced the fertility potential of spermatozoa from normozoospermic samples (13.40±22.73 vs. 9.00±13.87) and chromatin condensation (TB: 60.32±16.60 vs. 57.28±17.19) (AB: 38.32±8.14 vs. 34.35±6.87).
Conclusion: Vitrification had adverse effects on sperm parameters of motility and morphology. However, this technique did not increase the rate of vacuolization of sperm head or severe alteration in fertility potential. Since, the majority of human spermatozoa contained vacuolization in head region, it is highly recommended to use MSOME technology for assessment of sperm fine morphology during clinical microinjection procedure.
Key words: Sperm vitrification, Motile sperm organelle morphology examination (MSOME), Zona binding assay (ZBA).
P-127
Vitamin C attenuates detrimental effects of diabetes mellitus on sperm parameters, chromatin quality and rate of apoptosis in mice
Talebi AR1, Mangoli E1, Nahangi H2, Anvari M2, Pourentezari M2, Halvaei I1.
Introduction: Diabetes Mellitus (DM) may affect male reproductive functions at multiple levels. It is shown that intake of antioxidants such as vitamin C and E can reinforce the stability of testicular blood barrier and protect sperm DNA from oxidative stress.The main goal was to examine the protective effects of vitamin C on sperm parameters, sperm chromatin condensation and apoptosis in experimentally-induced diabetic mice.
Materials and Methods: 28 adult Syrian mice were divided into 4 groups. In group 1, the mice were diabetic that received a single dose of Streptozocin (STZ) (200 mg/kg) intra-peritoneally (ip). Group 2 included diabetic mice that received vitamin C (10 mg/kg/daily, ip). Mice in group 3 received vitamin C and group 4 was considered as control. After 35 days, sperm analysis was done accordingly. To asses sperm chromatin and DNA quality, we used aniline blue (AB), toluidine blue (TB), chromomycin A3 (CMA3), acridine orange (AO) and terminal transferase mediated deoxyuridine triphosphate biotin end labeling (TUNEL) tests.
Results: All of the sperm parameters (count, motility, morphology and viability) had significant reduction in diabetic mice but, the data showed a significant increase in all of the sperm parameters in diabetic + vitamin C when compared with diabetic and control animals (p<0.05). There were significant differences (p<0.001) between groups regarding TB staining (48.8±5.92 vs. 34.3±4.13), AO test (35.9±6.11 vs. 20.8±2.89) and TUNEL test (39.42±7.18 vs. 22.00±3.65) in diabetic and diabetic + vitamin C groups, respectively. Nevertheless, in CMA3 and AB staining assays, there were not any significant differences between different groups.
Conclusion: Vitamin C, as a potent antioxidant, can attenuate detrimental effects of diabetes mellitus on the sperm parameters, chromatin quality and apoptosis in an experimental model.
Key words: Sperm, Diabetes mellitus, Mice, Vitamin C, Chromatin, Apoptosis.
P-128
DNA fragmentation in mammalian spermatozoa and its relationship with male infertility
Vaseghi-Dodaran H1, Daghighkia H1, Atshan M2, Sobhani A3, Sobhani AGH4.
Introduction: It has been suggested that altered nuclear chromatin structure or damaged DNA in spermatozoa is implicated as a possible cause of increased infertility in males. Since the first reports on sperm DNA integrity, this subject has become the focus of numerous studies. Up to 8% of infertile men have been shown to have high levels of sperm DNA fragmentation despite a normal semen analysis. Currently, there are eight major tests of sperm DNA fragmentation, including the comet assay, sperm chromatin structure assay (SCSA), acridine orange test (AOT), tritium-labeled 3H-actinomycin D (3H-AMD) incorporation assay, terminal TdT-mediated dUTP-nick-end labeling (TUNEL) assay, in-situ nick translation (ISNT) assay, DNA breakage detection-fluorescence in-situ hybridizations (DBD-FISH) assay and sperm chromatin dispersion (SCD) test. New studies suggest that sperm with certain levels of DNA fragmentation serve as a strong predictor of reduced male fertility. Diagnosis of the fertilizing ability of a semen sample is important for consistently high reproductive efficiency. This paper aimed at investigating the potential use of sperm DNA fragmentation (SDF) to improve the routine screening of male infertility.
Materials and Methods: This article discusses some of the current techniques employed for evaluating chromatin structure or DNA damage in spermatozoa. A review of recent bibliography was carried out in PubMed, Google scholar and SID by the use of relevant keywords, in order to evaluate the possible correlation between the conventional seminal parameters and sperm DNA fragmentation assessment as diagnostic tools in male infertility evaluation.
Results: A negative correlation was found between sperm characteristics and the proportion of sperm showing DNA fragmentation. For fragmentation >30%, a significant decrease of the fertilization rate was observed.
Conclusion: The proportion of sperm with DNA fragmentation appears to be potentially useful as a predictor of infertility. Sperm DNA fragmentation is a parameter worth integrating in routine clinical practice. However, additional large scale studies based on the optimization of sperm DNA integrity are needed.
Keywords: Spermatozoa, Male, DNA damage, Infertility.
P-129
Establishment of spermatogonial stem cell line in mouse testicular culture
Azizi H1, Amirian M2, Skutella Th2.
Introduction: In the seminiferous tubules of the testis spermatogonial stem cells (SSCs), Sertoli cells and differentiating cells during spermatogonesis are present. Materials and Methods: Here we report the establishment of a new VASA/ DAZL/ PLZF/-, Sox9/ Nanog/ GFRa1 low and KLF4, SOX2, VIMENTIN, N-MYC, OCT4+ cell line both from neonate and old mice, which could be expanded on SNL feeders for more than a year.
Results: Unlike undifferentiated SSCs they have a smaller nuclear/cytoplasm ratio in EM. FACS analysis showed the expression of CD49, CD29, CD9, GFRa1 and E-CAD. In Fluidigm analysis the cells expressed pluripotency markers OCT4, NANOG, SOX2 and KLF4 but only partially expressed the typical germ cell profile of SSCs. One month after transplantation in busulphan treated NOD SCID mice we observed localization of GFP labelled cells in the basal compartment of the seminiferous tubule.
Conclusion: These more differentiated SSCs could provide an ideal cell system for studying both pluripotency and in vitro differentiation of SSCs to sperm and also provide a new strategy for isolation of SSCs from neonate and old mice by morphology based selection.
Key words: Germ cells, Spermatogonial stem cells, Testicular culture.
P-130
The association between TNF-alpha -308 polymorphism and susceptibility to spermatogenic failure
Khademi Bami M1, Dehghan Tezerjani M2, Vahidi Mehrjardi MY2, Ashrafzadeh Mehrjardi HR1, Ghasemi-Esmailabad S2, Montazeri F2, SabbaghNejad Yazd S2, Shadram Y2, Golzade M2, Karimi R2, Sheikhha MH2, Kalantar SM2.
Introduction: Approximately half of infertility reasons are related to male factors among which genetic etiology is the main cause. Signaling molecules can regulate spermatogenesis during regulation of germ cells maturation. Tumor necrosis factor-α (TNF-α) is one of the prominent cytokines that supports signaling molecules and it can affect spermatogenesis.
Materials and Methods: This case-control study includes 50 azoospermic men who referred to Yazd Research and Clinical Center for Infertility and 50 healthy controls. After sperm analysis, DNA was extracted and Restriction Fragment Length Polymorphic-Polymerase Chain Reaction (RFLP-PCR) was carried out for TNF alpha -308 polymorphism.
Results: The frequencies of A allele and G allele were 58% and 42% in azoospermic group, and 34% and 67% among controls, respectively. Among azoospermic patients, 42% presented AA homozygous genotype, 32% AG heterozygous genotype, and 26% GG homozygous genotype. In the control group, 20% presented AA homozygous genotype, 28% AG heterozygous genotype, and 52% GG homozygous genotype. According to our findings, A allele (p=0.001, OR=2.681) and AA genotype (p=0.030, OR=2.89) have a positive association with spermatogenic failure in the cases.
Conclusion: Our data suggest that the polymorphism might be associated with the risk of spernatogenic failure in Iranian azoospermic and it can be used as an infertility marker for screening as well as further treatment.
Key words: Azoospermic, Cytokines, TNF-α, Polymorphism, RFLP-PCR.
P-131
The role of FSH receptor gene alterations in poor ovarian response to gonadotropins
Masoudi N1, Zari Moradi S1, Mohseni Meybodi A1, Mansouri Z1, Totonchi M1, Mirghavam-Aldin N2, Ashrafi M2, Gourabi H1.
Introduction: Poor ovarian response is an infertility disorder in which women’s ovaries don’t have proper response to gonadotropins. Follicle stimulating hormone (FSH) has a critical role in the maturation of the ovarian follicles from the antral to the graffian stage. FSH will start a signaling cascade in the granulosa cells after sitting on its receptor (FSHR). Alteration of this receptor may change follicle maturation and therefore result in improper response to gonadotropins. We investigated the association of FSH receptor gene alteration in ovarian response of poor responder patients.
Materials and Methods: The presence of P.Ala307Thr, P.Ser680Ala, P.Ala665Thr and Mut.Val341Ala were analyzed in a case control study. 70 Iranian poor responder patients were selected as the case group. 60 Iranian fertile women were enrolled as the control group. The patients DNA were extracted from their peripheral blood and amplified by relevant primers. For determining allelic variant status all PCR products were analyzed by Sequencing.
Results: The results showed that the homozygous Ser680 and Ala307 variants seem not to be significantly associated with poor response to gonadotropins. The FSHR P. Ala665Thr genotype frequency was similar in all patients and controls. The number of oocytes retrieved was comparable between patients with different FSHR genotype.
Conclusion: Although data are accumulating with evidence suggesting that the ovarian response to gonadotropins may be mediated by different genetic alterations, the optimal biomarkers and the efficacy of the tests still remain to be evaluated.
Key words: Female infertility, Poor ovarian response, Follicle stimulating hormone receptor (FSHR).
P-132
A review of studies about vascular endothelial growth factor gene polymorphisms as genetic cause of infertility
Nabat E1, Farahzadi R2.
Introduction: Infertility is defined as an inability of couples to achieve pregnancy after one year of regular, non-contraception intercourse. Diverse factors such as environmental, immunological, endocrine and genetic predispositions are thought to be involved in the development of the infertility. Infertility may be due to male or female factors or both. Causes of female infertility may include ovulation disorders, uterine abnormalities and fallopian tube blockage. Recurrent miscarriage (RM) and endometriosis are the common cause of infertility in female. Genetic factors can play an important role in susceptibility to RM and endometriosis, such as polymorphisms in vascular endothelial growth factor gene (VEGF). Recently researchers have found that endothelial dysfunction and placental ischemia/hypoxia are important factors in miscarriage. VEGF plays a pivotal role in the formation of blood supply of the fetus through the placenta and development of endometriosis. Many recent studies have indicated that a reduced VEGF serum concentration was associated with increased risk of RM and endometriosis. Therefore, the aim of this study was to investigate the association of VEGF polymorphisms such as +405 G/C, -460 C/T in 5’-UTR, +936 C/T in 3’-UTR and -1154 G/A in promoter region, with RM and endometriosis.
Materials and Methods: This review is focused on the most recent laboratory and clinical findings to investigate the association of VEGF polymorphisms with RM and endometriosis.
Results: -1154A and +93 6T alleles are increased in RM, while only in the study of Hsieh et al +405 C/C and -460 T/T genotypes are highest in patients suffering from endometriosis.
Conclusion: According to many functional studies, A allele of -1154G/A, T allele of +936C/T, C/C genotype of +405 G/C and T/T genotype of -460 C/T are associated with reduced VEGF transcriptional levels. These could lead to inadequate angiogenesis thus, lower endometrial vascularization, impaired placentation and subsequent predispose to RM and risk of endometriosis.
Key words: Infertility, Endometriosis, Recurrent miscarriage, Polymorphism, VEGF.
P-133
Association study of GSTO1 gene polymorphism (E155del) among patients with recurrent miscarriage from NorthWest of Iran
Yasari S1, Bonyadi M1, Taghavi S2.
Introduction: Recurrent miscarriage (RM) is the occurrence of three or more consecutive pregnancies that end in miscarriage of the fetus before viability. About 1-5% of couples trying to conceive are affected by recurrent miscarriage. RM is considered as a multi-factorial disease and nearly half of RM cases cannot currently be explained clinically. Several studies have indicated that genetic polymorphisms could be factors for causing susceptibility of RM with unknown etiology. GSTO1 protein has a crucial role in detoxification metabolism and it is believed that GSTO1 gene could be involved in RM. In this study the possible association of GSTO1 gene polymorphism [E155del (rs11509437)] with recurrent miscarriage in NorthWest of Iran was evaluated.
Materials and Methods: In this case-control study, forty-eight patients with a history of RM and forty-eight referent women, who had at least two live births without other pregnancy complications, were included. Glutathione S-transferase Omega 1 (GSTO1) gene polymorphism (E155del) was screened using polymerase chain reaction with confronting two-pair primers (PCR-CTPP). Subsequently, PCR products were electrophoresed in 2% agarose gel and visualized by ethidium bromide staining.
Results: This study revealed that there was a significant association with the allelic frequency of del155 between the patient and control groups (p=0.04). However, analysis of genotype’s frequencies between patient and control showed no significant association (p=0.14).
Conclusion: Although some studies have indicated that the genetic polymorphisms of detoxification genes, encoding related enzymes, could be involved in RM diseases, current study showed the weak association of GSTO1 gene polymorphism (E155del) with susceptibility to develop RM in this population.
Key words: NorthWest of Iran, Recurrent miscarriage, GSTO1 gene, E155del polymorphism, Cellular detoxification.
P-134
Effects of in vitro maturation (IVM) on epigenetic changes in oocytes and early embryos
Allahveisi A1, Rezaei MJ2, Nikkhoo B3, Yousefian E4.
Introduction: In vitro maturation (IVM) of oocyte is an effective technique for avoiding ovarian hyperstimulation syndrome in patients with polycystic ovaries (PCOS) during in vitro fertilization (IVF). Pervious study indicated that more than 1000 children have been born from IVM technique, especially in patient with PCOS. However, IVM remains questions even today. Epigenetic reprogramming occurs in the periods of gametogenesis and embryogenesis, which regulates the gene activity without alteration of DNA sequences. This review discuss about effects of in vitro maturation (IVM) on epigenetic changes in oocytes and early embryos.
Material and methods: This article presents result of a systematic review about effects of in vitro maturation (IVM) on epigenetic changes in oocytes and early embryos.
Results: Different studies showed that IVM down regulated the protein expression of enzymes controlling histone acetylation such as histone acetyltransferase GCN5 (GCN5) and histone deacetylase 1 (HDAC1), as well as their common target, acetyl-histone H3 (Ac-H3), in metaphase II (MII) oocytes and two-cell embryos. The significantly decreased HDAC1 mRNA levels in oocytes and early embryos from IVM strongly suggested that IVM technique down regulated the transcription of HDAC1 gene in oocytes before and after fertilization.
Conclusion: Our studies indicated that IVM could affect the protein and gene expression level related to histone acetylation in oocytes and early embryos. Therefore, the impacts of IVM on epigenetic changes may be reasons for the lower rates of fertilization and early embryos in IVM.
Key words: IVM, Oocyte, Epigenetic.
P-135
Association of 14-bp insertion/deletion polymorphism of HLA-G gene with idiopathic recurrent miscarriage
Arjmand F.
Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: fateme.arjmand@yahoo.com
Introduction: HLA-G is supposed to play a pivotal role in tolerance of the semi-allogeneic graft in pregnancy by inhibiting the cytotoxic functions of T- and NK cells. A 14-bp insertion/deletion polymorphism in exon-8 has a possible role in HLA-G expression.
Materials and Methods: Genomic DNA from 200 recurrent miscarriage (RM) patients and 200 normal fertile control individuals were isolated using the routine salting out method. Exon-8 of HLA-G gene of the two groups were amplified using polymerase chain reaction and analyzed by electrophoresis on 10% non-denaturing polyacrylamide gel electrophoresis containing ethidium bromide and visualized under ultraviolet light. Statistical analysis used SPSS 19 software. HLA-G allele frequencies and genotypes in RM women and the fertile control group were compared using a Chi-square test.
Results: There was a difference in allelic frequencies of 14-bp insertion polymorphism between fertile controls and RM patients; frequency of +14/-14 bp heterozygotes was significantly increased in RM patients as compared with fertile controls.
Conclusion: A 14-bp insertion/deletion polymorphism in exon 8 plays a possible role in HLA-G expression in certain cases of recurrent miscarriage. However, additional studies are needed in this regard.
Key words: 14-bp insertion, Deletion polymorphism, HLA-G, Recurrent miscarriage.
P-136
Mutation analysis of exone 2 of Tnp2 gene in varicocele patients
Danafar AH1, Heidari MM2, Khatami M2.
Introduction: Varicocele is one of the main causes of male infertility that caused by inflammation of the veins of the pampiniform plexus. Several studies have given great evidences that revealed the relationship between sperm DNA damage and varicocele. Because of the crucial role of Transition Nuclear Proteins (TNPs) and Protamines in sperm DNA condensation and integrity, the mutations in these genes can increase the risk of sperm DNA damage and infertility in varicocele condition.
Materials and Methods: DNA was extracted from total blood of 78 infertile patients with varicocele and 75 fertile control men for PCR amplification and SSCP analysis. DNA from samples with altered band pattern in the SSCP was then sequenced to search for mutations.
Results: The results of sequencing showed one variant at position IVS1-26G>C (rs8043625) in the intronic region of this gene. Comparison of the genotypes between cases and controls showed significant differences in frequencies of GG and CC (p=0.002, p=0.01), but not in GC genotype of this polymorphism (p=0.41). Also it was found that varicocele risk in men who have the CC and GC genotypes is respectively 3.07 and 1.37 fold higher than those who don't have these genotypes (OR=3.07, OR=1.37).
Conclusion: High conservation of this SNP position during evolution can represent the effects of this nucleotide in some important processes associated with the expression of this gene like mRNA splicing; but the exact mechanism is not clear.
Key words: Varicocele, Infertility, Tnp2, SSCP.
P-137
Assessing the polymorphism frequency rate of insulin receptor gene in PCOS
Khazamipour A1, Nejatizade A1, Kalantar SM2.
Introduction: Polycystic ovarian syndrome (PCOS) is a common endocrine disorder with prevalence of 5-10% globally among women of reproductive age. PCOS is being characterized by chronic anovulation, polycystic ovaries, hyperandrogenism, hirsutism, overweight, insulin resistance and infertility. It is well-known that PCOS is a complex trait like type-2 diabetes where both genetic and environmental factors play a crucial role in pathogenesis of the disease. Some type-2 diabetes susceptibility genes including those for insulin secretion and action such as insulin receptor showed considerable contribution to genetic predisposition of PCOS. Since insulin receptor gene seems to be a strong candidate gene to PCOS, we aimed to investigate the role of insulin receptor gene polymorphism (T/C) located in a transcription enhancer element of the gene to the disease susceptibility.
Materials and Methods: We carried out a cross-sectional case-control study. Using simple random sampling 90 healthy women were selected. All 90 patients fulfilled the 2003 Rotterdam criteria of PCOS. The subjects were genotyped for insulin receptor gene polymorphism (T/C) using PCR-Sequencing. Differences in genotype distributions between case and control subjects were examined via a chi-square test.
Results: In the present study 180 women between 18-40 years old age were assessed. Clinical, biochemical and metabolic characters of women were compared. Among them, obesity (Waist round more than 85 centimeters), hirsutism and irregular menarche (monthly cycles more than 35 days), LH/FSH >2, BMI>25, (mean: 28.19) in the PCOS group were significantly higher than control group (p<0.05). There was no significant associated between two groups for diabetes mellitus type II, hypertension (>140/90 mmHg) and hormonal level (TSH >5 and prolactin >29.2). Similarly in PCOS and control group, 95% of women had TT genotype and 2.2% had CC genotype. Frequency of C allele in PCOS and control group were 3.3% and 2.2% respectively. Frequency of TC genotype in PCOS and control group were 2.2% and 4.4% respectively. Noted polymorphism had no significant difference between PCOS and control women (p=0.99).
Conclusion: Although PCOS showed high prevalence among women in Bandar Abbas, Iran, insulin receptor gene polymorphism (T/C) didnot show significant influence on susceptibility to PCOS.
Key words: Polymorphism, Insulin receptor gene, Polycystic ovarian syndrome.
P-138
Association of SNP rs.2414096 of CYP19 gene with polycystic ovarian syndrome in Iranian women
Mehdizadeh A, Kalantar SM, Sheikhha MH.
Department of Genetics, International Campus, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: anahita.mz@gmail.com
Introduction: Polycystic ovarian syndrome (PCOS) is the most common endocrinologic disorder of women in their reproductive age. Major characteristics of PCOS are clinical and/ or biochemical hyperandrogenism, ovulatory dysfunction and polycystic ovaries. The etiology of PCOS is unknown, however, it has been suggested that genetic factors play major role in development of PCOS. Of those the ones implicating in metabolism of androgens are important as they are impaired in PCOS. CYP19 gene encodes aromatase which has a crucial role in androgen synthesis and variants of this gene might implicate in the pathogenesis of PCOS. Investigations have indicated the role of single nucleotide polymorphism of CYP19 in hyperandrogenism and PCOS in some racial groups. There is no data on the variants of this gene in PCOS population in Iran.
Materials and Methods: In a case-control study 70 PCOS women and 70 normal controls were selected. Following informed consent, 5 ml blood was taken from each woman of which genomic DNA was extracted by salting out method. Then a set of PCR reactions for CYP19 gene was carried out using specific primers for SNP rs.2414096 followed by a subsequent enzyme digest (RFLP) with HSP92II.
Results: Genotype frequencies of SNP rs.2414096 in PCOS women were as follows: AA (14.4%), AG (44.3%) and GG (41.4%) while in normal group, these genotypes were 24.3%, 52.9% and 22.9%, respectively. While allele frequencies in PCOS group were 49.3% for A and 50.7% for G, normal group had a different percentage of A (36.4%) and G (63.6%). The calculations for both genotype and allele frequencies showed statistical significance.
Conclusion: Variants of SNP rs.2414096 in CYP19 could play a role in development of PCOS in Iranian women.
Key words: Polycystic ovarian syndrome, CYP19, Single nucleotide polymorphism.
P-139
Exposure to tertiary-butyl hydroperoxide (TBHP)- down regulates the expression of Ddx3y in mature mice testis
Mokhtari P, Fatemi N, Golkar Narenji A, Mohseni Meybodi A, Gourabi H.
Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email:gourabi@royaninstitute.org
Introduction: Approximately 40% of infertility is associated with male factors. Two major causative factors of male infertility are oxidative stress (OS) and genetic factors. OS damages the sperm plasma membrane, the genome integrity and alter the expression profile of genes involved in spermatogenesis. Ddx3y gene is one of the important azoospermia factor (AZF) genes in Y chromosome. AZF deletion causes a severe block in spermatogenesis which affects the proliferation of spermatogonia, and consequently male infertility. The expression profile of Ddx3y gene was evaluated in testis tissues of Balb/c mice after OS induction.
Materials and Methods: A model of oxidative stress in adult male Balb/c mice testis by injection of the 1:10 concentration of tertiary-butyl hydroperoxide (TBHP) was created. Case group included treated mice by TBHP for 2 weeks and control group treated only by injection of dH2O. Induced ROS levels in testes tissue samples of all mice were measured by flow-cytometry. Consequently the expression of Ddx3y gene was quantitatively measured in samples of both groups by real-time PCR.
Results: According to flow-cytometry results, an increase of oxidative stress in TBHP treated mice in comparison to control group was observed. The gene expression of Ddx3y in testis was significantly down regulated in OS-exposed and ROS induced mice.
Conclusion: Our results indicated that Ddx3y may be a major target gene of OS and the down regulated expression of Ddx3y can be closely related to male reproductive toxicity induced by TBHP.
Key words: Male infertility, Oxidative stress, TBHP, Ddx3y.
P-140
Association of +331G/A polymorphism and differential expression of PR-B isoform in endometriosis
Mousazadeh S1, Afsharian P1, Shahhoseini B1, Aflatoonian R2.
Introduction: It is well accepted that endometriosis is a progesterone resistance disease. The effects of progesterone are mediated by its two progesterone receptor (PR) isoforms, namely PR-A and PR-B. These isoforms are functionally different. The most widely studied polymorphism in the promoter region of PGR is the G to A substitution at position +331 (rs10895068). Recently, many studies have investigated the role of +331G/A polymorphism in the etiology of various types of cancers.
Materials and Methods: Blood samples were recruited from 98 women undergoing laparoscopy for endometriosis and 102 healthy fertile women at Royan Institute, Tehran, Iran in 2013-2014. After DNA extraction, allele and genotype frequencies were determined by PCR-RFLP. Then, RNA was extracted from selected eutopic tissue samples of endometriosis patients. Analysis of PR-B mRNA expression was performed using Real-time PCR.
Results:Our data showed the frequency distribution of GG, G/A and AA genotypes in +331G/A polymorphism was 98.04%, 1.96% and 0.0% in patients and 97.96%, 2.04% and 0.0% in control groups respectively (p=0.968). Although our data didn’t show any significant association with +331G/A in our groups, however, we were able to demonstrate higher expression level of PR-B in patients with G/A compared to patients with GG genotypes.
Conclusion: Many studies have been shown that the expression level of PR-B reduced severely during endometriosis, which can affect the function of progesterone. Our findings support this observation, patients with G/A genotypes have high expression level of PR-B compared to patients with GG genotypes. Therefore, +331G/A have been found to lead increased transcriptional activity of PR-B in patients by favoring G/A or AA genotypes that may be able to influence the function of progesterone and reduce the susceptibility and symptoms of endometriosis.
Key words: Endometriosis, PGR, Polymorphism.
P-141
Association between GSTT1 null mutation and endometriosis in an Iranian population
Rastegar M1, Seifati SM1, Sheikhha MH2.
Introduction: Endometriosis is a common gynecological disease that involves growth of endometrial tissue outside its normal location. It seems that genetic factors have an important role in the pathophysiology of endometriosis. In addition, there are reports suggesting a role for environmental pollutants in developing endometriosis. GSTTl is one of the phase II enzymes involved in detoxification of some environmental carcinogens.
Materials and Methods: Genomic DNA from 95 patients with endometriosis and 141 healthy controls was collected. GSTT1 null genotyping was performed using polymerase chain reaction (PCR) method. Results: Frequency of GSTT1 null deletion in both patients and controls was 22%, which shows no significant difference between two groups (p=0.983).
Conclusion: These results suggest that GSTT1 null mutation is not associated with endometriosis in our population.
Key words: Endometriosis, Glutathione s-transfers, GSTT1.
P-142
The low frequency of breast and ovary cancer protective allele (D302H) of CASP8 gene among Iranian patients
Rasti A1, Farashahi Yazd E2, Shiryazdi SM3, Nematzade H4, Rastegar M1, Sheikhha MH1.
Introduction: Suppression of apoptosis is one of the major mechanisms underlying the origin and progression of cancer. The CASP8 plays a key role in the initiation of apoptosis. Previous case-control studies have indicated that the D302H genotype of CASP8 is associated with a reduced risk of breast and ovarian cancer. However, the frequency of this polymorphism in the Iranian people has not been reported yet. Therefore, this polymorphism was genotyped among patients with breast cancer and healthy women.
Materials and Methods: PCR-RFLP technique was used to genotyping of CASP8 D302H polymorphism among 100 female patients with breast cancer and 100 healthy women in this case-control study. Fisher's exact test and SPSS software were used to analyze the data.
Results: The mean age of patients and healthy groups was 51.37±1.27 and 52.57±1.21 years respectively. The frequencies of the GG and GC genotypes in case group were 91% and 9%, respectively, and among the control group, the frequencies were 97% and 3%, respectively )p=0.2). CC genotype was not found in any of the groups. Furthermore, the C allele frequency was 4.5% among the cases and 1.5% among the control (p=0.14). There was no significant difference between cases and controls. In total, the frequency of the C allele was observed in about 3% of people in our study (breast cancer patients and healthy women).
Conclusion: Previous case-control studies have indicated that the D302H genotype of CASP8 was associated with a reduced risk of breast and ovary cancer. In the present study the frequency of allele C was detected 3%. Indeed, the frequency of C allele in Iranian study population is less than one-fourth the frequency of this allele in Caucasian populations (13.29%). According to the low frequency of this allele in Iranian population and the protective effect of allele C, one of the conclusions that can be raised is that the Iranian population is more prone to breast and ovarian cancer than Caucasian populations in relation to this polymorphism. Therefore further studies would be needed with appropriate sample size to investigate this matter.
Key words: CASP8 D302H, Polymorphism, Apoptosis, Breast cancer.
P-143
“Cell-free fetal DNA” a novel promising biomarker for prenatal diagnosis and complicated pregnancies
Sadeghi F1, Khalaj kondori M1, Sadeghi M2.
Introduction: Cell free fetal DNA (cff DNA) is a novel promising molecular biomarker that has been applied in various aspects of obstetrical research, notably in prenatal diagnosis and complicated pregnancies.
Materials and Methods: Data base was browsed using key words “cell-free fetal DNA” and “noninvasive prenatal diagnosis” for obtaining related reports. Articles were screened for relatedness and the recent progresses in the field.
Results: Cell-free fetal nucleic acids can be detected in the maternal circulation during pregnancy, potentially offering an excellent method for early non-invasive prenatal diagnosis (NIPD) of the genetic status of a fetus. Using molecular techniques, fetal DNA and RNA can be detected from 5 weeks gestation and are rapidly cleared from the circulation following birth. Cell-free fetal DNA comprises only 3-6% of the total circulating cell-free DNA, therefore diagnoses are primarily limited to those caused by paternally inherited sequences as well as conditions that can be inferred by the unique gene expression patterns in the fetus and placenta. Broadly, the potential applications of this technology fall into two categories: first, high genetic risk families with inheritable monogenic diseases, including sex determination in cases at risk of X-linked diseases and detection of specific paternally inherited single gene disorders; and second, routine antenatal care offered to all pregnant women, including prenatal screening/diagnosis for aneuploidy, particularly Down syndrome (DS), and diagnosis of Rhesus factor status in RhD negative women. Already sex determination and Rhesus factor diagnosis are nearing translation into clinical practice for high-risk individuals.
Conclusion: In conclusion, the discovery of Cell free fetal DNA (cff DNA) has revolutionized the field of non-invasive prenatal diagnosis (NIPD) and has opened a new avenue in the field of obstetrical research and may in future form part of national antenatal screening programmes for DS and other common genetic disorders.
Key words: Cell free fetal DNA, Noninvasive prenatal testing (NIPT), Prenatal diagnosis, Pregnancy complications.
P-144
Identification of a time window for spontaneous establishment of pluripotency in mouse spermatogonial stem cells in vitro
Azizi H1, Conrad S2, Hajizadeh Moghaddam A1, Baharvand H3, Skutella T4.
Introduction: Although testis-derived embryonic stem cell-like (ES-like) cells have been obtained in several studies, the time window for the shift to pluripotency is not clear yet.
Materials and Methods: Here we describe, that only during a special time window (41 until 125 days) after initiation of germ line stem cell (GSCs) cultures from neonate and adult promoter-reporter Oct4-GFP transgenic mouse the spontaneous appearance of germline-derived pluripotent stem (gPS) cells from both neonate and adult GSCs occurred.
Results: The isolated and long-term cultured (more than one year) GSCs which were isolated by a morphology based selection procedure expressed germ cells markers and exhibited a similar morphology with a high nucleus/cytoplasm ratio in comparison to undifferentiated SSCs (spermatogonial stem cells) in vivo. The generated gPS cells expressed pluripotency marker, in-vitro differentiated into all three germ lineages, formed complex teratoma after transplantation in SCID mice and produced chimeric mice.
Conclusion: Although the exact mechanism of the development of gPS cells from GSCs is still unclear, this new information could provide an ideal strategy for scheduling natural conversion mechanisms of ES-like cells from mouse testis.
Key words: Germ cells, Spermatogonial stem cells, Pluripotency.
P-145
Association between MspI polymorphism of CYP1A1 gene and endometriosis in an Iranian population
Babaki A, Seifati SM.
Department of Biology, Ashkezar Branch, Islamic Azad University, Yazd, Iran.
Email: babakiaz@yahoo.com
Introduction: Endometriosis is defined as the presence of endometrial tissue outside the uterine cavity. It is associated with dyspareunia, dysmenorrhea, pelvic pain and infertility and about 3-10% of women suffer from it in reproductive age. It seems that genetic and environmental factors play a role in the development of endometriosis. The relationship between genetic polymorphisms and endometriosis has been investigated in several genetic studies. CYP1A1 gene is a member of the cytochrome P450 gene family. It is involved in estrogen metabolism and in detoxification.
Materials and Methods: In a case-control study, DNA extraction was performed from the blood specimens of 93 cases of endometriosis and 139 healthy controls. The polymorphism was determined by PCR-based restriction fragment length polymorphism (RFLP) method.
Results: There was no significant difference in frequency of TT, TC, and CC genotypes of MspI polymorphism of CYP1A1 gene between case and control groups (p=0.961).
Conclusion: Results of this study revealed no association between the MspI polymorphism of the CYP1A1 gene and susceptibility to endometriosis in the studied population.
Key words: Endometriosis, MspI polymorphism, CYP1A1 gene.
P-146
Preimplantation genetic screening: Which stage? Which technique?
Bazrgar M.
Department of Genetics at Reproductive Biomedicine Research Centre, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: bazrgar.masood76@gmail.com
Introduction: Preimplantation genetic screening (PGS) techniques have a wide range of resolution, complexity and costs. Additionally each of biopsy stages has advantages and disadvantages.
Materials and Methods: Recent publications certainly systematic reviews and randomized clinical trials were considered in details.
Results: Polar body biopsy (PB) is the least invasive however provides the least capture of abnormalities. PB focuses on female meiotic errors, fails to identify approximately 40% of aneuploidies from postzygotic and paternal origins. PB is suitable in countries with legal restrictions for embryo biopsy. Cleavage stage biopsy (CB) of few cells (1 or 2) does not affect differentiation potential, however removal of 2 cells can result in an impaired implantation potential while mosaicism is not detectable in one cell biopsy. In trophectoderm biopsy (TB) several cells are available for analyses. In day 5 TB when the genetic results cannot be obtained within 24 hours, cryopreservation should be considered. The least and the most aneuploidy rates has been reported for TBs and CBs, respectively (1 versus more than 3). TB increases absolute implantation rate about 20% in comparison with CB. Blastocoel fluid aspiration is a new source of DNA for PGS with few publications. FISH-based PGS is low resolution due to limitation in number of both chromosomes and probes per chromosome which could be tested. Therefore it has not been effective in improvement of clinical outcomes while PGS methods with ability for detection of all 24 chromosomes (PGS-24) result in increased implantation and pregnancy rates and diminished effect of advanced maternal age. Concerning PGS-24 methods, digital PCR is mostly applied for PBs; real time qPCR is only applicable to TBs and limited by number of samples, two on each 384-well plate. Array CGH is robust and scalable, turnaround time as short as 12 hours, decreased cost per sample as increased case numbers. SNP arrays and NGS-based methods for copy number analysis are likely to be the most accurate and informative but complex and high-cost equipment. Low pass NGS-based PGS is moving to be cost effective in addition to performance same as array CGH.
Conclusion: Clearly, the largest reported increases in implantation and live birth rates to date have been with TB. TB is a good choice for good-prognosis patients particularly for those wishing to have elective single-embryo transfer. However, the cleavage-stage embryos of some poor-prognosis patients may implant and develop in utero but not develop to the blastocyst stage in vitro. At the moment, array CGH seems to be the most applicable method for PGS however future would be with higher resolution methods following decrease of related costs, hence ethical concerns on designer babies will need more serious considerations.
Key words: PGS, Biopsy, Stage, Technique.
P-147
Expression of CYP19A1 in patients with endometriosis and normal endometrium during the menstrual cycle
Khateri E1, 2, Shahhoseini M2, Mahdian S3, Aflatoonian R3, Afsharian P2.
Introduction: Endometriosis is an estrogen-dependent inflammatory disease defined by the growth of endometrial tissues outside of the uterus. Epidemiological and clinical studies show that estrogen is essential for the growth of endometriosis. Molecular studies have revealed the presence of aromatase P450, the key enzyme in the biosynthesis of ovarian estradiol, inside the endometriotic tissues, indicating local synthesis of estradiol. So, it is proposed that the enzyme aromatase P450, which is coded by CYP19A1 gene, will be expressed aberrantly in endometriosis.
Materials and Methods: To evaluate the expression profile of CYP19A1 gene in women with endometriosis during menstrual cycle, ectopic endometriotic lesions and eutopic endometrium samples were collected using laparoscopy from 20 women with endometriosis and endometrial biopsies were obtained from 10 healthy fertile women as a control group. For this respect, ethical approval and informed patient consent was gained for the use of tissue samples. Quantitative expression analysis was performed using real-time PCR technique.
Results: Data showed that mRNA expression of CYP19A1 was significantly higher in ectopic endometrium of patients with endometriosis, in both proliferative and secretory phase, in comparison with eutopic and control group. CYP19A1 expression in eutopic endometrium was significantly higher in comparison to control group in proliferative phase.
Conclusion: Higher expression of CYP19A1 mRNA in the endometriotic tissues, may contribute to the etiology and progression of endometriosis and it is involved in disease pathogenesis.
Key words: Endometriosis, CYP19A1, Menstrual cycle.
P-148
A headache in prenatal diagnosis: A case of vanished twin or hermaphroditism?
Mousavi F1, Mohammad Khani N2, Bagherizadeh E1, Hadipour F3, Hadipour Z3, Shafeghati Y4, Behjati F5.
Introduction: The finding of a mixture of both 46,XX and 46,XY cells in amniotic fluid culture has been frequently described. In the great majority of cases, the finding is followed by the birth of a normal male infant, leading to a consensus that the finding is the result of contamination with maternal cells in a normal male fetus. There are, however, several other possible explanations including the presence of cells from an undiagnosed twin pregnancy, cross contamination in the laboratory, the presence of cells from a “vanished” male twin, and true fetal chimerism. An accurate obstetric history and thorough sonography can be of great value in correct assessment of such situations.
Materials and Methods: A 29 Years-old woman was referred for genetic counseling. Her gestational age was 16 weeks and maternal biochemical serum screening test indicated high risk for Down Syndrome. She underwent amniocentesis for chromosome study using standard high resolution GTG banding technique.
Results: Karyotype result was 46,XY and sonography revealed a female fetus with normal internal genital organs. A normal baby girl was borned with 46,XX karyotype. Upon reviewing of amnio GTG slides both 46,XY and 46,XX cell lines were detected. The patient admitted that she had experienced a miscarriage and heavy bleeding early in pregnancy. The sonography at the time of amniocentesis had shown a shrinking cyst next to the fetus. These findings strengthened the vanished or resorbed twin as the reason for such a finding.
Conclusion: The fetus was most likely a non-identical twin. Most of grown cells were from the vanished male fetus which led to misdiagnosis. Thorough genetic counseling with the view of obstetric history is of great value in such situations.
Key words: Amniocentesis, Resorbed twin, Hermaphroditism, Genetic counseling.
P-149
+49A/G CTLA-4 gene polymorphism in NorthWest Iranian population with recurrent pregnancy loss
Parsa S, Jabbarpour bonyadi M, Zeinal zadeh N, Taghavi S.
University of Tabriz, Tabriz, Iran.
Email: sara.parsa6928@gmail.com
Introduction: Recurrent pregnancy loss is defined as three or more pregnancy loss before 20th gestational weeks. There are several factors which involved in pregnancy loss including anatomical, hormonal, immunological and infectious factors. Immunological factors are important due to the interaction between mother and fetus. In spite of allogeneic proteins encoded by paternal genes, it is clear that a series of regulatory mechanisms should exist in maternal immune system not to reject fetus during pregnancy. T regulatory cells which are one of the important component of humeral immune response plays role in the fetu-maternal interface. One of the regulatory ways for these cells is mediated by antigen independent co-stimulatory signals. Interaction of B7/ CTLA-4 is one of these signals. Cytotoxic T Lymphocyte Antigen 4(CTLA-4) is a glycoprotein transmembrane molecule which down regulate the activation and proliferation of T cells in a competitive interaction with CD28 to bind to B7.
Materials and Methods: We have studies 120 cases; patient group consist of 60 women with the experience of two or more pregnancy loss and control group consist of 60 women with at least two live births without any previous history of pregnancy loss and autoimmune diseases. Genomic DNA was extracted from whole blood using standard protocols. The CTLA-4 +49 A/G were detected using polymerase chain reaction- restriction fragment length polymorphisms assay.
Results: In this study CTLA-4 +49 A/G polymorphisms showed no significant differences between patients and controls. Polymorphic allele G showed the frequency of 39.16% among patients whereas its frequency for controls was 35.83%. The age of patient varied from 19 years to 39 years.
Conclusion: The results of the present study suggest that the CTLA-4 do not have association with recurrent miscarriage in NorthWest Iranian population.
Key words: Recurrent pregnancy loss, CTLA-4, Immunological factors.
P-150
Investigation of mitochondrial Gln tRNA molecular alterations in idiopathic repeated pregnancy loss
Vahidi Mehrjardi MY, Seyedhasani SM
Shahid Sadoghi University of Medical Sciences, Yazd, Iran.
Email: mmvahidi@gmail.com
Introduction: Recurrent pregnancy loss (RPL) is a critical medical problem in about 0.5-2% of women. The molecular genetics background for spontaneous abortion is being increasingly understood, and some polymorphisms associated with it have been reported
Materials and Methods: The nucleotide variations of threonine and proline were investigated in 96 women with idiopathic repeated pregnancy loss. The related mitochondrial area was amplified using a polymerase chain reaction (PCR). The PCR products were demonstrated by 2% agarose gel electrophoresis, and all the positive samples were purified and verified by an automated DNA sequencing method.
Results: The sequence analysis revealed 2 mutations in tRNA Gln . These mutations were 4343 in 1 cases and 4336 in 1 cases .
Conclusion: These tRNAs mutations can alter their steady state level and affect the structure of tRNAs. It results in protein synthesis defects and, in turn, mitochondrial dysfunction. The mutations of these genes may help in the assessment of RPL. Further study of an expanded series of these tRNA mutants is recommended to describe their etiologic role in idiopathic RPL.
Key words: tRNA, Mitochondrial mutation, Repeated pregnancy lo.
P-151
A novel mutation in morquio syndrome
Yavari M1, Asadollahi S2, Hasheminasab SH2, Harazi F3.
Introduction: The Mucopolysaccharidoses (MPS) are a group of inherited metabolic disorders caused by a deficiency or malfunctioning of lysosomal enzymes which are needed to break down complex carbohydrates known as mucopolysaccharides or glycosaminoglycans (GAGs). Accumulation of GAGs causes a cascade of events leading to the progressive damage of cells, tissue and organs. Morquio disease or Mucopolysaccharidosis Type IV (MPS IV) belongs to this group and has two sub-types, A and B. Type A is also known as Morquio A, GALNS deficiency, N-acetylgalactosamine-6-sulfate sulfatase deficiency or more simply MPS IVA. This lysosomal enzyme involved in the catabolism of keratan and chondroitin sulfate. Patients who inherit two mutated GALNS gene alleles have a decreased ability to degrade the glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate, thereby causing GAG accumulation within lysosomes and consequently pleiotropic disease. The objective was to report the results of clinical characteristics, enzyme activity determination and mutation analysis of GALNS gene in an Iranian patient with mucopolysaccharidosis (MPS) type IVA (Morquio A disease).
Materials and Methods: The 10 years old Iranian boy with MPSIV was firstly diagnosed by urin GALNS determination who w:as char:actraised by multiple skeletal abnormalities and dwarfism, kyphosis, hypermobility joints, difficultly in walking to 8 years old and now he can’t walk. But his intelligence was normal. We investigated for all coding exons and adjacent intron regions of GALNS gene by PCR sequencing method.
Results: We performed urin test (MS/MS) from patient. In this test total GAGS was high and MPS was likely. So lysosomal enzymes in dried blood were checked and MPS IVA was diagnosed. Two heterozygous missens mutation as c.135 G>T (S>I) and c.510 C>A (P>H), and a homozygous mutation as c.181 A>G (y>C) in GALNS gene were detected in this patient. The mutation c.181 (y>C) is a novel variant which is not reported yet.With the method of gene analysis of new variant, the mutation c.181 (y>C) was considered to be a pathogenic mutation.
Conclusion: The MPS IVA patient showed severe multiple skeletal deformities, normal intelligence, muscle weakness, short stature, who carries homozygous mutations c.181 A>G (y>C). The bioinformatics analysis in POLYPHEN predicted this mutation as being probably pathogenic.
Key words: Mucopolysaccharidoses (MPS), Morquio disease, GALNS gene.
P-152
A newborn with ambiguous genitalia and a complex X;Y rearrangement
Dehghani MR1, Hashemian Z2, Zuffardi O3.
Introduction: In most mammals, sex is determined at the beginning of gestation by the constitution of the sex chromosomes, XY in males and XX in females.
Materials and Methods: Array Comparative Genomic Hybridization (Array-CGH) analysis was performed by using oligonucleotide aCGH platforms (180K SurePrint G3 Human Kit, Agilent Technologies, Santa Clara, CA), as reported elsewhere. Changes in DNA copy number at a specific locus were observed as the deviation of the log2 ratio value from 0 of at least three consecutive probes, by using Genomic Workbench v. 5.0.14 software (Agilent, ADM-2 algorithm with a threshold of 5). Oligomer positions refer to the Human Genome GRCh37 (hg19) assembly.
Results: Array-CGH revealed an unbalanced rearrangement resulting in the deletion of the distal Xp and the duplication of the proximal Xp contiguous region with presence of the Y chromosome from Ypter to Yq11. Fluorescent in situ hybridization (FISH) showed that this portion of the Y was translocated to the tip of the abnormal X and that the duplicated portion of chromosome X was inverted. Altogether, the abnormal chromosome was a dicentric one with the centromere of the Y chromosome apparently inactivated.
Conclusion: The presence within the translocated Y chromosome of the SRY gene explains the devolopment of testes although it is not clear the reason for the genitalia ambiguity.
Key words: Ambiguous genitalia, 46, XX testicular DSD, Inverted duplication and Xp terminal Deletion (Invdup del), Rearrangement, Array Comparative Genomic Hybridization, FISH.
P-153
Effect of genetic variation of beta defensin 126 on ICSI and IVF outcome in unexplained infertile men
Abedini M, Mohseni Meybodi A, Sabbaghian M.
Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: maria.abedini@gmail.com
Introduction: Despite improved methods for evaluation of sperm quality, infertility remains unexplained in about 20% of affected couples. During sperm maturation, a Cysteine-rich secretory glycoprotein α-defensin 126 secreted by the epididymal epithelium adsorbed to the entire sperm surface. It remains on the sperm until sperm become capacitated in the female reproductive tract. Its removal from over the head of sperm is required for sperm zona recognition. A cytosine dinucleotide deletion in the open reading frame of second exon of DEFB126 gene generates an abnormal mRNA. Men homozygous for this mutation have reduced chances of successful fertilization.
Materials and Methods: Genomic DNA from the peripheral blood of 92 male partners of unexplained couples who underwent ICSI (n=74) and IVF (n=18), were extracted. PCR was performed and molecular genotyping for the DEFB126 variant was done by single strand conformational polymorphism (SSCP), tetra PCR and DNA sequencing. ELISA and immunocytochemistry by indirect immunofluorescence antibody performed for the assessment of this protein expression on sperm cells.
Results: In our study this allele frequency in Iranian men was 0.54. Statistical analysis shows, no significant differences were found between homozygote mutation and wild type carriers in fertilization rates, implantation rates and clinical pregnancy of IVF and ICSI. Our results by ELISA and immunocytochemistry showed that the protein expression was less in men with del/del genotype in comparing to other genotypes (p<0.005).
Conclusion: Although previous studies found that DEFB126 variation would affect sperm function and male fertility rate, in the present study, no significant differences were found between homozygote mutation and wild type carriers in fertilization rates, implantation rates and clinical pregnancy. Further confirmation in a larger scale study is needed.
Key words: Unexplained male infertility, Deletion, DEFB126, IVF, ICSI.
P-154
Association of TNFR1 36 A/G polymorphism with azoospermia in Iranian infertile males
Ashrafzadeh Mehrjardi HR1, 4, Dehghan Tezerjani M2, Khademi M1, Ghasemi-Esmailabad S2, Mossadegh AM3, Ghasemi N4.
Introduction: Infertility is the failure to achieve a pregnancy after one year of regular sexual intercourse without contraception. Half of the cases of infertility are due to male factors and nearly 60-75% of reasons of male infertility are unknown, due to unknown mechanisms of molecular defect. Tumor Necrosis Factor α (TNF-α) is a multi-functional cytokine that regulates cellular processes related to spermatogenesis. Tumor Necrosis Factor Receptor 1 (TNFR1) mediate TNF-α activity and changes in its structure can affect TNF-α activity. Studies show that variation in the TNFR1 gene may be associated with male infertility.
Materials and Methods: This case-control study includes 108 azoospermic men and 119 healthy controls. We investigated the association of TNFR1 36 A/G in the population with idiopathic azoospermia who referred to Yazd Institute of Reproduction Sciences. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR- RFLP) method was used to genotype the single nucleotide polymorphism in the both groups. PCR fragments digested by enzymes Mspa1I, and were separated by gel electrophoresis and then the frequency of A→G substitution in azoospermic males and fertile men were counted.
Results: According to our study, G/G genotype in the control group has higher frequency among men with azoospermia [p=0.01; OR=2.29 (1.248-4.229)]. Our findings also show that the allele frequency of G allele in azoospermic men is significantly different in comparison with control group [p<0.001; OR=2.302 (1.580-3.55)].
Conclusion: It seems that the G/G genotype and G allele are significantly associated with increased risk of idiopathic azoospermia.
Key words: Polymorphism, Male infertility, Sperm, Cytokines, Tumor necrosis factor alpha.
P-155
Epigenetic evaluation of histone methylation on HOXA1-5 genes in cumulus cells of polycystic ovary syndrome patients
Asiabi Kohneh Shahri P1, 2, Shahhoseini M2, Favaedi R2, Hassani F3, Movaghar B3, Karimian L3, Eftekhari Yazdi P3.
Introduction: Polycystic ovary syndrome (PCOS) is the major cause of an ovulatory infertility with uncertain etiology. Epigenetics and environment play critical roles in PCOS. Of important genes correlated with human reproductive system disorders are HomeoboxA (HOXA) cluster genes. Regarding the epigenetic role of histone modifications in regulation of gene expression, lysine (K) methylation of histone3 (H3K9me), as a repressive epigenetic mark, on the promoter regions of HOXA1-5 genes were evaluated in this study. For this respect, cumulus cells (CCs) which have critical roles during folliculogenesis, oocyte maturation, ovulation and fertilization, were aimed to monitor expression profile and epigenetic alterations of HOXA1-5 genes.
Materials and Methods: CCs were collected from 20 PCOS patients and 20 fertile women (18-36 year) with male infertility problems, referred to Royan Institute to have IVF-ICSI under GnRH antagonist protocol. Informed consents were obtained from the participants. After evaluation of genes expression by qRT-PCR, chromatin immune precipitation (ChIP) coupled with real-time PCR was performed to evaluate incorporation of H3K9me into regulatory regions of HOXA genes.
Results: Expression data, revealed significant decrease in mRNA level of HOXA1 (p<0.05) and significant increase in HOXA2, HOXA3, HOXA5 (p<0.05) in cumulus cells of PCOS patients vs. control group. There was no significant change in expression level of HOXA4 (p<0.05) among two studied groups .Obtained data from ChIP Real-time PCR verified well the results of gene expression; in the way that histone methylation level in regulatory region of HOXA1 does not show any alteration but there were significant decrease of H3K9me in promoters of HOXA2, HOXA3, HOXA4 and HOXA5 (p<0.05), in PCOS patients vs. control group.
Conclusion: These findings demonstrate the significant association of aberrant histone methylation with impaired oocyte maturation and confirm the functional role of epigenetics in pathogenesis of PCOS.
Key words: HOXA1-5, Histone methylation, Epigenetic, PCOS.
5 genes.
Materials and Methods: CCs were collected from 20 PCOS patients and 20 fertile women (18-36 year) with male infertility problems, referred to Royan Institute to have IVF-ICSI under GnRH antagonist protocol. Informed consents were obtained from the participants. After evaluation of genes expression by qRT-PCR, chromatin immune precipitation (ChIP) coupled with real-time PCR was performed to evaluate incorporation of H3K9me into regulatory regions of HOXA genes.
Results: Expression data, revealed significant decrease in mRNA level of HOXA1 (p<0.05) and significant increase in HOXA2, HOXA3, HOXA5 (p<0.05) in cumulus cells of PCOS patients vs. control group. There was no significant change in expression level of HOXA4 (p<0.05) among two studied groups .Obtained data from ChIP Real-time PCR verified well the results of gene expression; in the way that histone methylation level in regulatory region of HOXA1 does not show any alteration but there were significant decrease of H3K9me in promoters of HOXA2, HOXA3, HOXA4 and HOXA5 (p<0.05), in PCOS patients vs. control group.
Conclusion: These findings demonstrate the significant association of aberrant histone methylation with impaired oocyte maturation and confirm the functional role of epigenetics in pathogenesis of PCOS.
Key words: HOXA1-5, Histone methylation, Epigenetic, PCOS.
P-1
Fertility preservation in cancer patients
Abtahi NS, Ebrahimi B, Rezazadeh Valojerdi M, Fathi R.
Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: b.ebrahimi@royaninstitute.org
Introduction: Fertility preservation before gonadotoxic treatments is necessary in cancer patients. Ovarian tissue cryopreservation is the only available option for prepubertal girls and women who cannot delay cancer treatment or when ovarian stimulation is contraindicated.
Materials and Methods: Human ovarian tissue biopsies were obtained from six women (29-40 years old) who were candidates for oophorectomy for benign gynecologic conditions. Their medulla were removed, cortical parts were thinned and then cut into 10×5×1 mm strips. Cortical strips were vitrified in two steps by using vitrification solutions including; V1 [HTCM as base medium (BM)+ 7.5% DMSO+ 7.5% EG+ 20% HAS] and V2 (BM+ 15% DMSO+ 15% EG+ 0.25M sucrose+ 20% HSA) for 15 and 10 min respectively. Morphology of ovarian tissue and enclosed follicles were compared in control and vitrification groups by histological assessment.
Results: Ovarian stromal integrity, granulosa cells distribution, follicular population and morphology were well preserved in vitrified-warmed strips as fresh one. Oocyte degeneration, vacuolization and granulation were rarely observed in vitrified-warmed ovarian tissues. Primordial follicles were more distributed in cortical region in both groups. The mean percentage of intact follicles (primordial, primary and pre antral) are higher in control groups compared to vitrification one but the difference was not significant.
Conclusion: Accordingly, used vitrification method could be suitable for human ovarian tissue preservation in cancer patients.
Key words: Human, Fertility preservation, Vitrification, Ovarian tissue.
P-2
Effects of sesame-supplemented diet on the histology of adult rat reproductive system
Amini Mahabadi J1, Hassani Bafrani H1, Nikzad H1, Taherian AA2, Seyed Hoseini ES1.
- Gametogenesis Research Center, Faculty of Sciences, Kashan University of Medical Sciences, Kashan, Iran.
- Anatomical Sciences Research Center, Faculty of Sciences, Kashan University of Medical Science, Kashan, Iran.
Introduction: Studies show that antioxidants are important in improving male infertility. There are several antioxidant compounds in sesame seeds.
Materials and Methods: This experimental study was carried out on 30 adults Wistar rat (200 gr). Rats were randomly divided into experimental and control groups. The control group received standard diet and experimental group received a diet containing 70% standard diet and 30% sesame seed for 12 weeks. At the end of the study, body weight, testis weight and volume were measured and histology of testis, epididymis and prostate were evaluated. Serum FSH, LH and Testosterone levels were measured as well. SPSS software was used to calculate t-test and p<0.05 was considered as significant.
Results: The measured sperm count and motility, the number of epithelial cells and lumen diameter were increased significantly in the experimental group (p<0.0001). The number of spermatogonial cells, primary spermatocytes, spermatid and spermatozoa were increased significantly in the experimental group compared to the control group (p<0.0001). The epididymal diameter, lumen diameter and epithelial thickness did not change significantly. Fibromascular and epithelium diameter of seminal vesicle in treatment group were very significant differences and volume density epithelium significantly increased in treatment group compared to control group but volume density, Fibromascular and lumen significantly decreased in treatment group compared to control group. LH concentration increased significantly in the experimental group (p<0.03).
Conclusion: This is the first study, which evaluated the histology effect of sesame seed on reproductive system of adult Wistar rat. These results highly suggest that sesame seed can improve male reproductive parameters.
Key words: Rat, Male Sex hormones, Sesame seed, Testis, Prostate, Epididymis.
P-3
The effects of sperm preparation media on motility, viability and DNA integrity of human spermatozoa
Anbari F1, Halvaei I1, Nabi A1, Ghazali Sh1, Khalili MA1, Johansson L2.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Origio A/s, Knadrupvej 2, DK-2760 Malov, Denmark.
Introduction: The main goal was to compare the effects of three different sperm preparation media on sperm motility, viability and DNA integrity of semen samples from normozoospermic men.
Materials and Methods: A total of 15 normozoo spermic males were included in the study. The semen analysis was performed in accordance with the WHO guidelines (2010). After semen analysis, each sample was divided into three aliquots and swim-up performed with three different sperm preparation media (Sperm Preparation Media, Origio, Denmark, Ham’s F10, Biochrome, Berlin, Germany, and VitaSpermTM, Innovative Biotech, Iran). Sperm motility, viability and DNA fragmentation,were evaluated at 0, 1, 2, and 24 hr after swim-up.
Results: There were no significant differences, at any time intervals, in the totalsperm motility between the different sperm preparation media. However, the rate of progressive motility was significantly higher in spermatozoa prepared by the media from Origio in comparison to VitaSperm TM (68.3±11.5 vs. 58±9.8, p=0.03), whereas no significant difference was found against Ham’s F10. No significant differences in sperm viability were seen between the media products .One hour after swim-up therate of sperm DNA fragmentation was also significantly lower inthe medium from Origio versus VitaSpermTM (p=0.02).
Conclusion: The type of medium for preparation of semen samples from normozoospermic men significantly affects the performance of spermatozoain assisted conception.
Key words: Sperm preparation media, DNA fragmentation (SCD), Viability, Motility.
P-4
Investigating the underlying factors of preterm delivery prevalence preceded by preeclampsia among pregnant visitors of Shariati Hospital, Bandar-Abbas, Iran: 2011-2012
Aslavi N1, Fallahi S2, Nowroozi S1, Najafian A3, Ghanbarnejad A1.
- Student Research Committee, Hormozgan University of Medical Sciences, Bandar Abbas, Iran.
- Department of Molecular Medicine, Hormozgan University of Medical Sciences, Bandar Abbas, Iran.
- Department of Fertility and Infertility, Hormozgan University of Medical Sciences, Bandar Abbas, Iran.
Introduction: Along with blood loss and infection, disorders pertaining to blood pressure are the three deadly factors in preterm childbirth morbidities and disabilities. In other countries the prevalence rate of this crisis is maximally 10%, while in Iran, it is 7%.
Materials and Methods: The present descriptive/ analytic research is a cross-sectional retrospective study. It was carried out on 863 pregnant women (167 had a preterm delivery due to pre-eclampsia and 696 were diagnosed with preterm delivery due to other reasons). The data were collected via a checklist about mother’s age, blood group, pre-eclampsia history, systemic disease, preterm delivery pain, etc. SPSS 16 analyzed the data. T-test and Chi-squared tests were used too. Significance level was set at p<0.05.
Results: Participants with a preterm delivery after pre-eclampsia were found to have a higher average age than peers afflicted with preterm delivery for other reasons. Most of the women afflicted with pre-eclampsia were of the B+ blood type. Significant differences were observed in these variables: pre-eclampsia history, preterm delivery pain, preterm rupture in fetal membrane, vaginal bleeding, intrauterine growth restriction, mother’s systemic diseases, blood group, history of intrauterine death and amniotic fluid disorders.
Conclusion: There is an increase in the probability of preterm childbirth due to pre-eclampsia with intrauterine growth restriction, history of intrauterine death and mother’s background diseases. Timely medical and preventive attempts and instructions for pregnant mothers on pre-natal healthcare can help to decrease irrevocable a consequence that endanger mothers and baby’s health.
Key words: Pre-eclampsia, Premature childbirth, Disease prevalence, Bandar-Abbas.
P-5
Infertility and quality of life in women with polycystic ovary syndrome
Bahri Khomami M1, Ramezani Tehrani F1, Azizi F2.
- Reproductive Endocrinology Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
- Endocrine Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Introduction: Polycystic ovary syndrome (PCOS), the most common endocrine disorder, may cause infertility among affected women. Infertility is a life crisis and blamed for psychosocial distress and poor quality of life (QoL). Ethnicity and socio-cultural factors may have an impact on this negative influence on QoL.
Materials and Methods: The study procedure was completed by 796 women with polycystic ovary syndrome, aged 15-49 years. A reliable validated Persian version of the health related quality of life questionnaire (HRQoL) for polycystic ovary syndrome patients (PCOSQ) was filled for each participant. They were subdivided into 2 groups according to their fertility status: fertile and infertile. Using linear regression the association between infertility and health related quality of life in women with PCOS was assessed.
Results: Out of all participants 120 (15.1%) and 482 (60.6%) women were infertile and fertile, respectively. Infertility was associated with lower HRQoL score before and after adjustment for age, body mass index and other perceived PCOS symptoms (CI 95%: -16.76, -3.81; p=0.002 and CI 95%: -15.02, -3.67; p=0.001). Women who had longer duration of infertility, had got better scores.
Conclusion: We found that infertility causes poor quality of life in PCOS women. Care providers should pay attention to the effect of each PCOS symptoms on patients emotional health along with physical health and plan to treat them accordingly.
Key words: Quality of life, Health-related quality of life, Infertility, Polycystic ovary syndrome.
P-6
The effect of hydrostatic pressure on parthenogenetic activation of mouse oocytes
Bashiri Z, Azadbakht M.
Departeman of Biology, Faculty of Sciences, Razi University, Kermanshah, Iran.
Email: zbashiri88@gmail.com
Background: Parthenogenetic activation of mammalian oocytes using artificial stimuli is commonly used in various reproductive biotechniques. Hydrostatic pressure can act as a mechanical stimulator that rearranges egg contents. In this study, we investigated the effect of hydrostatic pressure on parthenogenetic activation of MП oocytes derived from superovulation and matured oocytes in vitro.
Materials and Methods: In experiment 1, immature oocytes were dissected from ovary of female NMRI mouse (8-week-old) and transferred to α-MEM medium for in vitro maturation. After 24 hr, MП oocytes were transferred to T6 medium. In experiment 2, mice were superovulated by injections of 10 IU of PMSG and 10 IU of HCG 48 hr apart. MП oocytes were collected 12 hr after HCG injection and transferred to T6 medium. Then oocytes from two groups (experiment 1, 2) were divided into experiment and control groups. Oocytes of experiment group were subjected to 20 mmHg pressure for 10, 20, 30 min (treatments I, П, III). Oocytes without exposure to pressure were considered as control. Oocytes were cultured for 72 hr and embryo development was assessed.
Results: In experiment 1, cleavage rate in treatments I, П, III and control was 21.42%, 15.71%, 8.52%, 5.71% respectively. The best cleavage rate were associated with treatment I which were significantly different with treatment III and control group (p<0.05). In experiment 2, cleavage rate in treatments I, П, III and control was 22.91%, 53.12%, 29.15% and 9.34% respectively. Oocyte activation rate in experiment group was higher than control group. The highest cleavage rate associated with treatment П which were significantly different with treatments I, III and control (p<0.05).
Conclusion: Exposure of MП oocytes derived from superovulation to hydrostatic pressure, on account of cumulus cells presence around oocyte, could improve embryonic development and by affecting on calcium channels, probably leading to increase rate of cleavage in the mouse oocyte.
Key words: Parthenogenetic activation, Hydrostatic pressure, Oocyte, Mouse.
P-7
Evaluation the knowledge and awareness of nulliparous women from adverse outcome of delayed childbearing
Behboudi-Gandevani S.
Reproductive Endocrinology Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Email: s.behboudi@hotmail.com
Introduction: Recently the numbers of women who delay childbearing in older age were rapidly increased. The aim of this study was to evaluate the nulliparous women’s knowledge and awareness from adverse outcome of delayed childbearing.
Materials and Methods: In this cross-sectional survey study, a total of 700 healthy nulliparous women 35 years and older that had self-choice delayed pregnancy were selected by cluster sampling in Tehran. To evaluate the knowledge and awareness of these women, a standard questionnaire about adverse pregnancy outcomes in older age were used. The questionnaire consists of three parts: demographic characteristics, assessment of maternal adverse outcomes (10 questions) and to assessment of fetal-neonatal adverse outcomes in maternal older age (6 questions). Data were analysis by SPSS software and the significance level was set at p˂0.05.
Results: The psychometric properties of questionnaire including forward and backward translation, face and content validity and reliability with cronbach's-alpha: 0.078 were done. The result of the study showed that women’s awareness about the risks of delayed childbearing including gestational diabetes, congenital anomaly, Down syndrome, caesarean section, low birth weight, stillbirth and long-term health problems like learning difficulties among neonate varies between 40%-72%. In this respect, most of the women were aware about the increased risk of infertility in older age (82.9%), but a little were aware about the possibility of a twin or multiple pregnancy and gestational hypertension in older ages of mother (35.2%). Awareness of delayed childbearing risks were independently related to the women’s educational levels (OR: 1.87; 95% CI: 1.25-2.78) and family income (OR: 3.12, 95% CI: 0.01-5.12).
Conclusion: It seems that women are largely unaware about the adverse consequences of pregnancy in older age among mother and child. Women’s should be more inform about these risks by health care professional.
Key words: Awareness, Delayed childbearing, Adverse outcome.
P-8
Study the protective role of jujube extract on teratogenic effects of Carbamazepine in Balb/c mice embryos
Doostabadi MR, Hasanzadeh Taheri MM, Afshar M, Hami J, Hosseiny M.
Birjand University of Medical Sciences, Birjand, Iran.
Email: doostabadim@yahoo.com
Introduction: Carbamazepine (CBZ) is an anticonvulsant medication that is consumed during pregnancy and can produce congenital anomalies including; neural tube defects, cardiac, skeletal and craniofacial abnormalities.
Materials and Methods: In this experimental study, 100 pregnant mice of Balb/c 25±3 gr body weight randomly divided into 8 experimental groups (E) and 2 control groups (C) (n=10). Experimental groups (E1, E5 and E6) and (E2, E7 and E8) received daily intraperitoneal injection (IP) of 50 and 100 mg/kg/ of CBZ respectively from gestational day zero (GD 0) to GD 15 and also groups (E5, E7) and (E6, E8) in addition to medicine, received jujube extract (AJE) at doses of 200 or 400 mg/kg/ from ten days prior to gestation, to GD 15. E3 and E4 groups gavaged only by 200 and 400 mg/kg of AJE respectively. Two control groups (C1, C2) received normal saline or tween -20. Dams underwent cesarean section on GD 18 and fetuses harvested from uterine. First, absorbed embryos were counted. Thereafter, morphological studies were done on the offspring by stereomicroscope. All malformed fetuses were stained with Alizarin red S and alcian blue for detection of skeletal anomalies. Data were analyzed by ANOVA, Tukey and c2 tests and using SPSS software version 18 and p<0.05 were considered significant.
Results: The findings of this study showed that administration of CBZ purely in pregnant mice induced various anomalies in their fetuses such as; limb defects, deformities of vertebral column, craniofacial malformations and etc in experimental groups so that their differences were significant when compared with control groups. But these anomalies decreased significantly in those experimental groups which received CBZ and AJE synchronously when compared with experimental groups that received CBZ only.
Conclusion: According to our findings it can be concluded that although administration of CBZ can induce several malformations in fetuses of pregnant mice, but consumption of AJE synchronously with CBZ, can prevent teratogenicity of CBZ. Therefore, probably AJE can play a protective role against CBZ induced anomalies.
Key words: Teratogenic, Jujube, Carbamazepine.
P-9
Depression and clinical markers in polycystic ovary syndrome
Enjezab B1, Ghadiri anari A2, Eftekhar M3, Mohajeri M1.
- Department of Midwifery, Faculty of Nursing and Midwifery, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Internal Medicine, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
Introduction: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among reproductive-age women. Previous studies have raised conflicting results of depressive disorders in PCOS compared with healthy people.
Materials and Methods: In this cross-sectional analytic study, 62 patients with PCOS diagnosed by Rotterdam criteria and 61 women without PCOS were selected after an initial survey conducted to determine demographic characteristic, systolic and diastolic blood pressure and reproductive status, the Beck depression questionnaire short form was used to assess depression in both groups. All women were introduced to determine fasting insulin and blood sugar and testosterone.
Results: The mean age, BMI, systolic and diastolic blood pressure of PCOS and Non PCOS was respectively (29.96±6.85, 29.49±7.44 years), (29.15±6.56, 25.65±5.84), (117.42±10.17, 117.18±66.57) and (75.41±9.27, 76.42±6.56) respectively that only mean of BMI was different (p˂0.05). The average Beck score in patients was 7.47±5.54 and in the control group was 7.57±5.79, (p˂0.05), which was not statistically significant. 37.1% of the patient group and 36.1% of the control group have degree of moderate to severe depressed mood (p˃0.05).
Conclusion: Despite non significant difference in prevalence of depression in both groups, due to the high prevalence of depression in both groups may be there is other reasons for depression in this population that overcomes on the psychological effects of this disease.
Key words: Depression, Polycystic ovary syndrome, BMI.
P-10
Comparison of metabolic syndrome in postmenopausal women with natural or surgical menopause: Community based cohort study
Farahmand M, Ramezani Tehrani F, Azizi F.
- Reproductive Endocrinology Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
- Endocrine Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Introduction: The ovaries produce female sex hormones that after natural menopause gradually and following surgical menopause abruptly end.
Materials and Methods: The study subjects were selected from 5019 women, 59-35 years old, which were participated in the Tehran Lipid and Glucose Study. They include 357 natural menopauses and 63 surgical menopauses during the follow-up that was 10 years. Data were collected by questionnaires in this project.
Results: Changes in metabolic and biochemical profiles of these two groups of women during follow-up were compared with each other. The findings of our study showed that the incidences of metabolic syndrome in surgical menopause and natural menopause subjects were 14.5 and 8.9% respectively. Mean serum low density lipoprotein in surgically menopausal women was significantly lower than natural menopause subjects. Also, mean systolic blood pressure in naturally menopausal women was significantly higher than surgical menopause women.
Conclusion: It seems the metabolic disorders associated with menopause differ in surgical and natural menopause women. The metabolic complications should be considered following menopause.
Key words: Surgical menopause, Natural menopause, Metabolic syndrome.
P-11
Medical staffs’ viewpoints on “ART nursing” working at Reproductive Institutes in Iran
Faramarzi A, Khalili MA, Borzouei Z, Hosseini A, Rahiminia T.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: faramarzi_90@yahoo.com
Introduction: As, assisted reproductive techniques (ART) advances, multifaceted changes are noted in the roles, responsibilities, and commitments of each members of the infertility team. The aim was to determine the level of knowledge and attitude of medical staff working at reproductive institutes about role of nursing in ART program.
Materials and Methods: In this descriptive cross-sectional study, knowledge and attitudes of 199 Bio-medical staffs working at reproductive institutes were investigated through a questionnaire including 20 questions. All participants based on work experience, education and communication with patients were classified. Chi-square test was used for data analysis and p<0.05 was considered as statistically significant.
Results: There was no significant relationship between educational levels and viewpoints on ART nursing. But, significant relationship between work experience, kind of relation to the patients and viewpoints on ART nursing were found. 52.5% of participants were familiar with ART nursing. By increasing experience, knowledge and attitudes of Bio- medical staffs enhanced. Laboratory technician, embryologist, ART Laboratory technician and nurses were more familiar with ART nursing; but, Urologists were less familiar with ART nursing. Over 97% of participants requested offering a master degree of ART nursing in Iran (p<0.05).
Conclusion: There is a need for nursing professionals to assume an ongoing, visionary, scientific, and academic approach to advancement. It seems that appropriate continuing education and opportunities are essential to support this group of nursing professionals.
Key words: Viewpoint, Nurse, ART, Medical staffs.
P-12
The effect of microtubule stabilizer pretreatment in IVM program
Fesahat F1, Khalili MA1, Khoradmehr A1, Anbari F1, Bazrgar M2, Taheri F1.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Genetics, Royan Institute for Reproductive Biomedicine, Tehran, Iran.
Introduction: It has been reported that in vitro maturation (IVM) increases the risk of abnormal spindles and chromosome configurations of oocytes compared with oocytes matured in vivo. This is one possible explanation for the reduced developmental potential of IVM oocytes compared with those matured in vivo. Paclitaxel is known to stabilize the microtubules that constitute the spindle. The aim was to investigate whether pretreatment by a microtubule stabilizer, paclitaxel, would improve IVF outcomes in IVM program.
Materials and Methods: In this ongoing experimental study, 75 GV oocytes were retrieved from 5 mice primed with rec-FSH for 48 hr. The immature oocytes were cultured 24-48 hr for IVM. After identifying mature oocytes by the presence of a first polar body extrusion under a stereomicroscope, Normal morphologically MII oocytes were divided in control and 5 experiment groups. Experiment groups were incubated in presence of 1μM paclitaxel for different times (30 min/group 1,1 hr/group 2,2 hr/group 3,3 hr/group 4,4 hr/ group 5 and group 6 as control) prior to IVF. Standard IVF program was performed for the 60 MII oocytes after pretreatment with paclitaxel and embryo development was followed until blastocyst formation.
Results: Total maturation rate of GV oocytes was 80% and fertilization rates of MII oocytes were 85% in groups 6, 83% in groups 1, 2 and 3 and 74% and 65% in groups 4, 5 respectively. Blastocyst formation rates of embryos were 95% in group 1, 2 and 6, 60% in group 3 and 40% in groups 4 and 5. The final results, which include probable relationship between paclitaxel pretreatment and the aforementioned variables, will be presented.
Conclusion: The preliminary results showed by performing critical concentration with optimized timing of microtubule stabilization procedure IVM, MII oocytes could efficiently develop to the blastocyst stage. Paclitaxel may cause cell toxicity with irreversible harm on normal fertilization and embryo development in higher concentrations.
Key words: Paclitaxel, IVM, IVF outcomes.
P-13
The effect of ovarian drilling on poly cystic ovary syndrome (PCOS) women
Ghasemi-Esmailabad S, Eftekhar M, Pourmasumi S, Rajabi Firoozabadi Sh, Sabeti P, Heidarian Meimandi H.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: Qasemisaeed@yahoo.com
Introduction: One of the most common causes of female infertility factors is ovulation disorder. Poly cystic ovary syndrome (PCOS) has the highest prevalence rate about 5-10% among women. There are different diagnostic methods and treatments for PCOS syndrome, including medical and surgical treatments. Laparoscopic surgical treatment is for the patients resistant to medical treatment, and could be done with ovarian drilling at some points.
Materials and Methods: In this retrospective study we evaluated 289 patients. All patients had proven clomiphene resistant PCOS with ultrasound and hormonal tests. Laparoscopic ovarian cautery was performed for all these women. Age, duration of infertility and assisted reproductive techniques in each patient were asked.
Results: Of the 289 patients, we access to the information of 135 patients. The mean age and the duration of infertility were 26.90 and 4.83 years respectively. Among these, 63 patients (46.6%) had not pregnancy and 72 patients (53.33) were pregnant, 52 patients (72.22%) were naturally pregnant and 20 patients (27.77%) used assisted reproductive techniques.
Conclusion: PCOS patients who resistant to clomiphene could be treated with ovarian drilling.
Key words: Polycystic ovary, Clomiphene, Ovarian drilling.
P-14
Comparison of two different embryo loading techniques for embryo transfer in IVF/ET cycles
Halvaei I, Mangoli E, Omidi M, Khalili MA.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: ihalvaei@gmail.com
Introduction: Embryo loading (EL) is one of main steps in embryo transfer (ET) technique that plays an important role in IVF success. This study was aimed to compare the effect of two different techniques for EL on rates of pregnancy and delivery in IVF/ET cycles.
Materials and Methods: In total 195 fresh ET and 171 frozen-thawed ET were included in this retrospective study in which 76 and 119 cycles in fresh ET and 76 and 95 cycles in frozen-thawed ET were placed in two groups of A and B, respectively. Embryo catheter loading techniques were divided into two groups of A and B. In group A, the whole catheter was flushed with a 1-ml air-tight syringe with Ham’s F10 medium. Then the embryos were drawn into ET catheter between two air brackets. In group B, 70 µl airs was held in the syringe and the catheter was flushed by Ham’s F10 medium. Then the media, air, embryos, and air were respectively drawn into catheter. The main outcome measure of the study was delivery rate.
Results: The groups were matched for number of fertilized oocytes, etiology of infertility, source of sperm, type of stimulation protocol, percent of conventional IVF or ICSI, high quality embryos, type of embryo loading catheter, fresh ET and ease of transfer. In fresh ET cycles, delivery rate showed an increasing trend in group B compared to group A (21% vs. 11.8%, respectively, p=0.1). In frozen-thawed ET cycles, the rate of delivery rate was also higher in group B compared to group A (16.8% vs. 13.1%, respectively), but the difference was not significant (p=0.5).
Conclusion: It seems different embryo catheter loading has no effect on delivery rate.
Key words: Embryo loading technique, Embryo transfer, Pregnancy.
P-15
Application of sonography in infertility treatment cycles: instructions for midwives and nurses
Irani Sh, Ahmadi F, Javam M.
Department of Reproductive Imaging at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: Irani_shohreh@yahoo.com
Introduction: Ultrasound has a pivotal role in imaging modality in the study of the female pelvis, and provides fundamental information in detecting and characterizing pelvic masses of uterine, ovarian, or adnexal origin.
Materials and Methods: A narrative review was performed within articles published at PubMed, Elsevier, SID and original text books to reach the aim.
Results: Every patient in the process of infertility treatment needs to go through four steps of sonography to roll out disorders and malformations in ovaries, uterus and tubes then to opt for the best treatment method and to follow up the results. Infertility Workup: 1) Base sonography is applied in examination of the following organs: -For overall evaluation of the pelvis and determining any pathologic condition in uterus such as polyps, fibroms, uterus malformations. -Ovarians are examined for ovarian cysts and Poly Cystic Ovary Syndrome (PCOS). 2) Pre-treatment sonography is applied in checking the result of operative surgeries- like Hysteroscopy, Laparoscopy, Myomectomy. 3) Monitoring sonography is done at different stages of treatment cycle, especially at ovulation stimulation period, in order to investigate ovarians and in picking up the eggs as well as endometrial thickness in response to drugs. 4) Post-Assisted reproductive techniques (ART) cycles is done for confirmation of pregnancy (gestational sac, fetal heart), and the side effects of treatment cycles such as ovarian hyper stimulation syndrome (OHSS), pregnancy complications such as ectopic pregnancy (EP) and mole.
Conclusion: Ultrasound has a key role in diagnosis and proposing of different options of treatment regimes, and the post-treatment follow-ups. It is important to know the application of the aforementioned four steps to improve the accuracy and efficiency of infertility treatment.
Key words: Ultrasonography, ART cycle, Infertility.
P-16
Evaluation of anxiety, depression and risk factor associated with them in women after tubal ligation
Jahanian Sadatmahalleh Sh1, Ziaei S1, Kazemnejad A2, Mohamadi E3.
- Department of Reproductive Health and Midwifery, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
- Department of Biostatistics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
- Department of Nursing, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Introduction: Tubal ligation is recommendable for women completed their family. The existence of anxiety and depression following this procedure has been the subject of debate for decades.
Materials and Methods: A historical cohort study was carried out on 200 subjects with tubal ligation and on 200 subjects using condom as contraceptive method. The two groups were matched in demographic and personal characteristics. Data collection tool was a questionnaire including questions regarding demographic and obstetrical characteristics. Anxiety and depressive symptoms were evaluated by the Hospital Anxiety and Depression Scale (HADS). All statistical analyses were carried out using software package used for statistical analysis (SPSS) version 20 (SPCC Inc., Chicago, IL, USA). Student’s t-test and chi-square test were carried out to reveal the statistical differences between the groups. Multiple linear regression was done to build a prediction model in anxiety and depression.
Results: The mean (SD) duration of tubal ligation was 4.1 (1.6) years. The overall prevalence of anxiety and depression in the two groups was 81.5% and 48.5% respectively (p<0.0001). Multiple linear regression analysis revealed a significant association between low education (p<0.0001), post-sterilization regret (p=0.03) and no consultation prior tubal ligation (p=0.03) with risk for anxiety and depression in women who have undergone tubal ligation.
Conclusion: Termination of fertility with tubal ligation may be a risk factor for anxiety and depression. We found significant differences in anxiety and depression between women with and without tubal ligation. Therefore, women should be informed by the health providers regarding the advantages and disadvantages of tubal ligation before the procedures.
Key words: Historical cohort study, Tubal Ligation, Anxiety, Depression.
P-17
Polymorphism in CGA affects the function of miR-1302 and increases the risk of men infertility
Jamalvandi M, Motevali bashi M.
Department of Biology, Faculty of Sciences, Esfahan University, Esfahan, Iran.
Email: mina.jamalvandi@yahoo.com
Introduction: Infertility occurs in 10-15% of couples worldwide and close to half of it is caused by male factors. Despite decades of efforts to clarify mechanism of male infertility, most of cases are still idiopathic. Many factors such as genetics and sexual problems can affect infertility. Among these problems, genetic disorders are the most common factors. A study has shown that one of genes that can affect male infertility is CGA. This gene is involved in miotic. CGA, α subunit of glycoprotein hormones, is the main part of thyrotropin glycoprotein hormone (pituitary TSH), lutropin (LH), follitropin (FSH), and Chorionic gonadotropin (human placental gonadotropin, hCG) that has essential role in development and function of tyroid and gonads. CGA gene is located on 6q14-q21. Rs6631 in CGA has strong association with men infertility. Studies have shown that miR-1302 can negatively regulate CGA and substitution of T with A may interfere this process. This miRNA can band with rs6631-A more strongly than rs6631-T.
Materials and Methods: Tetraprimer technique is an appropriate way to study this polymorphism; because it is faster and cheaper than ordinary PCR. Also laboratories with low equipment can use this method.
Results: Primers designed by the use of Primer1 and then checked by Oligo7 software. By the use of these primers and tetraprimer technique, this polymorphism can be studied for the first time in Iran.
Conclusion: SNP in genes specially the ones that are target of miRNAs, can have important role in complex disease such as infertility. In this study, miR-1302 negatively regulates CGA, and the substitution of T by A at rs6631 within the binding site disrupts its regulation. It is not exactly clear that what is the real role of this polymorphism in idiopathic male infertility; but it has been suggested that the variant allele of rs6631 may elevate the risk of idiopathic male infertility through up-regulation of the expression of CGA. It has been showed that α-subunit, that is produced by CGA, can has growth factor activity and can induce the differentiation of lactotrope and secretion of RPL. Because CGA and PRL play an important role in the development of gonads, the abnormal hormone level may elevate the risk of male infertility.
Key words: Men infertility, miRNA, SNP, CGA.
P-18
Evaluation of testosterone levels after GnRH agonist administration in the adult male rat
Khadivi B1, Peirouvi T1, Zirak Javanmard M2, Rasmi Y3, Mohammadbeigi H1.
- Department of Histology, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran.
- Department of Anatomy, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran.
- Department of Clinical Biochemistry, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran.
Introduction: Gonadotropin-releasing hormone (GnRH) is secreted from the hypothalamus in a pulsatile manner and stimulates the biosynthesis and release of gonadotropins, LH and FSH, via GnRH receptor located in gonadotropin cells. These gonadotropins regulate various gonadal functions such as gametogenesis and steroidogenesis.
Materials and Methods: 24 male adult Wistar rats were divided into three groups. In the first study group, 300 µg/kg buserelin, in the second study group, 600 µg/kg buserelin and in the control group, salin was injected subcutaneous for 5 days. Thirty day after the first injection, blood samples were collected from the heart, centrifuged and plasma was isolated from blood. Plasma testosterone level was measured by ELISA.
Results: The findings reveal no significant differences in plasma level of testosterone in the first study group (2.71±0.71) and second study group (2.12±1.74) compared with control group (3.14±1.20) (p>0.05).
Conclusion: Short-term administration of buserelin has no effect on the plasma testosterone level.
Key words: GnRH, Buserelin, Testosterone, Adult rat, ELISA.
P-19
Apoptotic cells and loss of follicle development were resulted after administration of Nano dioxide titanium on immature mouse ovary
Khoradmehr A1, Danafar AH1, 2, Hosseini M3, Halvaei I1, Golzadeh J1, Akyash F1, Anvari M1.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Young Researchers Club, Islamic Azad University, Yazd, Iran.
- Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran.
Introduction: Titanium dioxide (TiO2) is used as an antimicrobial and whitening agent in food and products such as chewing gums, candies, toothpastes, lip balms, shampoos, deodorants and sunscreens. Previous studies showed that ovary might be one of the target organs that TiO2 nanoparticles induced genotoxicity and cytotoxicity in ovary cells and exposure to TiO2 can effect on follicle development and fertility with changes the levels of sex hormones. The aim of the present study was to evaluate ovarian dysfunction by detect of follicle development.
Materials and Methods: 40 immature Balb/C female mice (4week ages) randomly divided into five groups. First, second and third groups were administrated by oral gavage 2.5, 5 and 10 mg/kg TiO2 for thirty days respectively. Forth group was gavaged normal saline as placebo and in control group we didn’t gavage anything. Finally female mice were dislocated and ovary was fixed in bouin's fluid. Ovaries were dehydrated with ethanol series, embedded in paraffin and then serially sectioned at 5μm thickness. Hematoxylin and Eosin used to evaluate primordial, primary, preantral and antral follicles by light microscope. TUNEL assay was used to detect cell apoptosis. Statistical significance was analyzed by Kruskal-Wallis test and the level of significance was determined to be at p≤0.05.
Results: According to data, primordial, primary, preantral and antral follicles were significantly decreased in third group in comparison with the controls and shams (p≤0.05) and also, there was increasing trend in primary and preantral follicles in first and second groups compared to control group during TiO2 exposure. Although, significant reduction of apoptotic cells was observed in third group compared to control and sham groups (p≤0.05).
Conclusion: Furthermore, there is little knowledge about physicochemical characteristics of TiO2 nano materials; our findings suggested that TiO2 exposure can defect follicle development and perhaps fertility potential in young mice.
Key words: Titanium dioxide, Ovary, Follicle development, TUNEL.
P-20
Correlation between 25-OH vitamin D in follicular fluid and implantation rate in infertile women undergo IVF/ICSI
Latifi Z1, Farzadi L2, Khayatzadeh Bidgoli H2, Fattahi A1, Nouri M2.
- Department of Clinical Biochemistry, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
- Women’s Reproductive Health Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Introduction: Vitamin D in combination with its receptors can be involved in implantation by regulating gene expression, endometrium immune response and stimulation of endometrium decidualization. So, it seems that the amount of vitamin D in follicular fluids (FF) may has an association with ART success.
Materials and Methods: In a prospective study, 80 infertile female candidates for IVF/ ICSI were enrolled. Blood samples (on the day of human chorionic gonadotropin) and follicular fluids were taken, and then levels of serum estradiol and follicular fluids 25-OH vitamin D were measured. Also clinical characteristics of patients (duration of infertility, causes of infertility, and menstrual status), number and quality of oocytes, number of fertilized oocytes, estradiol levels, and clinical pregnancy were evaluated.
Results: Concentration of FF 25-OH vitamin D in pregnant women was significantly higher than non-pregnant women (p=0.007) but there were no significant differences in age, body mass index (BMI), duration of infertility, menstrual status, number of oocytes, oocytes quality, number of fertilized oocytes and serum estradiol levels between the two groups (p>0.05). Statistically positive correlation was found between 25-OH vitamin D levels with patient age and implantation rate (r=0.264, p=0.018 and r=0.301, p=0.007 respectively).
Conclusion: The obtained results suggest that vitamin D without affecting the number and quality of oocytes can independently improve implantation rate and IVF outcome.
Key words: Embryo implantation, Vitamin D, IVF outcome.
P-21
The protective effect of N-acetyl-L-cysteine on spermatogenesis and sperm characteristics in mice following exposure to Para-Nonylphenol
Malmir M, Soleimani Mehranjani M, Naderi Noreini S.
Faculty of Science, Arak Univercity of Medical Scinecs, Arak, Iran.
Email: mehhdi@yahoo.com
Introduction: Para-Nonylphenol (p-NP) is an environmental contaminant with wide industrial applications which causes oxidative stress in different organs such as the reproductive system. The purpose of this study was to investigate the protective effect of N-acetyl-L-cysteine (NAC), as a powerful antioxidant, on spermatogenesis indices, sperm parameters, chromatin quality and tail length following treatment with para-Nonylphenol in adult mice.
Materials and Methods: 24 adult male NMRI mice (32±4 gr) were divided randomly into 4 groups (n=6), control, NAC (150 mg/kg/day), p-NP (250 mg/kg/day) and p-NP+NAC, and they were treated orally for 35 days. By the end of the treatment, mice were weighed and sacrificed, Their right testis was also weighed then the left caudal epididymis was cut in Ham's F10. Released spermatozoa were used to analyze the motility, viability and the abnormalities of the sperm. The sperm tail length was estimated by stereological methods. Sperm chromatin quality was also assessed by nuclear staining using acridine orange and aniline blue dyes.In continue, the right testis were taken out, fixed, sectioned, processed and stained using heidenhain azan method. Spermatogenesis indices including the tubular differentiation index (TDI), Sertoli cell index (SCI) spermatogenesis index (SI), meiotic index (MI) and repopulation index (RI) were studied. Data were statistically analyzed using one way ANOVA and means were considered significantly different at p<0.05.
Results: A significant decrease in the spermatogenesis indices, sperm motility, viability, sperm tail length and the number of sperms with normal morphology was observed in the p-NP group compared with the control (p<0.002), while no significant change was found in the sperm chromatin quality. The above parameters significantly increased in the p-NP+NAC group compared to the p-NP treated ones (p<0.01).
Conclusion: N-acetylc-L-ysteine, as an antioxidant, can prevent the adverse effects of para-Nonylphenol exposure on spermatogenesis indexes and sperm parameters in mice.
Key words: Para-Nonylphenol, N-acetyl-L-cysteine, Spermatogenesis indices, Sperm parameters.
P-22
The effect of vitamin C on human vitrified sperm parameters in normozoospermic men: raw semen and washed semen
Mangoli E1, Anvari M2. Talebi AR3, Taheri F1, Rahiminia T1, Vatanparast M1.
- Research and Clinical Center For Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Biology and Anatomical Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Andrology, Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: Vitamin C is a common component in seminal fluid. It can improve sperm parameters as an antioxidant. The objective was to evaluate the effect of Vitamin C on human vitrified sperm parameters in normozospermia men.
Materials and Methods: Semen samples were collected from 40 normozoospermic samples and divided into 4 groups. Group 1, raw semen was vitrified. Group 2, semen processed by swim up method and then vitrified. Group 3, vitamin C (600 µm) was added to raw semen and then vitrified. Group 4, vitamin C (600 µm) was added to prepared spermatozoa and then vitrified. The semen analysis was performed according to WHO criteria before and after vitrification.
Results: In this study data showed that progressive motility and immotile spermatozoa after vitrification were significantly different between groups (p=0.014, 0.005 respectively). All groups were compared, and the results revealed that progressive motility and immotile spermatozoa after vitrification were statistically significant in washed semen+vitamin C group compared with wash semen and raw semen groups (p=0.05, 0.01 for progressive motility and p=0.04, 0.007 for immotile spermatozoa respectively).
Conclusion: Adding vitamin C, as an antioxidant, to washed semen has shown beneficial effect on sperm motility after vitrification.
Key words: Sperm parameters, Vitamin C, Vitrification.
P-23
Review of fertility diet and its effect on infertility among women from the viewpoint of Iranian traditional medicine in comparison with modern medicine
Tork Zahrani Sh1, Mirahi A1, Tansaz M2, Alaeddini F3, Hekmatdoost A4.
- School of Nursing and Midwifery, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
- School of Traditional Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
- Ministry of Health, Tehran, Iran.
- Institute and Faculty of Nutrition Sciences and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Introduction: In the recent years the infertility diagnosis and treatment methods have undergone a radical change and development but still, the success rate of modern treatments like in vitro fertilization (IVF) even in the best centers is between 30-40%.
Materials and Methods: The available texts on the Iranian traditional medicine were reviewed. Besides, the relationship between fertility diet factors and infertility among women was investigated through reviewing databases on the world-wide web.
Results: Iranian traditional medicine investigates the relationship between nutrition and infertility from a perspective postulating that complying with fertility diet principles helps maintain healthiness and improve fertility. From this view, body members have their special temperaments; if balanced, body members function normally. Any kind of abnormal temperament happened to the semen or uterus results in infertility or hardship in fertility. These are seen as disorders in sanguine, choleric, melancholic, and phlegmatic temperaments. It should be noted that the bad temperaments are mainly caused by not following the health and fertility diet.
Conclusion: The different viewpoint of the Iranian traditional medicine towards the relationship between infertility and nutrition and its focus on the nature of food and nature of a person could be of interest to researchers in the field and if approved, help to prevent infertility or treat infertile couples.
Key words: Infertility, Iranian traditional medicine, Infertility diet.
P-24
The in vitro fertilization outcome and luteal phase GnRH antagonist administration
Eftekhar M1, Miraj S1, 2.
- Department of Obstetrics and Gynecology, Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Obstetrics and Gynecology, School of Medicine, Shahrekord University of Medical Sciences, Shahrekord, Iran.
Introduction: Genital tissues (ovary, endometrium and placenta) express GnRH receptors. GnRH plays essential roles in embryo implantation, invasion of trophoblastic tissue and steroid synthesis in the placenta. In IVF-ICSI cycles, the use of GnRH antagonists is limited to the last days of ovulation. The aim of this study was to evaluate the effects of GnRH antagonist at pharmacological doses given in the early implantation period on pregnancy.
Materials and Methods: This retrospective study was performed in Yazd Research and Clinical Center for Infertility, 2014-2015. Women under 40 years old, with >20 follicles (>11mm) and risk of OHSS were included. Participants with history of endometriosis, hysteroscopy and history RIF were excluded. The treatment for all of participants was antagonist protocol. Twenty seven patients did not receive Cetrotide in luteal phase, and 67 patients received Cetrotide. Pregnancy outcomes were assessed based on chemical and clinical pregnancy.
Results: Totally 94 patients were included. The mean age of participants was 28.40±4.25 years old. There were no differences in ART and demographic characteristics (p>0.05). The most frequent causes of infertility were: male factor (45.2%) and polycystic ovary syndrome (19.4%). The pregnancy outcome was not significantly different between Cetrotide and non Cetrotide group (p=0.224).
Conclusion: The present study proposed that luteal phase GnRH antagonist administration does not influence the chance of successful pregnancy outcome. The incidence of chemical and clinical pregnancy in two groups was not significantly different.
Key words: GnRH antagonist, Pregnancy outcome, IVF.
P-25
Hazardous effect of acrylamide on development landmarks in rat offspring and the role of Glycyrrhiza Glabra
Mirgalili A1, Yadegari M1, Talebi AR2, Dortaj H1.
- Department of Biology and Anatomy, Faculty of Medical Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Research and Clinical Center for Infertility, Faculty of Medical Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: Acrylamide (AA) is a chemical substance used mainly in certain industrial process. Monomer of Acrylamide formed in food stuffs containing carbohydrates during high temperature cooking.
Materials and Methods: 24 pregnant rats were selected for this study. The pups of these mothers divided into four groups: group A:(control group); group B: (ACR administration 10 mg/kg/day orally); group C: (GG administration 150 mg/kg/day orally); group D: (ACR+GG). At day 21 the pups were evaluated for developmental study. The results were analyzed by SPSS software (15) and p<0.05 was significant.
Results: The result demonstrate that eye opening and fur development appeared occurred slightly later in AA administration group.
Conclusion: These results showed that AA treatments induce delay in developing offspring and GG as an antioxidant reduce these changes.
Key words: Acrylamide, Glycyrrhiza glabra, Eye opening, Fur appearance, Offspring.
P-26
The effect of long-term exercise via TNF-α level on the type-1 diabetic rats in testis tissues
Moghaddami Z1, Sheikhzadeh Hesari F1, Banan khojasteh SM1, Hemmati AR2, Dastranj A3.
- Department of Animal Biology‚ Faculty of Natural Sciences‚ University of Tabriz‚Tabriz‚ Iran.
- Department of Anatomical and Histologyical, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
- Department of Pathology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz‚ Iran.
Introduction: Approximately 90% of diabetes patients often show sexual abnormalities and impotence infertility. Damages caused by hyperglycemia and oxidative conditions result in inflammatory mediators increasing such as cytokines in tissues of sex organs. The TNF-α is the first locally produced cytokines in chronic inflammation pathway. TNF-α level in diabetics blood is higher than normal people that probably histological studies of TNF-α could determine source of this increasing. Also exercise can reduce complications of diabetes especially in tissue by reducing of oxidative elements and establishing of Hypoglycemia.
Materials and Methods: In this study, 40 male rats weighing 200-250 gr were used and randomly were classified into four groups; each group consist 10 rats (control, diabetic, healthy with period of 60-day exercise, diabetic with period of 60-day exercise). Treadmill exercise daily was 1 hr with 22 m/min speed. Diabetes was induced by injection intraperitoneal of streptozotocin with the amount of 60 mg/kg. After expelling of the testis under general anesthesia, samples were homogenized and TNF-α protein levels were measured according to ELISA kit (special rat TNF-α).
Results: The level of TNF-α showed no significant changes among diabetic groups in testicular tissue compare to control groups. Also, exercise didn’t have any negative effects on the level of TNF-α in exercise control group compare with control group. Therefore, we didn’t show any significant change of TNF-α level between our groups.
Conclusion: In this study, the period of disease time may be is not enough to determine the level of TNF-α via exercise treatment in testis tissues. It seems that duration of diabetic developing, severity and type of exercise could be many important reasons to change inflammatory mediators.
Key words: Cytokine, Hyperglycemia, ELISA Kit, Sexual dysfunction.
P-27
Evaluation of progesterone and estradiol levels after GnRH agonist administration in the adult female rat
Mohammadbeigi H1, Peirouvi T1, Rasmi Y2.
- Department of Histology, Faculty of Sciences, Urmia University of Medical Sciences, Urmia, Iran.
- Department of Clinical Biochemistry, Faculty ofSciences, Urmia University of Medical Sciences, Urmia, Iran.
Introduction: Gonadotropin-releasing hormone (GnRH) is secreted from the hypothalamus in a pulsatile manner and stimulates the synthesis and release of gonadotropins, LH and FSH, via GnRH receptor located in adenohypophysis. These gonadotropins, regulate various gonadal functions such as folliculogenesis and steroidogenesis. Various data are reported of GnRH agonists effects on ovary steroidogenesis.
Materials and Methods: 24 female adult Wistar rats were divided into three groups. In the first study group, 300 µg/kg buserelin, in the second study group, 600 µg/kg buserelin and in the control group, the same amount of salin was injected subcutaneous for 5 days. 30 days after the first injection, blood samples were collected from the heart, centrifuged and plasma was isolated from blood. Plasma esteradiol and progesterone levels were measured by ELISA.
Results: The findings reveal a significant incereas in plasma level of estradiol in the second study group (165±104.89 pg/ml) in comparison with control group (55.47±50.32 pg/ml) and in the second study group compared with the first study group (47.28±32.95 pg/ml) (p<0.05). But no significant differences were observed in plasma level of progesterone in the three groups (p>0.05).
Conclusion: Short-term administration of high doses buserelin increase plasma estradiol level, while has no effect on the plasma progesterone level.
Key words: Buserelin, Progesterone, Estradiol, Adult rat.
P-28
A comparative study on the effect of face-to-face or group education during the pregnancy period on sexual function of the couples under coverage of selected clinics in Isfahan in 2013
Mohammadi M1, Khani MB1, Bahadoran P2.
- Department of Nursing and Midwifery, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Nursing and Midwifery, Isfahan University of Medical Sciences, Isfahan, Iran.
Introduction: Pregnancy can conflict with sexual function that can be affected by physical and psychological changes during Pregnancy.
Materials and Methods: In this quasi-experimental pre-post-test study, 64 couples with pregnant women were selected and randomized in two groups in Isfahan. The data were collected via the triangulation of FSFI, BFSI and demographic characteristics questionnaires. SPSS -18 was used to analyze the data by descriptive and inferential statistics.
Results: No significant difference was found in the demographic characteristics between two groups. Education was effective on sexual function in two groups of women (p<0.001), but no significant difference was found between two groups (p=0.61). Also education was effective on sexual function of the men in both groups (p<0.001), and there was a significant difference between the two groups (p=0.003). Meanwhile, there was no significant difference between couples regarding the education (p=0.104).
Conclusion: The results of the study showed that type of education plays a role in improvement of sexual function in pregnancy. In addition, sex education is effective on prevention of sexual disorders in pregnancy. Therefore, having a special approach toward sex education classes during pregnancy is inevitable for the health providers, particularly midwifery professionals.
Key words: Group education, Face to face education, Sexual function, Pregnancy, Iran.
P-29
Assisted reproductive technology outcomes in couples with hepatitis virus infection
Mohsenzadeh M, Omidi M, Nabi A, Khalili MA.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, iran.
Email: mohsenzade@sums.ac.ir
Introduction: Currently, many hepatitis positive infertile couples attend assisted reproductive technology (ART) program, so there is concern as this viral infection may be deleterious to human fertility. Some have suggested that hepatitis virus have an adverse effect on pregnancy outcomes.
Materials and Methods: The study was conducted in two groups of patients: A) seropositive for hepatitis B virus (HBV) and hepatitis C virus (HCV) (n=46); and B) patients lacking these viruses as a control group (n=32). Patients included couples seeking ART from January 2010 to October 2014. ART cycle characteristics and clinical outcomes were assessed between two groups.
Results: Demographic characteristics and age were similar between two groups. The mean±SD age was 30.19±5.54 and 34.31±6.86 years in female and male, respectively. Regarding to oocyte number and embryo score, there was no significant differences between groups A and B. The rate of pregnancy was, however, significantly higher in group B in comparison with A (p=0.03).
Conclusion: Although, the data on embryo formation was similar in both groups of infertiles, but the pregnancy outcomes were noticeably reduced in infertile patients with hepatitis virus infections.
Key words: Hepatitis virus infection, ART, Pregnancy outcome.
P-30
The influence of the Corpus Luteum (CL) on hormonal and biochemical metabolites composition of follicular fluid from different sized follicles and their relationship to serum concentrations in dairy cows
Moradi kor N.
Research Center of Physiology, Faculty of Medicine, Semnan University of Medical Sciences, Semnan, Iran.
Email: moradikor@semums.ac.ir
Introduction: Metabolic changes in blood serum may be rejected in the biochemical composition of follicular fuid (FF) and could indirectly influence oocyte quality. In addition, the levels of hormonal and biochemical metabolites in the FF were related to follicular size and interestingly to the presence or absence of a corpus luteum. The purpose of this study were to examine the influence of the corpus luteum on hormonal and biochemical metabolites composition of follicular fluid (FF) harvested from different sized follicles and their relationship to blood serum in dairy cows.
Materials and Methods: Ovaries were recovered from 30 female adult cows (Holstein Friesian) 4-7 years of age with clinically normal reproductive tracts after slaughtering. Blood samples were collected from the jugular vein before slaughter from each cow. The stage of the cycle in the cows slaughtered was diestrus determined post mortem. The ovaries collected per cow were classified with corpus luteum (CL+) and without corpus luteum (CL-). Visible follicles on the surface of the ovaries were classified, based on their diameter, into (I) small (3-5 mm), (II) medium (6-9 mm) and (III) large (10-20 mm) categories. Follicular fluid was aspirated from different sized follicles in CL+ and CL- ovaries. Serum and FF samples were analyzed for hormones (estradiol-17β, progesterone, testosterone, T3 and T4) and biochemical metabolites (glucose, cholesterol, triglyceride, total protein, albumin and globulin).
Results: Results showed that the FF concentration of estradiol-17β, progesterone and testosterone in different size follicles categories (small, medium and large follicles in CL+ and CL- ovaries) were significantly higher (p˃0.05) when compared with the serum. The FF concentration of estradiol-17β, testosterone, glucose and cholesterol in same follicle size categories of CL- ovaries were significantly higher (p˃0.05) when compared with CL+ ovaries. In the present study, the serum concentration of glucose, cholesterol and triglyceride were significantly higher (p˃0.05) when compared with the fuid from different sized follicles categories in CL+ and CL- ovaries. The differences between follicle size categories in CL+ ovaries were only significant for concentration of estradiol-17β and in CL- ovaries were significant for concentrations of estradiol-17β, glucose and triglyceride.
Conclusion: According to the results of the present study, the levels of the biochemical metabolites in the FF were related to follicular size and interestingly to the presence or absence of corpus luteum.
Key words: Dairy cow, Follicular fluid, Corpus luteum, Hormone, Metabolite.
P-31
Health related quality of life in women with polycystic ovary syndrome
Moridi M1, Rahebi SM1, Seyf Zadeh Z2.
- Guilan University of Medical Sciences, Rasht, Iran.
- Alzahra Hospital, Rasht, Iran.
Introduction: Polycystic ovary syndrome (PCOS) is associated with symptoms that affect psychological wellbeing and health-related quality of life (HRQoL).
Materials and Methods: This was a retrospective case-control study which was conducted on 116 women from April-August 2014 in Rasht, Iran. Cases were 60 newly diagnosed women with PCOS, according to the Rotterdam criteria while the controls were 60 healthy women and groups were matched on sociodemographics characteristics. Data needed to determine health-related quality of life were collected via convenience sampling using modified PCOS quality-of-life questionnaire (MPCOSQ) that contains 6 subscales: emotional disturbances, hirsutism, infertility, weight, menstrual and acne. Data collected were analyzed using statistical T-test, chi square and Kolmogorov-Smirnov.
Results: Finding demonstrated that groups were matched in age, body mass index, marital status, occupation, educational status and socioeconomic status. The mean HRQoL score was significantly lower among women with PCOS (20.96±7.4) than among controls (29.8±4.5) (p<0.001). The mean scores of all subscales in cases (32.01±13.56 for emotional disturbances, 23.34±9.35 for hirsutism, 12.02±6.6 for infertility, 21.25±10.16 for weight, 16.89±6.71 for menstrual and 20.27±7.25 for acne) were significantly lower than controls (47.53±8.28 for emotional disturbances, 31.81±6.68 for hirsutism, 25.83±4.19 for infertility, 20.23±7.70 for weight, 21.15±7.27 for menstrual and 24.26±6.17 for acne) (p<0.001).
Conclusion: According to the results of this study, the average of HRQoL and all the six subscales of MPCOSQ in the case group was significantly lower than in the control group. The psychological implications of PCOS are easily underestimated and have been largely ignored. Clinicians has a pivotal role in recognizing these concerns and implementing therapy to improve quality of life in women with PCOS.
Key words: Polycystic ovarian syndrom, Quality of life, Infertility.
P-32
Counseling during pregnancy and women with precedent infertility
Naeimi N.
Department of Biology, Faculty of Science, University of Sistan and Baluchestan, Zahedan, Iran.
Email: nnaeimi@rocketmail.com
Introduction: Delivery is physiological phenomenon and among the beautiful events in the life of every woman and yet stressful reality. In the present era considering the increasing of caesarean sections, it require using consulting services for informed, correct decisions.
Materials and Methods: In this descriptive-analytical study, 271 pregnant women, completed a researcher-made questionnaire including, demographic characteristics (age and precedent of infertility) and kind and rates of counseling. To analyze the obtained data, chi-square test was used through applying SPSS19 software.
Results: Our results showed that 7 pregnant women (2.6%) had a precedent of infertility while 259 pregnant women had not (95.6%). In total 53 (19.2%) subject did consulting during pregnancy and 213 (80.8%) did not. From these, 6 women with precedent infertility (85.7%) and 47 (14.3%) without, perform counseling during pregnancy. There was a significant relationship between counseling during pregnancy and fertility (p<0.05).
Conclusion: The results showed that women with precedent infertility because of the importance of pregnancy and because of the history of information which were obtained during the course of treatment were more acquainted with the role of consultant, and therefor used consultant services mostly during pregnancy. Ferthermore, considering to the high prevalence of elective cesarean, counseling during pregnancy can informe the pregnant women about the advantage of normal delivery, causing the reduction in cesarean section prevalance.
Key words: Counseling, Precedent infertility, Pregnancy termination.
P-33
Overweight and obesity of patients: Blind spot in assisted reproductive technology treatment
Naghibzadeh M.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: naghib330@yahoo.com
Introduction: The effects of lifestyle on health were investigated via several studies. But, the impacts of overweight and obesity on fertility and reproductive system are poorly understood. Overweight and obesity may reduce fertility rate via reducing the IVF success rate in both women and men undergoing treatment via assisted reproductive technology (ART). Improve lifestyle, including physical exercise and weight loss program, may improve fertility and ART outcome.
Materials and Methods: The results of the related epidemiological and randomised control trials (RTCs) sited on Pubmed and ISI database were selected as references.
Results: The results indicated that the patients with a BMI above 30 have about 68% less chance to have a live birth following their first ART cycle compared with women with a BMI less than 30. Also, overweight and obesity of the patients were associated with both lower implantation and more risk for cycle cancellation. A correlation between obesity and the risk of spontaneous abortion (22% increased risk) was observed in obese women compared with normal weight women. The body composition, specific fat mass, was adversely associated with the success of the IVF procedure. Finally, some studies indicate that weight loss is associated with a higher spontaneous pregnancy rate.
Conclusion: Lifestyle behaviours may have a significant impact on pregnancy rates in women with infertility problems. The results support the clinical recommendation of advising overweight and/ or obese women to lose weight prior to ART. Nevertheless, it seems that the prospective randomized controlled trials are required to prove efficient evidence-based guidelines for weight loss interventions in overweight and/ or obese women before the ART procedure.
Key words: Lifestyle, Obesity, Fertility, ART.
P-34
Role of nitric oxide in the developmental process of ovarian follicles in pregnant rats
Naseri Sh, Khanehzad M, Noori Moogehi SMH.
Department of Histology, Faculty of Medicine, Tehran University of Medical Sciences (TUMS), Tehran, Iran.
Email: naserishafighe@yahoo.com
Introduction: Nitric Oxide (NO)- as one of the smallest active products, involved in control of a number of physiological processes including cell growth, apoptosis, regulation of blood pressure, defense mechanism and especially in the reproductive process.
Materials and Methods: We used 40 Wistar pregnant rats between 200 and 250 grams in weight and aged eight weeks. Based on observation of vaginal plug, pregnant mice were divided into five groups. The first group was received 2 mg/kg normal saline and the others were received respectively 200 mg/kg L-Arginine, 20 mg/kg L-NAME and a mixture of the same doses of L-Arginine and L-NAME on 3, 4 and 5th gestational days via intraperitoneal. The control group did not received any injection. Ovaries were removed on 18th gestational days, and after fixation and tissue preparation via staining by the routine H&E method, studied by Light microscopy.
Results: Comparing the groups using ANOVA, there was a significant difference in reducing the number of primary follicles and increasing atretic follicles in the L-Arginine group in comparision with the other groups. The histological changes were also observed in the L-Arginine group.
Conclusion: The study results showed that nitric oxide during pregnancy has damaging effects on ovaries and is recommended to be used with caution during pregnancy.
Key words: Ovarian follicles, Nitric oxide, L-Arginine, L-NAME.
P-35
Comparison of ovarian stimulation protocols base on AMH level in patients undergoing intracytoplasmic sperm injection
Nikmard F1, Hosseini E1, Bakhtiyari M1, Ashrafi M2, Aflatoonian A3, Aflatoonian B4, Aflatoonian R2.
- Anatomy Department, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
- Department of Endocrinology and Female Infertility at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Obstetrics and Gynecology, Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Yazd Centre for Stem Cell Biology, Research and Clinical Center for Infertility, Shahid Sadoughi University for Medical Sciences, Yazd, Iran.
Introduction: Controlled ovarian hyperstimulation (COH) plays important role in reproductive medicine because selection of appropriate ovarian stimulation strategy can improve assisted reproductive technology outcomes. Antimullerian hormone (AMH) is a predictor of ovarian response, which can help to select the best treatment strategies in women undergoing agonist and antagonist protocols to optimize safety and clinical pregnancy rates.
Materials and Methods: This study is a retrospective study conducted in a private assisted reproductive unite and a total of 243 patients with tubal factor infertility are selected.
Results: In both of GnRH agonist and antagonist protocol, with increasing of AMH level, oocyte and good embryo number is increased. On the other hand in AMH˂1.1, ˃ 2.8 ng/ml levels, GnRH agonist leads to higher oocyte and embryo number that is significant. On the contrary, pregnancy rate with increasing of AMH level is not increased and the highest rate of pregnancy is observed in AMH 1.1-2.8 ng/ml levels that it observed with antagonist protocol.
Conclusion: Based on AMH levels we can predict assisted reproductive outcomes. In three range of AMH levels, GnRH agonist protocol can lead to better results. In the women with poor prognosis and low and high levels of AMH, it should focus on improving results with increasing of endometrial receptivity or embryo quality.
Key words: Antimullerian hormone, Ovarian response, ovarian stimulation protocols.
P-36
Prenatal exposure to a single dose of testosterone leads to appearance of polycystic ovary syndrome in female rat’s offspring in adulthood
Noroozzadeh M1, Ramezani Tehrani F1, Zahediasl S2, Piryaei A3, Azizi F2.
- Reproductive Endocrinology Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
- Endocrine Physiology Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
- Department of Biology and Anatomical Sciences, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Introduction: Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women, affecting 8-12% of reproductive-aged women. PCOS is associated with reproductive and metabolic disorders, including infertility, hyperandrogenism, luteinizing hormone hypersecretion, polycystic ovaries, insulin resistance and dyslipidaemia, with an increased risk of cardiovascular disease and type 2 diabetes in those affected. PCOS is known as a genetic disease. Beside genetic factors, environmental factors may contribute to appearance of this syndrome.
Materials and Methods: Pregnant rats were divided into two groups, experimental and control groups (n=10 in each group). Experimental group were subcutaneously injected with 5 mg free testosterone on gestational day 20 and control rats received only solvent at the same time. The female offspring of these mothers were examined for the functioning of their reproductive system in adulthood.
Results: Levels of testosterone and luteinizing hormone and the luteinizing hormone/follicle-stimulating hormone ratio were increased in prenatally androgenized offspring compared with control rats (p<0.05). The numbers of preantral and antral follicles in the ovaries of prenatally androgenized offspring were also increased compared with control rats (p=0.07 and p<0.01, respectively). The number of corpora lutea was decreased in prenatally androgenized offspring compared with control rats. Cystic follicles were observed in the ovaries of prenatally androgenized offspring.
Conclusion: Prenatal exposure to a single dose of testosterone during the critical period of fetal development leads to appearance of PCOS phenotype in female rats with minimal morphological disorders in their reproductive system in adulthood. Production of a functional rat model that resembles many features of PCOS may contribute to a better understanding of this syndrome.
Key words: Fetal life, Testosterone, Polycystic ovary syndrome, Rat.
P-37
Advanced paternal age does not influence the outcomes of ART cycles
Omidi M, Halvaei I, Khalili MA, Ghazali Sh, Tahajjodi S, Sabeti P.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: omidi.marjan@ymail.com
Introduction: The reports in assisted reproductive technology (ART) cycles noted that the fertilization rates were significantly decreased in men over 50 years of age. However, data have shown that there was no adequate substantiation to prove the effects of paternal age on fertility outcomes.
Materials and Methods: In a retrospective study, the outcome of intracytoplasmic sperm injection (ICSI) in ART cycles in two groups of patients was studied. Patients regarding etiology of infertility were divided into two groups: A) male factor infertility (n=47), and B) female factor infertility (n=16). Sperm parameters, ART cycle characteristics and clinical outcomes were assessed between two groups.
Results: There were no significant differences for rates of sperm count and morphology between two groups, but rate of spermatozoa with progressive motility were higher in group B in comparison with A (p=0.002). The rates of high quality embryos, pregnancy and live birth showed no significant differences between the groups (p=0.4, p=0.4, p=0.2, respectively).
Conclusion: It appears that the paternal age has no detrimental effect on rates of pregnancy and live birth in ART program.
Key words: Paternal age, ART, Live birth.
P-38
Effect of non-polar extract of Phaleria macrocarpa (Mahkota dewa) on serum testosterone level and libido behavior in male rats
Aziz Dollah M1, Parhizkar S2, Ferdaus Binti N1.
- Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University Putra, Malaysia.
- Medicinal Plants Research Centre, Yasuj University of Medical Sciences, Yasuj, Iran.
Introduction: Phaleria macrocarpa (PM) has been claimed to overcome male fertility problems related to reduction of testosterone level.
Materials and Methods: Non-polar hexane extract of PM was prepared using soxhlet extraction technique, dried to powder using rotary evaporator and been kept for later use. In total 30 male adult Spraque Dawley rats weight about 250 gr were divided into five groups. For libido study, 30 female rats weight about 250 gr each were used for the purpose of mounting latency and mounting frequency study. The male rats were kept in individual cage while for female rats, there were five rats for one cage. Three groups of rats were given three different concentration of PM non-polar extract [High dose (60 mg/kg), Medium dose (12 mg/kg) and Low dose (6 mg/kg)]. One group was given tween 20 solution (negative control) and another group was given commercial testosterone hormone (positive control) orally for seven weeks. At the end of experiment period each male rat was introduced to one female rat to determine the libido behavior. Blood sample was then collected using cardiac puncture and the testosterone level was determined by radioimmunoassay kit (TESTO-CTK P3093).
Results: Administration of non-polar hexane extract of PM showed no significant changed for the serum testosterone level and mounting latency of the rats. While, there was a significant change (p<0.05) in the mounting frequency in different groups; 6, 14, 14, 4 and 7 times for the negative control, low dose, medium dose, high dose, and positive control respectively.
Conclusion: PM non-polar hexane extract did not show a potential value as an alternative way to improve the sexual strength. However it showed a potential for the improvement of the mounting frequency.
Key words: Phaleria macrocarpa (PM), Testosterone level, Libido behavior.
P-39
Effect of vitamin C on sperm parameters and serum malondialdehyde levels in mice fallowing treatment with sodium arsenite
Pirozmanesh H, Soleimani Mehranjani M, Naderi Noreini S.
Faculty of Science, Arak University of Medical ASciences, Arak, Iran.
Email: hp457@yahoo.com
Introduction: Arsenic as an environmental toxicant is able to exert malformation in male reproductive system by inducing oxidative stress. Ascorbic acid with potent antioxidant property is able to restrict oxidative stress.
Materials and Methods: In this experimental study, 24 NMRI mice were divided into four groups (n=6): control, sodium arsenite (7 mg/kg), ascorbic acid (150 mg/kg) and ascorbic acid + sodium arsenite. Oral treatment was performed for five weeks. At the end, left cauda epididymis was removed and used to analyze sperm viability, morphology, motility and sperm tail length. Serum MDA levels, as lipid peroxidation index, was measured by spectrophotometeric method. Data were analyzed using one way ANOVA and means difference was considered significant at p<0.05.
Results: The mean of sperm viability, morphology, motility and sperm tail length significantly decreased in sodium arsenite group compared to control (p<0.04). A significant increase in the serum MDA levels was found in sodium arsenite group compared to control (p<0.05). In ascorbic acid+ sodium arsenite group, a significantly reversed in adverse effect of sodium arsenite on these parameters was observed when compared with sodium arsenite group (p<0.02). In addition sperm viability, motility and sperm tail length was significantly increased in the mice treated with ascorbic acid alone in comparing to the control ones (p<0.001). Also, a significant reduction in MDA levels was found in ascorbic acid group compared with control group (p<0.001).
Conclusion: Ascorbic acid could compensate the adverse effects of sodium arsenite on viability, morphology, motility, sperm tail length and serum MDA levels in adult mice. Hence, this study suggests that ascorbic acid may improve the male fertility by improving sperm parameters.
Key words: Sodium arsenite, Ascorbic acid, Viability, Motility, Sperm tail length.
P-40
The predictors of spiritual growth and interpersonal relations in infertile couples referring to the Infertility Center, Al-zahra Hospital, Tabriz, Iran.
Rahimi M, Sehhatie Shafa'I F, Mirghaforvand M.
Tabriz University of Medical Sciences, Tabriz, Iran.
Email: marieh.rahimi@yahoo.com
Introduction: In all cultures, involuntary childlessness is recognized as a crisis that has the potential to threaten the stability of individuals, relationships, and communities. Every society has culturally approved solutions to infertility involving, either alone or together with alterations of social relationships (e.g., divorce or adoption), spiritual intercession (e.g., prayer or pilgrimage to spiritually powerful sites), or medical interventions.
Materials and Methods: This study is a descriptive cross-sectional study on 322 infertile couples referring to the Infertility Center at Al-zahra Hospital in Tabriz. Samples were selected through simple random sampling method. Questionnaires used in the study were demographic data questionnaire and Lifestyle Profile II (HPLP-II) questionnaire with 52 questions. The multivariate linear regression analysis method was used for defining the social-individual predictors of Spiritual Growth and Interpersonal relations. Data were analyzed by SPSS Win/13.
Results: The findings showed that the mean (SD) score of Spiritual Growth and Interpersonal relations in couples was 2.6 (0.5) out of 1-3 grade range. The variable of job, family member, history of contraception, reason of infertility, and education level were the predictors of Spiritual Growth and Interpersonal relations.
Conclusion: According to the results of the study, the mean score of Spiritual Growth and Interpersonal relations in infertile couples is average, therefore we should focus on predictive variables for recovering the infertile couples’ Health-promoting behaviors status.
Key words: Health-promoting behaviors, Spiritual Growth, Interpersonal relations.
P-41
Comparing in-vitro maturation of oocytes in presence of mature or immature cumulus cells in mice
Rahiminia T, Faramarzi A, Khoradmehr A, Khalili MA.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: tahere_1980@yahoo.com
Introduction: Cumulus cells (CCs) are somatic cells which are coupled with oocyte during maturation. During process of maturation, the secretions by CCs influence oocyte to maintain the functional competence.
Materials and Methods: Superovulated mice were killed and ampulla was ruptured to release cumulus oocyte complex (COCs) in IVM culture media. COCs were incubated for 30 min before removing oocytes. Then, denuded mature oocytes were removed and remaining mature CCs were collected for culturing germinal vesicles (GVs) for exp II. After dissecting ovaries, denuded GVs were considered as control group and put in IVM medium. In exp I, collected GVs with intact immature CCs were put in IVM medium and observed for maturation after 24 and 48 hr. In exp II, GVs were cultured with mature CCs and maturation was checked as mentioned for exp I group.
Results: The rate of maturation was (77.36±14.4) in control group. In exp I and exp II, the rate of maturation was (91.32±22.5) and (63.33±7.4) respectively. Difference in maturation rate was significant between two groups of exp I and exp II (p=0.04) at 24 hr. However, maturation rate did not increase after 24 hr. The average of maturation rate in metaphase I (MI) oocytes was as low as about 15% in different groups at 24 hr and did not increase by 48 hr. Degeneration rate was increased from time 0-48 hr in exp II more than this rate in the other two groups.
Conclusion: Presence of mature CC did not improve maturation of GVs comparing to exp I with immature CCs, or control group as conventional IVM.
Key words: In vitro maturation, Cumulus cells, Mice.
P-42
Investigating the effect of culture pH, on Chinese hamster ovary (CHO) cells growth and recombinant human chorionic gonadotropin (hCG), expression
Rahnama M, Sanati MH, Amiri Yekta A, Gourabi H.
Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: mhsanati@yahoo.com
Introduction: The demand for production of recombinant therapeutic proteins by mammalian cell lines, such as Chinese hamster ovary (CHO) cells, continues to grow. Significant achievements in process optimization including development of cell culture strategies for large-scale, cost effective production have been made. One of the key parameters, that has effect on mammalian cell growth, metabolism and productivity, is pH. Human chorionic gonadotropin (hCG), is a member of pituitary glycoprotein hormone family, which has an important role in regulating human reproductive functions and is widely used in assisted reproductive technologies (ART).
Materials and Methods: Equal number of rCHO cells were cultured in four DMEM media with different pH ranging from 6.8-7.7 (6.8, 7, 7.3 and 7.7). The cells were harvested at confluence and viable cell concentration was determined by trypan blue exclusion. The rhCG production was assessed using SDS-PAGE, Bradford and Western blotting techniques. ImageJ software was used for quantitating Western blotting results.
Results: Results demonstrated that maximum viable cell concentration was at pH=7. Bradford assay showed that total protein concentration reached its maximum level at pH=7 and its minimum at pH=6.8. hCG expression was confirmed by SDS-PAGE and western blotting methods. According to ImageJ analysis, maximum hCG expression level was found at pH=7, and minimun expression at pH=7.7 but no significant difference was found at pH=7.7 and pH=6.8. Overall, pH=7 was assessed as optimum pH for culturing rCHO cells.
Conclusion: Several strategies including culture condition optimization, have developed to improve productivity of a recombinant cell line. Findings suggest that optimizing simple parameters such as culture pH, had the potential to increase the viability and productivity of recombinant CHO cells.
Key words: Chinese hamster ovary (CHO) cell, Optimization, pH, Human chorionic gonadotropin (hCG), Assisted reproductive technology (ART).
P-43
Noscapine induced cell death in human endometriotic epithelial cells
Rashidi Z1, Khazaei M2, Khazaei MR2.
- Department of Anatomy, Faculty of Medicin, Tehran University of Medical Sciences, Tehran, Iran.
- Department of Anatomy, Faculty of Medicin, Kermanshah University of Medical Sciences, Kermanshah, Iran.
Introduction: Endometriosis is the presence of a functional endometrial tissue outside the uterine cavity and defines as cancer like model. Noscapine is a safe cough suppressant which has been introduced as cancer suppressor.
Materials and Methods: Human endometriotic biopsies (n=7) were digested by enzymatic method (collagenase, 2 mg/ml). Epithelial glands were collected by sequential filtration through nylon meshes (70 and 40 µm). The cells were divided to five groups: control and 10, 25, 50 and 100µM concentration of noscapine and cultured for 24, 48 and 72 hr.Viability of cells was assessed by MTT assay; cell morphological analyses with Acridine orange (AO)-ethidium bromide (EB) double staining also cell death was assessed by TUNEL assay.
Results: Viability of endometrial epithelial cells were decreased in 10, 25, 50 and 100 µM noscapine concentration (84.1%, 80.8%, 72.7%, 67.1%) compared to control group (91.0%) in 48 hr respectively. Cell death increased in high concentrations noscapine and were increased TUNEL positive cells in 10, 25, 50 and 100 µm noscapine concentration (9.04%, 13.07%, 15.15% and 17.82%) respectively compared to control group (4.96%) in 48 hr (p<0.05).
Conclusion: Noscapineas inducer cell death decreased endometriotic epithelial cells viability and decreased apoptotic index in dose dependent manner. It can suggest for endometriosis treatment.
Key words: Apoptosis, Epithelial Cell, Endometriosis, Noscapine.
P-44
The effect of chronic prenatal stress on insulin secretion from Langerhans isolated islets in male offspring rats
Rostamkhani F1, Zardooz H2, Shirvani H3.
- Young Researchers and Elite Club, Yadegar-e-Imam Khomeini (RAH) Branch, Islamic Azad University, Tehran, Iran.
- Neuroscience Research Center and Department of Physiology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
- Exercise Physiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Introduction: A large number of studies have reported associations between prenatal stress and offspring lifetime consequences. Chronic gestational stress alters maternal glucocorticids and subsequently disturbs intrauterine environment which may lead to metabolic disorders in the offspring.
Materials and Methods: In this study the effects of gestational 8 and 20 days foot-shock and psychological stress on body weight, plasma corticosterone, insulin, glucose, concentrations and also insulin secretion from isolated pancreatic islets of rats' offsprings were examined. Stress was induced by Communication Box twice a day (1 h/session) for 8 consecutive days beginning on E8 in 8-day stressed group and for 20 consecutive days beginning on E1 in 20-day stressed group.
Results: The results obtained from this investigation indicate that 8 and 20-day chronic foot-shock stress arises maternal plasma corticosterone concentration. Prenatal stress induces lower birth weight and body weight gain in offspring. Insulin secretion from isolated pancreatic islets of offspring in 8 and 20-day foot-shock stress groups in presence of 8.3 and 16.7 mM glucose increased as compared to the control and psychological groups. Furthermore, prenatal stressed offspring had significant elevation in plasma glucose concentration without marked alteration in plasma insulin and corticosterone concentrations.
Conclusion: These data suggest that prenatal stress could result in impaired glucose metabolism in the offspring which is independent of timing of the stress exposure.
Key words: Prenatal stress, Corticosterone, Insulin, Glucose, Langerhans isolated islets.
P-45
In vitro culture of vitrified mouse ovarian tissue derived preantral follicles in two and three dimensional systems
Sadr SZ1, Ebrahimi B1, Shahhoseini M2, Fatehi R1, Favaedi R2, Maroufizadeh S3.
- Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Epidemiology and Reproductive Health at Reproductive Epidemiology Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: Cryopreservation is the best choice to preserve fertility in patients exposed to premature ovarian failure. Designing an appropriate system for in vitro follicle culture that resembles in vivo conditions is really valuable and could improve the usage of cryopreserved tissue.
Materials and Methods: Ovaries of 12-day-old female NMRI mice were exposed to EG and DMSO combination (7.5% and 15%) in two steps for 15 and 30 minutes respectively, then cryopreserved by needle immersed method (NIV). Afterwards, middle sized preantral follicles (110-130 µm) were mechanically isolated and were distributed into two different groups: two dimensional culture system (2D) and three dimensional culture system (3D; alginate encapsulation). Both groups were cultured for 12 days in α-MEM that supplemented with 10 mIU FSH, 1% ITS, 50 ng/ml activin A and 5% FBS. Finally, follicular morphology, survival, growth rate and also quantitative expression of oocyte maturation genes (Gdf9, Bmp15 and Bmp6) were studied on the first and last days of culture.
Results: At the end of culture period, although follicular morphology in 3D culture system was better preserved as compared to the 2D one, survival rate of cultured preantral follicles was significantly (p<0.05) lower than in 2D group. Expression analysis of oocyte maturation genes indicated a reduction process in both groups during 12 days of culture. In the last day of culture period, genes expression level in 3D culture system was significantly lower than 2D group.
Conclusion: Although the better survival rate was seen in 2D culture system, morphologic characteristics including antrum formation and follicle development was extremely better preserved in 3D culture system. Overall, as three dimensional culture system mimic in vivo conditions, so could be used as a more appropriate system for follicle culture.
Key words: Ovarian tissue, Preantral follicle, Two dimensional culture system, Three dimensional culture system, Vitrification.
P-46
Routine use of EmbryoGlue® as embryo transfer medium does not improve the ART outcomes
Safari S1, Razi MH1, Safari S2, Razi Y3.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Qom University of Medical Sciences, Qom, Iran.
- Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: The assisted reproductive technology (ART) success rates have been significantly improved over last decades; however, embryo implantation still remains a major limiting factor. The composition of an embryo transfer (ET) medium is important for interaction between embryo and endometrium at the time of implantation. Some modifications to the embryo culture media are developed to mimic the in vivo conditions. One of the examples for that is supplementation of ET media with hyaluronan (HA, hyaluronic acid), a major glycosaminoglycan in uterine fluid, which assumed to improve the process of implantation.
Materials and Methods: A cohort of total 229 patients was retrospectively enrolled for the present study. They were subjected for ET on day 2 either in EmbryoGlue® (n=117) as study group or in conventional ET medium with low concentration of HA as control group (n=112).
Results: Patients in the both groups, in regards to the mean level of day 3 FSH, the etiology of infertility, the history of implantation failure and the rate of good quality embryos showed similar characteristics. There were no significant differences between two groups in terms of clinical and ongoing pregnancies, implantation, delivery and live birth rates. In spite of a decreased abortion and increased multiple pregnancy rates in the study group compared to the control group (15.8 vs. 19% and 20.6 vs. 15.6 respectively), the differences were not statistically significant.
Conclusion: Routine use of EmbryoGlue® as a HA enriched ET medium for cleavage stage embryos does not have advantage to the conventional one for infertile patients undergoing ART.
Key words: Hyaluronan, EmbryoGlue®, Embryo transfer, Pregnancy outcome.
P-47
The relation between vitamin C in follicular fluid with the morphology of oocyte and the quality of embryo in IVF patient
Saffari S1, Bahadori MH2, Sharami SH3, TorabZadeh P1, Goudarzvand M1.
- Department of Developmental Biology, Karaj Branch, Islamic Azad University, Karaj, Iran.
- Cellular and Moulicular Research Center, Guilan University of Medical Sciences, Rasht, Iran.
- AlZahra Reproductive Health and Medical Research Center, Rasht, Iran.
Introduction: Oxidative stress and the inappropriate effect of reactive oxygen in the body or laboratory circumstances can decrease the quality of embryo and sexual cells. Ascorbic acid is a natural antioxidant which has a protective effect in the body.
Materials and Methods: This is a cross-sectional analytical study which was conducted on 50 women with IVF referred to Al Zahra Hospital, Rasht, Iran. 20-45 years old women with infertility caused by tubal dysfunction, ovarian factors or male factors were included. Patients underwent same protocol for stimulating; the ovulation and injection of 10,000 units of hCG. After 36 hr, follicles were suctioned and matured oocytes were separated for fertilization. Vitamin C was assessed biochemically and the morphology of oocyte and the quality of embryo were evaluated by by inverted optical microscope.
Results: Among patients 583 oocytes and 275 embryos were assessed. There was no significant relation between, age, BMI and duration of infertility with vitamin C (p>0.05). Also there was no significant relation between vitamin C, the maturity of oocyte and the quality of embryo (p>0.05). But MII oocytes were more in patients with vitamin C less than 1 in comparison with ≥1 (p=0.038). Also the mean 2PN embryos in 0-0.05 level of vitamin C was higher than other groups but no significant relation was noted (p=0.719).
Conclusion: According to our results vitamin C at special level can improve the morphology of oocytes and the quality of embryos.
Key words: Infertility, Follicular fluid, IVF, Vitamin C, Oocyte, Embryo, Antioxidants.
P-48
Polscope analysis of meiotic spindle and zona pellucida birefringence of metaphase ΙΙ oocytes in polycystic ovarian syndrome patients
Safian F1, Khalili MA1, 2, Ashourzadeh S2, 3, Omidi M2.
- Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Afzalipoor Clinical Center for Infertility, Afzalipoor Hospital, Kerman University of Medical Sciences, Kerman, Iran.
Introduction: Polycystic ovarian syndrome (PCOS) is one of the most common causes of infertility in women of reproductive age. Currently, one of the best therapeutic options for PCOS patients is ICSI. Moreover, IVM can be a useful technique for women with PCOS who are at the risk of ovarian hyperstimulation syndrome (OHSS). On the other hand, the oocyte quality can be a determining factor for outcome of ICSI cycles.
Materials and Methods: This prospective study included immature oocytes (30 GV and 5 MI) undergoing IVM, and MII oocytes obtained from PCOS patients (29.64±5.31 years) in ICSI program. Using a PolScope, the presence of MS and ZP birefringence was assessed in both in vivo-matured (n=32) and matured oocytes after IVM (n=24). Oocytes were classified as high birefringent (HB) ZP and low birefringent (LB) ZP. Furthermore, the rates of fertilization and embryo development were evaluated.
Results: The maturation rate was 68.5% after IVM. Analysis revealed that the percentage of a HB ZP was significantly higher in the IVM oocytes than in vivo-matured ones (58.3% vs. 31.2%, p=0.04). There was insignificant relationship between spindle detection and either in vivo-maturation or IVM oocytes (p=0.53). Likewise, there were similar outcomes for the rates of fertilization and embryo development after ICSI between two groups (p=0.80 and p=0.13, respectively).
Conclusion: Clinical IVM is a safe technology for the maturation and maintenance of oocytes integrity. Furthermore, the use of non-invasive PolScope is recommended for the detection of healthy oocytes in ICSI.
Key words: PCOS, IVM, PolScope, ZP birefringence, Meiotic spindle.
P-49
History and application of public bank of umbilical cord blood
Seyfipoor Naghne E1, Bolorytabar F1, Khoshravash S2.
- Department of Nursing and Midwifery, Islamic Azad University, Felavarjan Branch, Isfahan, Iran.
- Department of Nursing and Midwifery, Isfahan University of Medical Sciences, Isfahan, Iran.
Introduction: Umbilical cord blood is rich of hematopoietic stem cells. Today, umbilical cord blood constitutes about 20% of stem cells transplant. 75% of umbilical cord blood banks in the world are public and the remaining are private (trade). 27 years after the first successful umbilical cord blood transplant, more than 555,000 umbilical cord blood units have been made available around the world after performing qualitative study in public bank of umbilical cord blood in order to treat patients who need hematopoietic stem cells transplant. The first program of public umbilical cord blood bank was implemented by the New York in 1991 and in Asia in 1999 in Japan. In Iran in late 2008, the Stem Cells Association has started its activity by receiving financial supports. In this type of storing, umbilical cord blood does not only belong only to the family and public organizations are responsible for paying related costs. Cells in this banking are kept safe and number of their alive cells remains unchanged during the freezing process. Samples saved in bank in terms of health indices such as viral and microbial contaminations are evaluated by colony measurement method. With regard to high prevalence of blood malignancies and thalassemia in Iran, it has been very important to establish public bank of umbilical cord blood and support it spiritually and materially.
Materials and Methods: Subjects of this review study are derived from articles and sites related to collecting, maintain and transplanting umbilical cord blood.
Results: Development of umbilical cord blood banks to develop transplantation centers and treat patients is of unavoidable necessities in the society health system. In societies that the government has ability to establish public bank and supports this bank, development of public banks has preference to development of private banks.
Conclusion: In year 2011, research and development department established public bank of umbilical cord blood in order to scientific and practical promoting and proceeded to amniotic membrane, standardization of tissue adhesive, production and standardization of amniotic extract and platelet extract by establishing bank. Collecting this bio garbage and transforming it to biologic bands, eye drop to improve cornea wounds and mixing hydrogen with amniotic membrane extract to treat wounds for therapeutic consumptions is economical and is effective to decrease therapeutic costs. Also fibrin glue is produced only via umbilical cord blood. Fibrin glue as cell carrier and tissue adhesive is widely used in surgery and cell therapy. Transplant of stem cells of umbilical cord has opened bright and promising horizons for medical society to treat patients with different chronic and acute disorders. So, saving umbilical cord blood finds high importance and its necessity feels further.
Key words: Umbilical cord, Blood bank
P-50
Protective effect of alcoholic extract of Garden cress (Lepidium sativum) seeds on the histopathological changes of epididymis and fertility in diabetic rats
Seyed Hoseini ES1, Nikzad H1, Amini Mahabadi J1, Haddad Kashani H2.
- Gametogenesis Research Center, Kashan University of Medical Sciences, Kashan, Iran.
- Anatomical Sciences Research Center, Kashan University of Medical Sciences, Kashan, Iran.
Introduction: Diabetes mellitus (DM) is a frequent and serious metabolic illness all over the world and plants have been a desirable source of medicine recently. Diabetes has unpleasant effect on male reproductive system and it may lead to male infertility. It causes erectile dysfunction and reduces ejaculate volume by affecting the health of small blood vessels and the small nerves that control ejaculation and also decrease libido by decreasing testosterone levels. Current study evaluated the possible protective efficiency of Garden cress (Lepidium sativum) seed extract on fasting blood sugar (FBS) and then assessed epididymal histopathologic changes in streptozotocine (STZ)-induced diabetic rats.
Materials and Methods: 50 male adult Wistar rats were randomly categorized into five groups (each 10 rats). Groups 1 were control placebo group receiving only 0.1 ml normal saline via gastric gavages, Group 2 as control diabetic rats received an intraperitoneally injection of STZ 60 mg/kg body weight. Rats with FBS >250 mg/dl were considered as diabetic. Group 3 were diabetic rats receiving insulin in dose 3U/100 g body weight and Groups 4 and 5 were diabetic rats that received 0.1 cc of 200 and 400 mg/kg‚ ethanol extract of Lepidium sativum seed by gavages daily. One day after the last gavages, rats were anesthetized by chloroform. Epididymis duct was removed from abdomen and weighed with a digital scale. Afterwards, samples were putted in Bouin's solution for histological measurement.
Results: Administration of 200 and 400 mg/ml doses of Lepidium sativum seed extract increased epithelium height and decreased interstitial volume density and fibro muscular thickness significantly. Also, volume density of epithelium, fibro muscular, lumen and interstitial were decreased significantly. Tubular and lumen diameter did not change significantly in different groups.
Conclusion: It seems Lepidium sativum seed extract has benefit as a supplementary protective agent against bad effects of diabetes on reproductive system in diabetic male.
Key words: Diabetes‚ Lepidium sativum seed extract, Epididymis‚ Streptozotocine‚ Insulin.
P-51
New insights into the effect of aflatoxin on infertility of animal models
Sheikhpour R, Jabbari S.
Yazd Medical Science Branch, Islamic Azad University, Yazd, Iran.
Email: R.sheikhpour@yahoo.com
Introduction: Aflatoxins are produced via Aspergillus flavus and Aspergillus parasiticus, species of fungi. Toxic and carcinogenic properties of Aflatoxin are determined. After entering the body, Aflatoxins may be metabolized by liver to a reactive epoxide intermediate or hydroxylated to become the less harmful. Nowadays, infertility properties of Aflotoxine have been known in studies.
Materials and Methods: Literature review was conducted using the following databases: SciELO, Lilacs, Medline, PubMed, Scopus and science direct from 2000-2014. The key-words used were Aflatoxin, infertility, fertility, animal, human.
Results: When Aflatoxin level increased in diet of male goats, glucose and total protein concentration were decreased in the testes and testosterone level was significantly reduced. The study suggests that exposure of male goats to dietary Aflatoxin will reduced testicular biochemical and testosterone with resultant depression in sperm storage capability and daily sperm production in the animals. Chronic Aflatoxin exposure in animals can result in impaired reproductive efficiency. One study showed that Aflatoxin decreased fertility, abortion, lowered birth weights and caused disturbances in hormonal metabolism in sheep. Another study showed that the fertility of treated mice with Aflatoxin at a daily dose of 50 µg/kg body weight was reduced drastically. Sperm concentration in the epididymis and sperm motility decreased whereas sperm abnormalities increased.
Conclusion: The results of studies showed that Aflatoxin can cause infertility in animal models, but these results need to be evaluated by more clinical trials.
Key words: Aflatoxin, Infertility, Animal.
P-52
ART cycles outcome in donor oocyte recipient couples: A case-control study
Tahajjodi S, Khalili MA, Nabi A, Omidi M.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Science, Yazd, Iran.
Email: s.tahajjodi@yahoo.com
Introduction: Oocyte donation is an acceptable alteration in women infertility treatment for different indications. Several factors including the age of the oocyte donor and recipient, oocyte and embryo quality and characteristics of endometrium can influence on ART outcome.
Materials and Methods: Outcome of 202 cycles, consist of 113 DO cycles and 89 AO cycles, were compared. Both DO and AO groups were divided to two subgroups according to fresh and frozen embryo transfer. First DO and AO groups included the fresh embryo transfer (fDO, fAO) and another DO and AO groups included the frozen embryo transfer (fzDO, fzAO). Comparison was performed in regard to chemical pregnancy, clinical pregnancy, implantation rate, live birth and abortion rate.
Results: DO (mean=28.29 years) and AO (mean=29 years) groups were significantly matched by age. No significant differences were observed between fDO and fAO groups and also between fzDO and fzAO groups with regard to chemical pregnancy, clinical pregnancy, implantation rate, live birth and abortion rate. But, there was a significant increase in oocyte number, MII oocyte number and embryo number in DO compared with AO groups (p<0.001).
Conclusion: Despite of increasing in total number of oocyte, MII oocyte and embryo, our findings showed that oocyte donation program has no impact on ART pregnancy outcome.
Key words: Oocyte donation, ART outcome, Women infertility.
P-53
Dose stress always has a negative effect on sperm cell?
Vaseghi-Dodaran H1, Zhandi M2, Daghighkia H1, Sobhani A3.
- Department of Animal Sciences, Faculty of Agriculture, Tabriz University, Tabriz, Iran.
- Department of Animal Sciences, College of Agriculture and Natural Resources, University of Tehran, Tehran, Iran.
- Department of Animal Sciences, Faculty of Agriculture, Mohaghegh Ardabili University, Ardabil, Iran.
Introduction: Cryopreservation of semen requires optimized condition to minimize the harmful effects of various stresses. Oxidative stressors are known to be major mediators of cell damages during cryopreservation. An important perspective is that free radicals are not exclusively beneficial or exclusively detrimental. Rather, they need to be maintained at appropriate levels to ensure physiological function, while preventing pathological damage. Recent studies showed that placing the cells in a controlled stress conditions could affect cell survival in later stages.
Materials and Methods: This article discusses some of the new approaches employed for improve spermatozoa before and after freezing-thawing. A review of recent bibliography was carried out in PubMed, Google scholar and SID by the use of relevant keywords.
Results: Osmotic, oxidative, hydrostatic and mechanical stressors before freezing induce expression a series of heat shock proteins (HSPs), decrease apoptotic sperm amount and increase live sperm during treatment and high mitochondrial activity observed in treatments. Cell's reaction to environmental stresses is depended on domain, concentration and acts time of this reactions.
Conclusion: Although cellular and sub-cellular mechanisms, supposedly contributing to these processes, require further research, the new principle may outline a completely new strategy in mammalian embryology, as well as cryopreservation of other cells and tissues with remarkable theoretical and practical consequences.
Key words: Cryopreservation, Sperm, Stress, Sublethal.
P-54
The effect of KIT ligand (KITL) and leukemia inhibitory factor (LIF) on maturation and apoptosis of follicles after vitrification of mouse ovary and three dimensional culture
Abdi Sh1, Salehnia M1, Hosseinkhani S2.
- Department of Anatomy, Tarbiat Modares University, Tehran, Iran.
- Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Introduction: In vitro ovarian follicle culture techniques not only provide a model for research into the mechanism of folliculogenesis but also, in combination with cryopreservation of ovarian tissue, it may have clinical applications in preserving of fertility.
Materials and Methods: This experimental study is carrying out on 7-days-old female mice (NMRI). In the first step, the ovaries were vitrified with a solution containing ethylene glycol and then their morphology, ultrastructure and apoptosis were compared with non-vitrified ovaries. In the second step, the non-vitrified and vitrified ovaries were cultured in base medium α-MEM supplemented with KL and leukemia inhibitory factor (LIF) for 7 days then their morphology, area, apoptosis, hormone assay were analyzed. In third step, mechanically isolated preantral follicles were cultured in two dimensional and three dimensional systems in α-MEM supplemented with 5% FBS, 100 mlU/ml, 1% ITS, 10 ng/ml rEGF and KL for 12 days then the ovulation were induced. At the end of culture survival and maturation rate, apoptosis and hormones were assayed. In the fourth step, the level of reactive oxygen species (ROS) and ATP, distribution of mitochondria and fertilization rate in MII oocyte were assessed.
Results: The results of first step showed that the morphology, ultrastructure and apoptosis of vitrified ovaries were similar to non-vitrified ovaries. The results of second step showed that the percentage of preantral follicles, ovarian area, production of hormone in the non-vitrified ovaries cultured in medium supplemented with KL were significantly higher and the level of caspase 3/7 was lower in comparision to other groups (p<0.001). The results of next step showed that the survival, maturation rates and production of hormone of follicles in KL supplemented in three dimensional system groups were higher than other groups. The level of caspase-3/7 activity was lower in three dimensional culture systems (p<0.05). The results of fourth step showed that the level of ROS and ATP in MII from in vitro culture of follicle had significant different with control in vivo group and the rate of embryo development were significantly higher in three dimensional culture system.
Conclusion: Our results demonstrated that the culture of ovarian tissue and three dimensional culture of ovarian follicle in combination of KL improved the growth and development of ovarian primordial follicle.
Key words: Vitrification, In vitro maturation, Apoptosis, Mitochondria, KIT Ligand (KITL).
P-55
Evaluating the levels of expression of TLR 9 in women with endometriosis and its comparison with normal endometria
Aflatoonian Ali1, Janan A2, Ramazanali F2, Moieni A1, 2, Aflatoonian R2.
- Tehran University of Medical Sciences, Tehran, Iran.
- Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: Endometriosis is a benign condition which endometrial glands and stroma appear outside the uterine cavity which presents by pelvic pain and infertility. Endometriosis is associated with changes in cellular and humeral immunity and impaired immune response, leading to inefficient removal of debris after a menstrual cycle. Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system. Based on recent studies TLRs are increased in endometriosis and initiate immune responses.
Materials and Methods: This case-control study assessed and compared expression of Toll like Receptor 9 in the endometrium of three type tissues: 1) Eutopic endometria of women with endometriosis 2) Ectopic endometria women with endometriosis 3) Endometrium of women without endometriosis. Eutopic and ectopic endometrial samples were taken from 10 patients with endometriosis (Case Group). Also endometrial samples were taken from 10 patients without endometriosis or infertility history whom operated for other gynecological cause. The level of TLR9 gene expression in ectopic samples, eutopic and controls were evaluated by Real Time-PCR. Patients were chosen randomly from Arash Hospital. Data was analyzed by SPSS.
Results: Real Time-PCR showed that TLR9 expressed in all three groups of eutopic, ectopic and control. According to statistical analysis, level of TLR9 was higher in ectopic group, but not significantly (p=0.13). There was no significant difference between eutopic and control groups.
Conclusion: Considering the role of TLR9 in the innate immune system, such as pathogen detection and set up a cascade of inflammatory response associated with cell proliferation and inhibition of apoptosis, this study approves the association between TLR9 and endometriosis. Because of lack of information about this new issue, we suggest further researches and more studies.
Key words: TLR9, Endometriosis, Inniate immune system.
P-56
Congenital uterine malformations: A widespread phenomenon among infertile women with recurrent miscarriages and IVF failure
Javam M, Ahmadi F.
Department of Reproductive Imaging at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: f_ahmadi@royaninstitute.org
Introduction: Uterine malformations are a various group of congenital uterine disorders found in women with recurrent abortions or IVF failure.
Materials and Methods: A narrative review was performed within articles published at “PubMed”, “Elsevier”, "EBSCO", original text books and etc. to reach the aim. Several unique high-quality images were provided in this article, using the archive of infertile patients referred to imaging department of Royan Institute.
Results: Congenital uterine anomalies are originated from development defects of mullerian ducts during fetal growth. They are also associated with higher incidences of infertility, preterm birth, intra uterine fetal death and etc. However, manifestations and severity of the obstetric/ gynecologic complications and treatment procedures vary depending on the type of anomaly. Thus, accurate diagnosis of uterine malformations and differentiation between various types of them, have a vital role in decision about treatment procedures and management of these patients. Several imaging modalities are used to investigate women suspected to have uterine anomalies. In this article, we described about various types of uterine malformations and imaging evaluation of them prior to IVF treatment cycles.
Conclusion: Congenital anomalies of the uterus are a major cause of recurrent miscarriage and pregnancy failure. Several imaging methods help midwives and obstetricians evaluate this group of infertile women to detect suspected uterine anomalies.
Key words: Congenital uterine malformation, Infertility, Recurrent miscarriages.
P-57
Study on the impacts of anti-histamine dimenhydrinate on testicular tissues and sperm production of male mature rat
Ahmadifar M1, 2, Vahidi-eyrisofla N3, Taghvaei F4, Jafarzade Rastin S4, Yousef Zadeh AR6, Daliri L5, Gharanfoli M1.
- Department of Biology, College of Science, University of Science and Culture, ACECR, Tehran, Iran.
- Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Biology, Damghan Branch, Islamic Azad University, Semnan, Iran.
- International Campus, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Epidemiology and Reproductive Health at Reproductive Epidemiology Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Scientific Promotion, Tehran, Iran.
Introduction: In this study we did a research about the effects of AHDH on testicular tissues and sperm production of rats. Dimenhydrinate (DH) is used as anti-nausea as well as an anti-gastric reflux in cases of nausea caused during travelling. It is easily taken orally and it will be excreted by the kidneys within 24 hr.
Materials and Methods: This survey is conducted in laboratory on experiment basis among 30 mature male rats categorized in 3 different groups; two experimental groups and one control group. The control group was watered normally for 60 days and the first experimental group was watered with 200 mg DH blended in their water equal to one kg of their body weight for 60 days. In the end, after the rats were anesthetized, testicles were removed, sectioned and painted with Hematoxilin and Eozine and were investigated.
Results: The obtained results have revealed that in the experimental groups, testicles weight decreased substantially in comparison to those of the control group (p<0.001). The reduction of the number of sperm cells within the experimental group, in comparison to those of the control group was significant (p<0.001). The reduction of the Leydig cell was very substantial in the second experimental group (p<0.001). The difference of the Sertoli cells was not noticeable in neither of the experimental groups.
Conclusion: DH has impact on testicular tissues and weakens their mechanism.
Key words: Dimenhidranate, Testis.
P-58
Investigating association of G103T polymorphism of Coagulation factor XIII and recurrent pregnancy loss
Ahmadpour Nazm S1, Bonyadi M2, Taghavi S3, Ajamian F1.
- Biology Group, Science faculty, Guilan University, Rasht, Iran.
- Biology group, Science Faculty, Tabriz University, Tabriz, Iran.
- Department of Obstetrics and Gynecology, Medical faculty, Tabriz University of Medical Sciences, Tabriz, Iran.
Introduction: Recurrent miscarriage (RM) occurs in 1-3% of couples that attempting to bear children. Thrombophilia is one of the suspected cause of recurrent miscarriage. At the end of coagulation cascade there is Factor XIII that makes blot stable. The polymorphism G103T of factor XIII gene is the most common polymorphism that affects FXIII’s activity.
Materials and Methods: The study groups consisted 50 patients with two or more consecutive miscarriage. The control group included 50 women with at least two successful delivery and no history of pregnancy loss. By using PCR-RFLP, DNA from both groups analyzed for carrying mutation of FXIII.
Results: 4% in the case group were homozygote (TT) for 34 Leu mutation whereas no homozygote (TT) was found in control group (p<0.05). 28% patients in the case group and 26% women in the control group were found to be heterozygote for G103T polymorphism (p>0.05). No significantly differs was observed between patients with RPL and healthy women for G103T mutation.
Conclusion: No statistically differs between case group and control group was observed.
Key words: Recurrent miscarriage, Thrombophilia, F XIII.
P-59
Effect of pumpkin seed and ginger extracts on adult rat’s sperm characteristics, biochemical parameters and epididymal histology treated with cyclophosphamide
Amini Mahabadi J1, Nikzad H1, Aghaie S2, Taghizadeh M3, Azami-Tameh A2, Taherian SS2.
- Gametogenesis Research Center, Faculty of Sciences, Kashan University of Medical Sciences, Kashan, Iran.
- Anatomical Sciences Research Center, Faculty of Sciences, Kashan University of Medical Science, Kashan, Iran.
- Research Center for Biochemistry and Nutrition in Metabolic Disorders. Faculty of Sciences, Kashan University of Medical Science, Kashan, Iran.
Introduction: Infertility is one of the problems of human society and 10-15% of couples have experienced some forms of infertility problems. Reproductive toxicity is one of cyclophosphamide (CP) side effects in cancer treatment.
Materials and Methods: Male adult Wistar rats were randomly divided into six groups. Group 1 as control received an isotonic saline solution injection intraperitoneally (IP). Group 2 was injected intraperitoneally with a single dose of CP (100 mg/kg) once. Group 3 and 4 received CP plus 300 and 600 mg/kg combined pumpkin seed and Zingiber officinale extract (50:50). Group 5 and 6 received only 300 and 600 mg/kg combined pumpkin seed and Zingiber officinale extract. Six weeks after treatment, sperm characteristics, histopathological changes and biochemical parameters were assessed.
Results: Results showed that in CP treated rat’s sperm characteristics were diminished significantly. Biochemical analysis showed that the administration of combined extracts could increase the Total Antioxidant Capacity (TAC) level significantly in groups 3, 4, 5 and 6. Also, in these groups, the sperm viability, motility, count, normal sperm morphology, epididymal epithelium and fibromascular thickness were improved compared to control and CP groups. Interestingly, the mixed extract could improve histopathological changes such as vacuolization, disorganization and separation of epididymal tissue in CP treated rats as well.
Conclusion: Overall our findings indicated that the combined extracts might be used as a protective agent against CP-induced reproductive toxicity.
Key words: Pumpkin seed extract, Zingiber officinale extract, Cyclophosphamide, Total antioxidant capacity (TAC), Sperm parameters, Rat epididymis.
P-60
Effect of vitamin D insufficiency treatment on fertility outcomes in frozen-thawed embryo transfer cycles: A randomized clinical trial
Aflatoonian A, Arabjahvani F, Eftekhar M, Sayadi M.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: F_arabjahvani@yahoo.com
Introduction: Frozen- thawed embryo transfer is an essential part of ART treatment and outcomes of this procedure are associated with several clinical factors. Several studies have showed an increase level of IVF outcomes in women with sufficient vitamin D. whether treatment of vitamin D insufficiency can improve pregnancy rates in frozen-thawed embryo transfer cycles.
Materials and Methods: This is an interventional, randomized clinical trial. Serum 25-(OH) vitamin D level of 128 women who had undergone IVF/ ICSI with cryopreservation of embryos was checked. One hundred fourteen infertile women with insufficient serum vitamin D (less than 30 ng/ml) were included in the study. Fifty seven women were treated with supplementary vitamin D, 50000 IU weekly, for 6-8 weeks and 57 women were received no supplementation. 106 women completed frozen thawed embryo transfer cycles and included in the final analysis. Primary and secondary outcomes were chemical and clinical pregnancy respectively.
Results: Our study did not show any significant difference between vitamin D insufficient and treated women in term of chemical (29.40% vs. 29.10% respectively, p=1.00) or clinical (25.50% vs. 21.80% respectively, p=0.81) pregnancy rates.
Conclusion: Vitamin D insufficiency treatment is not associated with higher pregnancy rate in frozen-thawed embryo transfer cycles.
Key words: Vitamin D, Embryo transfer, Pregnancy rate.
P-61
Association of rs10954213 polymorphism of IRF-5 gene with idiopathic recurrent miscarriage
Arjmand F1, Ashrafzadeh Mehrjardi HR2, 3, Ghasemi N3.
- Department of Immunology, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Faculty of Medicine, Yazd, Iran.
- Department of Biology, Faculty of Science, Islamic Azad University, Ashkezar Branch, Yazd, Iran.
- Recurrent Abortion Research Center, Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: Interferon regulatory factor (IRF) 5 is a transcription factor that can induce transcription of IFN-α mRNA. Recent studies have shown that IRF-5 promotes the proliferation of T cells and the activation of TH1 and TH17 cells but does not induce TH2 or Treg differentiation. TH17 cells produce inflammatory cytokine IL-17 which plays an important role in the induction of inflammation via neutrophil infiltration and stimulation of inflammatory cytokines. There are many reports that inflammatory processes play a major role in recurrent miscarriage in a manner that an uncontrolled and persistent inflammatory response during pregnancy can harm placental growth. We analyzed the rs10954213 polymorphism of IRF-5 gene in normal pregnancy and recurrent miscarriage patients in order to discover a possible mechanism for the genetic control of immune regulation in patients with recurrent miscarriage.
Materials and Methods: Genomic DNA from 100 recurrent miscarriage (RM) patients and 100 normal fertile control individuals using the routine salting out method were isolated. DNA fragments were then analyzed by real time PCR with SYBR Premix Ex Taq II master mix. Statistical analysis used SPSS 19 software. IRF-5 allele frequencies and genotypes in RM women and the fertile control group were compared using a Chi-square test.
Results: Our results so far indicate that there is a detectable relationship between rs10954213 polymorphism of IRF-5 and susceptibility to recurrent miscarriage.
Conclusion: IRF-5 gene plays a possible role in the induction of inflammation and therefore leads to recurrent miscarriage. However, additional studies are needed in this regard.
Key words: Rs10954213, IRF-5, Recurrent miscarriage.
P-62
Role of 45,X mosaicism in couples with fertility problems
Bahman I1, Bagherizadeh I1, Abdi A1, Shajarehpour L1, Hadipour F1, Hadipour Z1, Saremi A2, Shafaghati Y1, 3, Behjati F1, 3.
- Department of Medical Genetics and Sarem Cell Research Center (SCRC), Sarem Women’s’ Hospital, Tehran, Iran.
- Department of Perinatology and Sarem Cell Research Center (SCRC), Sarem Women’s’ Hospital, Tehran, Iran.
- Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran
Introduction: Infertility is typically defined as the biological inability of a person to contribute to conception after one year of attempting to have a child. Genetic factors such as chromosomal abnormalities are one of the major causes of infertility and spontaneous abortions. The objective of this study was to establish chromosome X abnormality rate among women referred for infertility problems to Sarem Women's Hospital in Tehran between October 2007 and January 2015.
Material and methods: A total number of 1764 women with the age range of 12-60 years were referred for chromosomal investigation. Referral reasons included recurrent abortions, primary infertility, secondary infertility, Turner syndrome, premature ovarian failure, amenorrhea and ART failures. Cytogenetic analysis was performed on cultured peripheral blood lymphocytes stimulated with phytohaemagglutinin M, using standard techniques. Karyotype was done in all patients using high resolution GTG banding technique. For each patient, at least 15 chromosome spreads were examined by light microscopy, and extended to 100 in the case of mosaicism.
Results: Cytogenetic investigation was performed on 1764 patients. The overall chromosome X abnormality rate in infertile and subfertile women was 4% (74 out of 1764). The cytogenetic result for these patients with fertility problems is as follows: 74 patients had a numerical chromosome abnormality and 65 (85%) of them had mosaicism of 45,X with other different cell lines. The age range of women with 45,X mosaicism was 12-53 years old.
Conclusion: Accurate genetic diagnosis is the most important prerequisite for genetic counseling in patients with fertility problems. Finding of chromosome X numerical abnormality are of great value in better management of the patients. However, the attribution of chromosome X instability due to age should be considered in Genetic counseling of these couples.
Key words: Fertility problems, Chromosome X abnormality, 45,X cell line.
P-63
Effects of mitochondrial gene deletions on human sperm motility
Bahrehmand Namaghi I, Vaziri H.
Department of Biology, Faculty of Science, Guilan University of Medical sciences, Rasht, Iran.
Email: i_man_bahrehmand@yahoo.com
Introduction: Asthenozoospermia is an important cause of male infertility. It has been primarily characterized by reduced sperm motility owing to a variety of factors, including ultra-structural abnormalities, abnormal semen liquefaction, anti-sperm antibodies, varicocele and endocrine abnormality, etc. The mitochondrion is the major energy provider for sperm motility. Mitochondrial DNA contains several genes encoding for proteins that play an important role in oxidative phosphorylation and in ATP production. Mutations in sperm mtDNA result in either functionless or malfunctioning proteins, subsequently affecting sperm motility leading to asthenozoospermia.
Materials and Methods: To detect 4977 bp deletion in spermatozoa mtDNA, semen samples of 42 asthenozoospermic infertiles and 50 controls from northern Iran were collected. After extraction of spermatozoa total DNA, Gap-PCR was performed.
Results: 4977 bp deletion was observed in 73.80% of patients with asthenozoospermia, compared with 36% in controls (OR=5.0101, 95% CI: 2.0408-12.2998, p=0.0004).
Conclusion: Large-scale mtDNA deletions in spermatozoa may induce bioenergetic disorders and cause subfertility or infertility in men. It is concluded that there is a strong association between sperm mtDNA 4977 bp deletion and asthenozoospermia-induced infertility in the population examined. Nevertheless, to validate our results broader research may be needed.
Key words: Asthenozoospermia, mtDNA deletion, Male infertility, Motility.
P-64
Effects of Nano TiO2 on chromatin, apoptosis and parameters of sperm in mice
Danafar AH1, 2, Khoradmehr A1, Hosseini M3, Halvaei I1, Golzadeh J1, Anvari M1.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Biology, Ashkezar Branch, Islamic Azad University, Ashkezar, Iran.
- Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran.
Introduction: Titanium dioxide nanoparticles (TiO2 NPs) are manufactured worldwide and used in a broad range of applications. In mammals, there are only limited reports regarding the effect of TiO2 NPs on male reproductive system. It has been demonstrated that TiO2 NPs taken up by mouse Leydig cells reduced the viability and proliferation of these cells. Also it is improved that titanium compounds can disrupt the mouse blood-testis barrier. In this study we decide to evaluate the effects of nano-TiO2 on chromatin, apoptosis and sperm parameters of male mice.
Materials and Methods: 35 NMRI male mice were classed in five groups: control, sham and three treated groups. Low, medium and high treated groups were gavaged 2.5, 5 and 10 mg/kg doses of TiO2 respectively for thirty five days. Sham group only received saline instead. The mice were weighted before and after administration. Sperm count, motility, morphology and viability were analyzed accordingly. Sperm DNA integrity and apoptosis were assessed using acridine orange, aniline blue, toluidine blue, CMA3 and TUNEL assay.
Results: Non-significant decrease in body weight gain was observed. Sperm analysis showed no count, motility and viability changes but morphological changes were significant. Most of the morphological abnormalities were observed in sperm neck and tail. Tail morphological changes in treated groups were significant in comparison to non-treated ones (p<0.001). Compared to non-treated groups, sperm neck changes in medium and high treated groups were significant but not in low treated group (p<0.001). Results of acridine orange, aniline blue, toluidine blue and CMA3 were non-significant in all groups. Also increasing apoptotic cells in treated groups were discovered by TUNEL assay but there were non-significant statistically.
Conclusion: TiO2 significantly may increase sperms with abnormal morphology especially in tail and neck part. Also apoptosis boosting occurred but the exact mechanism is still controversial.
Key words: Nano-TiO2, Sperm parameters, Apoptosis.
P-65
Protective role of vitamin C on hazardous effects of acrylamide in rat offspring
Dortaj H, Anvari M, Yadegari M.
Shahid Sadoughi University of Medical Scienses, Yazd, Iran.
Email: hengameh.dourtaj@gmail.com
Introduction: Acrylamide (ACR) is a substance chemical used in industrial and laboratory procedures. Acrylamide according to the method of cooking foods are increasingly used and its adverse effects on multiple organ systems have been described sporadically in the literature. The purpose of this study was to evaluate the effects of ACR during pregnancy and lactation on the development and changes in cortical layer of the cerebellum and cerebellar Purkinje cells in fetal and neonatal rat by using histological and morphometric and stereological technique.
Materials and Methods: In this study 20 adult female Wistar rats weighing 180 gr and aged two months were used. Animals were randomly divided into four groups. Female pregnant rats were orally administered 10 mg/kg ACR and/or 200 mg/kg vitamin C (vit C). Pregnant rats were sacrificed on the 15th day of gestation and mother’s weight was measured. After that, their fetuses were taken out and were evaluated for fetus number, weight, crown-rump length (CRL) and cerebellar development. To study the neonatal period, 6 infants at day 21 were randomly selected and placed under deep anesthesia and transcardial perfusion. The cerebellum was taken out and fixed and cerebellum changes were evaluated by crysel violet and Hematoxylin-Eosin staining method. The cerebellar cortex layers volume were investigated by Cavalieri's principle method. Data were analyzed by SPSS software and by ANOVA and LSD Test. P<0.05 considered as statistically significant.
Results: The results showed that ACR decreased fetal weight and CRL, but this reduction in weight and volume of the cerebellum p˂0.001 and the number of embryos with p<0.05 was significant. Histological and stereological examinations revealed that the cerebellar volume was decreased in ACR and ACR+vitC group vs control (p˃0.001). While in vitamin C group the cerebellar volume was inversed (p˃0.05). ACR in newborn decreases body weight, brain weight, thickness of cerebellum with p˃0.001. The extent of this reduction in the weight of the cerebellum was significant with p˂0.05.
Conclusion: ACR exhibits a harmful effect on the development of the cerebellar cortical layers, which may be prevented by administration of vit C as an anti oxidant.
Key words: Acrylamide, Vitamin C, Cerebellum, Development, Rat.
P-66
Robust stem cell isolation from human dental pulp
Ebrahimi B, Banaei M, Namiranian G, Tabatabaei SA.
Yazd Cardiovascular Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: bhnmebrahimi@yahoo.com
Introduction: Dental pulp stem cells (DPSCs) are a population of clonogenic and highly proliferative cells derived from enzymatically digested Dental pulp tissue. Providing scaling-up of stem cells at early passages is of importance for regenerative medicine purposes. Typically, two protocols are employed to isolate stem cells from human dental pulp: tissue explants culture and enzyme digestion of pulp tissue and culture of released cells.
Materials and Methods: Dental pulp was extracted from third molars of 60 healthy subjects. In the first method pulp was digested with 1 mg/mL collagenase/ dispase (Roche) for 30 min and released cells obtained using a 70-µm cell strainer for culture, in the second method intact pieces of pulp were cultured and in the third method digested pulp pieces were immobilized and cultured. The cells and tissues maintained in alpha-MEM supplemented with 20% fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, and 25 ng/mL amphotericin B and incubated in humidified incubator with 5% CO2 at 37oC. In each group cells and colonies were counted and compared.
Results: Results showed that treating pulp segments with enzymes and culturing them (combinatory method) increased the efficiency of cell isolation up to 60% significantly in 3-4 days of culture compared with other methods which this value was <20% in 10-15 days.
Conclusion: According to the small size of pulp tissue and its low stem cell contents, acquiring substantial quantities of cells in primary culture will facilitate the in vitro expansion and providing adequate production of the stem cells at early passages with minimum risk of losing their ‘stemness’ and aberrant genetic changes for use in research, tissue engineering and regenerative medicine. Optimized method increase efficiency of cell isolation and provides significant quantities of stem cells in primary culture more than other methods.
Key words: DPSCs, Cell isolation, Cell culture.
P-67
Evaluation of exon 8 of DPY19L2 gene in total globozoospermic patients referred to Royan Institute
Ebrahimi Nasab M1, Totonchi M2, Sabbaghian M3.
- Department of Biology, Faculty of Basic Sciences and Advanced Technologies, Science and Culture University, ACERCR, Tehran, Iran.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Andrology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: Lack of acrosome decreases the capacity of the sperm for penetrating in the oocyte which consequently results in infertility. Globozoospermia is a rare and severe teratozoospermia characterized by round-headed spermatozoa lacking acrosomes. DPY19L2 is one of the genes which are dominantly expressed in the testis and it has been shown that is involved in the cause of this phenotype. Recent studies have shown that in large majority of globozoospermia patients a 200 kb deletion including DPY19L2 gene occurs. Different mutations in exon 8 of this gene have been also observed.
Materials and Methods: In total 24 men with total globozospermia and 24 men with normal spermogram referring to Royan Institute were selected. Then we sequenced exon 8 and intron boundaries in the non-deleted patients using specific primers and PCR technique.
Results: In our results no mutations were detected in exon 8 of the patients without the gene deletion, which were 29.17% of the total globozospermia patients.
Conclusion: According to our data, exon 8 mutations or polymorphisms has no effect on globozospermia but whereas 70.83% of globozospermia patients had whole DPY19L2 deletion and on the critical role of DPY19L2 protein in acroplaxome attachment to the nucleus, it can be concluded that the absence of this protein is one of the major causes of globozoospermia in Iranian infertile men. In addition, exploring other coding exons of this gene is our next aim.
Key words: DPY19L2 gene, Globozoospermia, Male infertility.
P-68
Anti-oxidant effects of herbal supplements on seminal parametmeters
Eghbal Eftekhaari T, Fallahi S, Malekzadeh K.
Molecular Medicine Research Center, Hormozgan University of Medical Sciences, BandarAbbas, Iran.
Email: tasnim.eftekhaari@gmail.com
Introduction: Varicocele is one of the leading causes of male infertility, causing a decrease in sperm parameters such as motility, count, concentration, and abnormal sperms such as azoospermia and asthenospermia.
Materials and Methods: A herbal compound capsule containing 4 herbs was prescribed 5 times perday (3 times during morning and evening and 2 times at night), and because of the warm nature of these herbs, 3 herbal extracts including Cichorium intybus L, Salix Aegyptiaca and Fumaria Parviflora 3 cups/day were added to this medication.
Results: After 4 weeks of consuming these medications, the body warmness decreased , ejaculated sperm volume and other sperm parameters increased including motility, concentration and morphology of sperm changed form astenia and oligospermia to normal morphology.
Conclusion: The herbal compound containing antioxidant supplements and enriched in minerals and fat soluble vitamins showed promising effects on improving sperm parameters and increase the chance of male fertility.
Key words: Varicocele, Increased sperm motility, Increased sperm count, Sperm paramteters, Antioxidants.
P-69
The effect of brown algae Sargassum extract on freezing of sperm
Fardmanesh H1, Fallahi S2, Shakerizadeh S3, Masoudi M3, Fadaei- Arsanjan M4.
- Department of Student Research Committee , Hormozgan University of Medical Sciences, Bandar Abbas, Iran.
- Department of Fertility and Infertility of Hormozgan, Hormozgan University of Medical Sciences, Bandar Abbas, Iran.
- Department of Molecular Medicine, Hormozgan University of Medical Sciences, Bandar Abbas, Iran.
- Department of Biology, Islamic Azad University, Arsanjan Branch, Fars, Iran.
Introduction: Oxidative stress process resulting from freezing and thawing of sperm, influences parameters of sperm and decreases its fertility. Antioxidants play a conservative role against oxidative damages during freezing of sperm. Using an appropriate freezing bank effects on fertility and motility of sperm, so we decided to evaluate effect of antioxidant extract of brown algae Sargassum on reactive oxygen species (ROS) and parameters of frozen sperm.
Materials and Methods: 11 normal semen samples were divided in to 3 groups, including A treated with 250 µg/ml and B with 500 µg/ml of extract of algae Sargassum and C group without any treatment (as a control group) then froze throughout rapid freezing method. Using CASA software and oxisperm kit, the motility/morphology and ROS levels of sperm were measured, respectively. Finally data were analyzed with SPSS software assumed significant level of p<0.05.
Results: The analysis of sperm parameters demonstrated that general motility (p=0.006) and advanced motility (p=0.007) significantly increased in both A and B groups, after treatment compared with C group. Moreover, the level of ROS notably declined in both treated group. However this treatment revealed any changes in sperm morphology.
Conclusion: The brown algae Sargassum extract is rich in antioxidant component that leads to decrease oxidative damage via neutralizing of ROS and improve sperm motility. Our results suggested that the extract can be used as a potential antioxidant factor in sperm freezing bank.
Key words: Sperm motility, Antioxidants, Reactive oxygen Species, Brown algae sargassum.
P-70
Immunohistochemical evaluation of a testis-specific histone demethylase, JMJD1A, in tissue samples of infertile men referred to Royan Institute
Favaedi R1, Shahhoseini M1, Kordestani shargh E2, Sodeifi N3, Sadighi Gilani MA3.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
- Department of Andrology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: Post-meiotic stages of mammalian spermatogenesis require unique and dynamic epigenetic events leading to histone removal followed by chromatin condensation. Through these events, histone demethylases such as JMJD1A play an important role in compaction of sperm chromatin due to regulation of histone methylation dynamics and alteration of chromatin structure. As “histone methylation” is one of the best-characterized modifications in the study of germ cell development, evaluation of presence/ absence of JMJD1A protein as in impaired spermiogenesis were aimed in this study.
Materials and Methods: For this respect, consent was obtained from azoospermic infertile men referred to Royan Institute according to local ethical approval, and then testis tissue samples were collected from three groups including complete maturation arrest, Sertoli cell only syndrome, and hypo spermatogenesis as positive control. Immunohistochemical analysis of paraffin embedded tissue samples was performed qualitatively using anti-JMJD1A antibody to elucidate presence/ absence of this protein in nucleus of germ cells.
Results: Immunohistochemical analysis data showed absence of JMJD1A protein in nucleus of germ cells in groups with spermatogenesis impairment (complete maturation arrest and Sertoli cell only syndrome groups) compared to control group.
Conclusion: It can be concluded that there is an obvious association between absence of histone demethylation as a chromatin condensing state with impairment of spermatogenesis and male infertility.
Key words: Spermatogenesis, Male infertility, Epigenetic, JMJD1A, Demethylation.
P-71
Pregnancy outcome of intracytoplasmic injection with epididymal and testicular sperm retrieval in azoospermic patients
Moein MR, Ghasemi-EsmailabadS, Ghasemzadeh J, Fesahat F, Tabibnejad N.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: qasemisaeed@yahoo.com
Introduction: Intra cytoplasmic sperm injection (ICSI) is an assisted reproductive technique for treatment of infertility in azoospermic men. It seems that pregnancy outcome in ICSI following percutaneous epididymal sperm aspiration (PESA) is better than using testicular sperm retrieving from testis due to greater motility and better morphology of epididymal spermatozoa. To determine clinical pregnancy outcome following ICSI with epididymal sperm comparing to that of testicular sperm in men with azoospermia.
Materials and Methods: 60 men with azoospermia who were candidate for ICSI have been selected. Sperm retrieval was performed using PESA (n=30) or testicular sperm extraction (TESE) (n=30). The number of embryos and live births were analyzed and evaluated between two mentioned groups and also thawed sperms after freezing in TESE group (n=30).
Results: No difference was seen in age and duration of infertility between groups. The number of embryos were not different significantly between TESE and PESA groups (p>0.05), but the different was significant between PESA and TESE freeze groups (3.67±2.89 vs. 2.10±1.53 respectively; p<0.05). The live birth rate was higher in the PESA group compared with the TESE group p<0.05).
Conclusion: Intracytoplasmic sperm injection using sperm from epididymis is more effective than testicular sperm injection and can successfully be performed to treat men with azoospermia.
Key words: ICSI, TESE, PESA, Freeze, Live birth rate.
P-72
Prevalence and risk factor of congenital malformation in Kashan
Haddad Kashani H1, Seyed Hoseini ES2, Dadkhah M3, Nikzad H2.
- Anatomical Sciences Research Center, Kashan University of Medical Science, Kashan, Iran.
- Gametogenesis Research Center, Kashan University of Medical Science, Kashan, Iran.
- Faculty of Medicine, Kashan University of Medical Sciences, Kashan, Iran.
Introduction: Congenital malformation (CM) is a major childhood health difficulty. Treatment and rehabilitation of children with congenital malformations is costly and complete recovery is usually impossible. The prevention and treatment of congenital malformations are basic concerns for child health. The purpose of this study was to define rate of CM in Beheshti Hospital in Kashan, Iran to find out if there has been any variation in the rate and types of CM in this area.
Materials and Methods: This descriptive-observational study carried on 2700 births delivered from Beheshti Hospital in Kashan during 2012 and determines the prevalence of CM and type of it in this city.
Results: Prevalence of CM was 1.851% (2% in male and 1.61 % in female). Out of the 50 cases, 28 (56%) were males and 21 (42%) were females and 1 with uncertain genitalia. 13 members of the family were CM positive in musculoskeletal system (26%). Overall, anomalies of the cardiovascular system were second in frequency which involved 10 out of 50 patients (20%), also Down syndrome and skin malformation were the lowest rate anomaly in this study in Kashan (4% and 2% respectively). There wasn't statistical difference between prevalence of CM and neonatal gender and mother’s age, father’s age, mother’s number of abortion, and mother’s number of live children. But there was statistical difference between prevalence of CM with gestational age and Apgar number.
Conclusion: In this study the overall prevalence of congenital malformation among the newborn was less than those previous reported in Tehran and Yazd and higher than the rate of malformation in Gorgan and Arak. This difference determining the needs of more extensive studies.
Key words: Congenital malformation, Newborn, Beheshti Hospital, Kashan.
P-73
Relevance between testicular tissue vitrification and short term culture with degeneration and apoptosis genes expression
Hajiaghalou S1, 2, Ebrahimi B2, Shahverdi AH2, Sharbatoghli M2.
- Department of Developmental Biology, Faculty of Basic Sciences and Advanced Technology in Biology, University of Science and Culture, ACECR, Tehran, Iran.
- Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: Cryopreservation of testicular tissue has been recommended as a promising technique for fertility preservation in pre-pubertal boys who scheduled to undergo gonadotoxic treatment. Our aim was finding the quota of apoptosis genes involved in extrinsic pathway in testicular cell death after vitrification and during short term culture.
Materials and Methods: Testes were obtained from 7 days old NMRI male mice, divided and randomly distributed into control and vitrification groups. Vitrification was performed in 3 step by increasing concentration of vitrification solution (DMSO, EG). Both fresh and vitrified-warmed testes was cultured in RPMI and 10% KOSR for 20 hr. Real-time PCR, flow cytometery and light microscopy were used respectively for evaluation of gene expression, cell death and tissue integrity at 0, 3 and 20 hr of culture.
Results: Decreasing of tissue integrity was obvious in vitrification group as compared to the control one at all times of culture. Mean percentage of cell death was significantly (p<0.05) higher in vitrification group in comparison with control during culture period. Although expression of Fas was significantly (p<0.05) higher in vitrification group at 0 and 3 hr of culture, it was significantly lower at 20 hr of culture as compared to the control group. Significant (p<0.05) increase of Fas ligand was found in vitrification group at 3 and 20 hr of culture. Mean percentage of Caspase 3 was significantly (p<0.05) higher in vitrification group than control group.
Conclusion: Concurrent increment of cell death and apoptosis genes expression in vitrification group during culture period, could be a reason for extrinsic pathway involvement in degeneration of testis tissue after vitrification and also during culture period.
Key words: Extrinsic pathway of apoptosis, Tissue degeneration, Vitrification, Short term culture.
P-74
Magnetic activated cell sorting and its application for selection of human non apoptotic spermatozoa in ART
Hassani Bafrani H1, Catt S2, Pangestu M2, Smith Peter T2.
- Kashan University of Medical Sciences, Kashan, Iran.
- Monash University, Melbourn, Austuralia.
Introduction: Hydrogen peroxide has been implemented in literature to induce a significant increase in caspase. Activation of caspase 9 triggers a cascade of caspase activation, including caspase 3, which promotes cellular apoptosis. Magnetic cell sorting (MACS) using annexin V-conjugated microbeads eliminates apoptotic spermatozoa with annexin V-positive.
Materials and Methods: A total of 20 semen samples were obtained from male partners of couples for analysis. One aliquot (0.5ml) of the sperm suspension was subjected to MACS. Motility and concentration was checked, and a sample taken for tunnel before and after MACS. The remaining sperm suspension was divided into 6 tubes (2x control, 2x peroxide, 2x peroxide/ melatonin). DNA fragmentation was evaluated using the TUNEL assay (Roche, Indianapolis, IN, USA) (14), with some modifications. Data were analyzed using Graph Pad Instat Ver. 3.10.
Results: Results of the TUNEL assay in pretreatments of human spermatozoa with 100uM peroxidase for 24 hrs revealed that the percentage of sperm with fragmented DNA was significantly lower on the sored sperm after sorting MACS (p˂0.001 vs. control). Following the pretreated human spermatozoa with peroxidase for 24 hr, percentage of sperm motility and progressive motility were significantly reduced (p˂0.001 vs. control). Pretreatments of human spermatozoa for 24 hr revealed that the percentage of sperm motility and progressive motility were significantly reduced by 100 µM peroxidase and peroxidase with MACS, versus zero hour in control group (p<0.001).
Conclusion: The sperm was treated with 100 µM peroxidase sorting using MACS retain appropriate spermatozoa and select sperm good quality. The use of MACS will select only sperm with intact this result in high percentage motility and progressive motility.
Key words: Apoptosis, Human spermatozoa, MACS.
P-75
Bibliometric mapping and clustering analysis of Iranian-based research on reproductive medicine
Bazm S1, Ramezani A2.
- School of Public health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Faculty of Management and Information Sciences , Iran University of Medical Sciences, Tehran, Iran.
Introduction: Nowadays infertility is a major problem in the world. Increasing the rate of infertility in the world and Iran led our study to assess the trends in Iranian research output related to reproductive medicine through the year of 2010-2014.
Materials and Methods: We used bibliometric mapping and clustering analysis method to visualize representing bibliographic data of Iranian production. All publication data from Scopus was retrieved for 2010-2014.
Results: Analysis of data showed that a total number of 3035 papers had been indexed in Scopus through the period of study.
Conclusion: The study indicated that scientific production in the field of reproductive medicine had increased in the past 5 years.
Key words: Reproductive medicine, Bibliometric analysis.
P-76
Menstrual pattern following tubal ligation: A historical cohorts
Jahanian Sadatmahalleh Sh1, Ziaei S1, Kazemnejad A2, Mohamadi E3.
- Department of Reproductive Health and Midwifery, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
- Department of Biostatistics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
- Department of Nursing, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Introduction: Tubal ligation (TL) is recommendable for women completed their family. The existence of the menstrual disorders following this procedure has been the subject of debate for decades.
Materials and Methods: A historical cohort study was carried out on 140 women undergoing tubal ligation and on 140 women who used condom as the main contraceptive method. They aged between 20 and 40 years and were selected from a health care center, during 2013-2014. The two groups were comparable in demographic and personal characteristics. Data collection tool was a questionnaire including questions regarding demographic, menstrual and obstetrical characteristics. A validated Pictorial Blood Loss Assessment Chart (PBLAC) was also used to measure the menstrual blood loss. All statistical analyses were carried out using software package used for statistical analysis (SPSS) version 20 (SPCC Inc., Chicago, IL, USA). Student’s t-test and chi-square test were carried out to reveal the statistical differences between the groups. Logistic regression was done to build a prediction model in menorrhagia.
Results: Women with TL had more menstrual irregularity than those without TL (24.3% vs. 10% respectively, p=0.002). Women with TL had more polymenorrhea (9.3% vs. 1.4%; p=0.006), hypermenorrhea (12.1% vs. 2.1%; p=0.002), menorrhagia (62.9% vs. 22.1%; p<0.0001) and menometrorrhagia (15.7% vs. 3.6%; p=0.001) than those without TL. There is a significant difference in the PBLAC score between women with and without TL (p<0.0001). According to logistic regression, age (OR=1.08, CI:1.07-1.17; p=0.03), TL (OR=5.95, CI:3.45-10.26; p<0.0001), and cesarean section (OR=2.72, CI:1.49-4.97; p=0.001) were significantly associated with menorrhagia.
Conclusion: We found significant differences in menstrual disorders between women with and without TL. Therefore, women should be informed by the health providers regarding the advantages and disadvantages of TL before the procedures.
Key words: Historical cohort study, Tubal ligation, Menstrual disorders, PBLAC.
P-77
Effect of nano silver on morphological and chromosomal abnormality of NMRI mouse fetus
Mozafari M1, Kalantar SM2, Khoradmehr A2, Danafar AH2, 3, Miresmaeili M4.
- Department of Biology, Islamic Azad University, Ashkezar Branch, Yazd, Iran.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Young Researchers Club, Islamic Azad University, Yazd, Iran.
- Yazd Academic Center of Education, Culture and Research Higher Education Institute, Yazd, Iran.
Introduction: Development of nanotechnology caused the use of the materials in nano sizes. Nowadays, special biological properties of nano silver are playing an important role in our life. Some studies suggest a potential for adverse effects of nano silver on fetal development of mammals, but additional research is needed. In this study we decide to evaluate the effects of nano silver on fetus chromosomal structure and its development.
Materials and Methods: 24 pregnant mice were divided into four groups. Nano silver (1 mg/kg, 70 nanometer) were gavaged to the first, second and third group, from the 1th-7th, 8th-14th and 1th-14th gestational days respectively. Nothing was gavaged to the control group. On 14th day the pregnant mice were dislocated and the liver of fetus was used for karyotyping analysis. The fetuses were weighed and their crown-rump length and head circumference were measured by caliper.
Results: According to the effects of nano silver, the results indicated that body weight of embryos were significant decreased in third group in comparison with the others (p<0.001). Also there were significant reduction of crown-rump length of fetus in third and second group compared to other groups (p<0.05). There was lower fetus head circumference among nano silver treated and controls (p<0.05). Karyotyping analysis of fetal liver was normal in all groups.
Conclusion: It seems that abuse of nano silver during pregnancy can reduce weight, crown-rump length and head circumference in mice fetus, but its mechanism is not completely clear.
Key words: Pregnant mice, Nano silver, Karyotype.
P-78
Neuronal markers expression of induced human adipose-derived stem cells in alginate hydrogel
Khosravizadeh Z1, Khosravizadeh Z1, Razavi Sh2.
- Tehran University of Medical Sciences, Tehran, Iran.
- Isfahan University of Medical Sciences, Isfahan, Iran.
Introduction: Hydrogels provide appropriate three-dimensional environment for cell cultures and cell encapsulation in hydrogels is a promise plan for tissue engineering applications. Alginate is a biocompatible hydrogel that provides a supportive system for the encapsulated cells. Moreover human adipose derived stem cells (hADSCs) might be a suitable source of cells for use in autologous cell therapy; also these cells can differentiate into neuron-like cells. Therefore, in this study we evaluated effect of alginate hydrogel on the neural potential of induced hADSCs.
Materials and Methods: Isolated hADSCs were induced in neural medium and encapsulated in alginate hydrogel. Using Immunocytochemical and real-time RT-PCR analysis, some neural markers were evaluated in differentiated hADSCs.
Results: The expression of Nestin, GFAP and MAP2 markers significantly increased in alginate cell cultures relative to monolayer induced cells (p<0.001).
Conclusion: These findings showed that alginate hydrogel can provide a suitable environment for neural differentiation of hADSCS.
Key words: Human adipose-derived stem cells, Alginate hydrogel, Neural differentiation, Tissue engineering.
P-79
Protective effect of N-acetyl-L-cysteine on testicular tissue in mice treated with Para-Nonylphenol
Malmir M, Soleimani Mehranjani M, Naderi Noreini S.
Faculty of Science, Arak Univercity of Medical Scinecs, Arak, Iran.
Email: mehhdi66@yahoo.com
Introduction: Para-Nonylphenol (p-NP) is an alkylphenol considered as an environmental pollutant with estrogenic and toxic effects. It can also cause morphological and functional alterations in the male reproductive system inducing infertility. The purpose of this study was to investigate the protective effects of N-acetyl-L-cysteine (NAC), as a powerful antioxidant, on p-NP-induced testicular toxicity in mice.
Materials and Methods: Adult male NMRI mice (32±4 gr) were divided randomly into 4 groups (n=6), control, NAC (150 mg/kg/day), p-NP (250 mg/kg/day) and p-NP+NAC, and they were treated orally for 35 days. Finally, mice were killed, their right testis were removed and fixed followed by sectioning, tissue processing and Heidenhain azan staining. Testicular tissue sections were then evaluated using stereological method. The level of Malondialdehyde (MDA), a lipid peroxidation index, was also measured in the testis of mice. Data were statistically analyzed using one way ANOVA and means were considered significantly different at p<0.05.
Results: A significant decrease in the mean total volume of testis, diameter, length and the volume of the seminiferous tubules, height of the germinal epithelium and the thickness of the basement membrane along with a significant increase in the MDA level was observed in the p-NP group compared to the control (p<0.01) while the above parameters were compensated to the control level in the p-NP+NAC group (p<0.01).
Conclusion: Our findings indicate that N-acetyl-L-cysteine can protect the testicular tissue against the tissue damage and oxidative stress induced by para-Nonylphenol in mice.
Key words: Para-nonylphenol, N-acetyl-L-cysteine, Stereology, Testis.
P-80
Effects of dietary canola oil on sperm quality parameters at Afshari Ram Breed
Mansouri S, Pirestani A.
Islamic Azad University, Khorasgan Branch, Isfahan, Iran.
Email: nmansorrin@yahoo.com
Introduction: In mammalian and non-mammalian spermatozoa, there are natural fatty acids, cholesterol, phospholipids (mainly lecithin, cephalin and sphingomielin) and glycolipids. Phospholipids of mammalian sperm cell membranes characteristically contain very high proportions of long-chain polyunsaturated fatty acids, particularly n-3 series
Materials and Methods: 18 Kurdish rams were selected with weight average 54.47±2.58 kg and with the age of 3-4 years approximately. They were divided to two experimental groups randomly. Experimental groups were control and canola oil (2.5% of DMI). Before of study, 10 day was considered as adaptation period and then Sperm was collected by electro ejaculation at 6 week and 11 week after begging of adaptation period and sperm motility was analyzed by using CASA software at zero time and 24 hours post ejaculation. Also, percentage of live/dead sperm and morphology was evaluated by staining of Eosin-Nigrosin and Papanicolaou, respectively.
Results: The results showed that motility parameter wasn’t significantly different between whole experimental groups at first time (week 6) but PM% and TM% was significantly different in canola oil group compare to control group at second time (week 11), separately. Percentage of live/dead sperm and morphology was higher significantly in canola oil group compare to control group.
Conclusion: It was concluded that canola oil can improve sperm motility, morphology and viability in Afshari ram.
Key words: Canola oil, Motility, Afshari Ram, Sperm.
P-81
Mutation analysis of bone morphogenetic protein-15 gene in Iranian patients with polycystic ovarian syndrome
Mehdizadeh A, Kalantar SM, Sheikhha MH.
Department of Genetics, International Campus, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: anahita.mz@gmail.com
Introduction: With the prevalence on the order of 6-10%, polycystic ovarian syndrome (PCOS) is considered the most common endocrinologic disorder affecting women in their reproductive age. Major criteria that have been proposed for the diagnostic characteristics of PCOS are clinical and/ or biochemical hyperandrogenism, ovulatory dysfunction and polycystic ovary in ultrasound. It has been suggested that genetic factors participate in the development of PCOS. Follicular development has been considered as one of the impaired processes in PCOS. BMP-15 gene is a candidate gene in follicular development and its variants may play role in pathogenesis of PCOS. Previous investigations have revealed controversial results on BMP-15 mutations in PCOS women among various racial groups.
Materials and Methods: A cross-sectional study was carried out on 70 PCOS patients. Following taking the informed consent, 5ml venous blood was taken from each participant. Genomic DNA was extracted from the blood sample by salting out Method. Then a set of PCR reactions for BMP15 gene was performed using specific primers followed by genotyping with direct sequencing.
Results: As for exon1 in BMP-15, 20 heterozygote (G/C) and 2 homozygote (G/G) cases were found in 70 PCOS patients. Also, one -9 C>G polymorphism in 5’UTR and 3 cases of A308G mutation (A/G) were discovered in our patients.
Conclusion: Result of this study indicates that variants of BMP-15 gene could be related to susceptibility of development of PCOS and may be used as genetic markers for detecting PCOS in the future.
Key words: Polycystic ovarian syndrome, Bone morphogenetic protein 15.
P-82
Effect of adding human chorionic gonadotropin to frozen thawed embryo transfer cycles with history of thin endometrium
Davar R1, Farid Mojtahedi M1, Miraj S1, 2.
1. Department of Obstetrics and Gynecology, Research and Clinical Center for Infertility, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Department of Obstetrics and Gynecology, School of Medicine, Shahrekord University of Medical Sciences, Shahrekord, Iran.
Email: miraj.sepideh@gmail.com; dr.s.miraj@gmail.com
Introduction: Embryo implantation process is a complex phenomenon and depends on fetal and maternal factors interaction. Endometrial thickness is needed for successful implantation. Increasing endometrial thickness, raise the chance of clinical pregnancy. The triple line pattern, with thickening more than 7mm, is indicating the greater chance of successful implantation. We designed this study to assess adding HCG to the conventional protocol in endometrial preparation in women with thin endometrium and history of IVF-ET failure.
Materials and Methods: This non-randomized clinical trial study (quasi experimental design) was performed in Yazd Research and Clinical Center for Infertility on 28 patients. Participants were women who candidate for frozen thawed embryo transfer and had 2 previous failed ET cycle because of thin endometrium. All patients received 8 mg estradiol valterate on second day of menstrual cycle and continued during the study. HCG was administrated (150 IU, IM) from 8th days of cycle. In 12th-13th day Trans-vaginal sonography was done, when endometrial thickness reached at least 7mm, HCG was discontinued and frozen thawed embryo transfer was done.
Results: Totally 28 patients were included. The mean age of participants was 30.39±4.7 years. The mean of endometrial thickness before and after HCG were 5.07±0.43 and 7.85±0.52 mm which were significantly different (p=0.00). After HCG administration 100% patient's endometrial thickness reached more than 7mm. The frequency of 20% improvement after HCG was 89.3% (25 patients). Also there were 5 (17.8%) clinical and chemical pregnant women after HCG.
Conclusion: Our study findings suggest that adding HCG to the conventional preparation method is an effective protocol and significantly improved endometrial thickness and pregnancy outcomes in women with previous embryo transfer failure because of thin endometrium.
Key words: Human chorionic gonadotropin, Frozen thawed embryo transfer, Thin endometrium.
P-83
Glycyrrhiza glabra and vitamin C can reduce toxic effects of acrylamide on sperm parameters in rat
Mirjalili A1, Talebi AR2, Mangoli E2, Ghasemzadeh J2.
- Shahid Sadoughi University of Medical Science, Yazd, Iran.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: Acrylamide (AA) is a chemically reactive substance used in various industries. Recently, acrylamide was discovered in a variety of human foods including heat-processed starchy foods such as potato chips and bread. AA is able to induce sperm damage in male mice.
Materials and Methods: In this experimental study, 30 male Wistar rats of 28 days of age were divided in to five groups: Acrylamide, Acrylamide+ Vitamin C, Acrylamide+ Glycyrrhiza glabra, Acrylamide+ Vitamin C+ Glycyrrhiza glabra and Control. All treatments were administered (oral Acrylamide 10 mg/kg, Vitamin C 200 mg/kg and Glycyrrhiza glabra 150 mg/kg) daily for two months. Thereafter, the cauda epididymis of each rat was dissected and placed in 1 mL of pre-warm Ham’s F10 culture medium for 30 min. The swim-out spermatozoa were analyzed for count, motility, morphology and viability.
Results: The results showed that almost all of the sperm parameters except non progressive motility were significantly different between groups (p=0.001). Also, the mean of sperm parameters in Acrylamide+ Vitamin C+ Glycyrrhiza glabra group was higher than other groups.
Conclusion: This study showed that the co-administration of vitamin C and Glycyrrhiza glabra as antioxidant can reduce the detrimental effects of acrylamide on sperm parameters in rats.
Key words: Glycyrrhiza glabra, Vitamin C, Acrylamide, Sperm, Rat.
P-84
Infertility in male by neurological disorders
Mollaei AR, Seihei E.
Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
Email: ptalireza@yahoo.com
Introduction: Normal sexual and reproductive functions depend largely on neurological mechanisms. Neurological defects in men can cause infertility through erectile dysfunction, ejaculatory dysfunction and semen abnormalities.
Materials and Methods: Science Direct, PubMed, Cochrane, CINAHL, Embase, ProQuest Dissertations, Scopus, (2000-2015) were searched for English-language studies using a list of keywords. The books about physical therapy and medical and neurological were studied too.
Results: Among the major conditions contributing to these symptoms are pelvic and retroperitoneal surgery, diabetes, congenital spinal abnormalities, multiple sclerosis and spinal cord injury. Erectile dysfunction can be managed by an increasingly invasive range of treatments including medications, injection therapy and the surgical insertion of a penile implant. Retrograde ejaculation is managed by medications to reverse the condition in mild cases and in bladder harvest of semen after ejaculation in more severe cases.
Conclusion: An ejaculation might also be managed by medication in mild cases while assisted ejaculatory techniques including penile vibratory stimulation and electro ejaculation are used in more severe cases. If these measures fail, surgical sperm retrieval can be attempted. Ejaculation with penile vibratory stimulation can be done by some spinal cord injured men and their partners at home, followed by in-home insemination if circumstances and sperm quality are adequate. The other options always require assisted reproductive techniques including intrauterine insemination or in vitro fertilization with or without intracytoplasmic sperm injection. The method of choice depends largely on the number of motile sperm in the ejaculate.
Key words: Ejaculation, Electro ejaculation, Infertility, Nervous system diseases.
P-85
Adenosine deaminase G22A polymorphism and recurrent spontaneous abortions
Mohammadi A1, Bonyadi M1, Khalaj kondori M1, Taghavi S2.
- Department of Animal Biology, Faculty of Natural Sciences, Tabriz University, Tabriz, Iran.
- Maternal Fetal Medicine Unit, Department of Obstetrics and Gynecology, Alzahra Hospital, Tabriz University of Medical Sciences, Tabriz, Iran.
Introduction: Adenosine deaminase (ADA) is an enzyme of purine salvage pathway and has two important isoenzymes ADA1 and ADA2. The adenosine deaminase G22A polymorphism (ADA*2) increases the level of adenosine. Adenosine may play a protective role against recurrent spontaneous abortions (RSA), since it regulates blood flow into the uterus and placenta.
Materials and Methods: A total of 100 women were classified in two groups: First group, with a history of recurrent spontaneous abortions (n=50), and second one, without a history of abortions (n=50). Genomic DNA was extracted from peripheral blood with a commercial kit and PCR-RFLP analysis was used to identify the G22A genetic polymorphism.
Results: The frequency of homozygotes (AA) was 2% in control group, whereas no homozygote (AA) was found in the case group (p>0.05). The frequency of heterozygotes (AG) was 20% in the control group and 8% in the case group (p<0.05). The frequency of homozygotes (GG) was 78% in the control group and 92% in the case group (p<0.05). A significant increase in the frequency of AG genotype in controls (p<0.05, OR=0.348) relative to women with the history of RSA demonstrates the protective effect of AG genotype in controls.
Conclusion: The data suggest that women carrying the ADA*2 allele which is associated with the lower enzymatic activity are better protected against recurrent spontaneous abortions. Because decreased enzymatic activity increases adenosine levels. High adenosine levels may play a protective role against recurrent spontaneous abortions, since it regulates blood flow into the uterus and placenta.
Key words: PCR-RFLP, Recurrent spontaneous abortion, Adenosine deaminase.
P-86
Seminal bacterial contaminations: Probable factor in unexplained recurrent pregnancy loss
Nabi A, Khalili MA, Halvaei I, Ghasemzadeh J, Zare E.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: ali.nabi67@yahoo.com
Introduction: It is estimated that about 50% of causes of recurrent pregnancy loss (RPL) cases remain unknown. Sperm factors are suggested to have probable role in cases with RPL. The goal was to determine the possible relationship between semen bacterial contaminations with unexplained RPL. Also, the correlation between number of bacterial colony and sperm chromatin condensation was examined.
Materials and Methods: This study consisted of 30 fertile men (group A) and 30 infertile men (group B) with unknown RPL in their wives. Semen collection and analysis were done according to WHO manuals. Sperm count and motility were evaluated by Makler chamber. Eosin-Nigrosin and Papanicolaou staining methods were applied for viability and morphology assessment, respectively. The semen samples from both groups were cultured for aerobic bacteria. Aniline blue (AB) and toluidine blue (TB) staining methods were applied for evaluating sperm chromatin condensation.
Results: The numbers of colonies were significantly higher in group B when compared to group A. Also, S. aureus and E. coli contaminations showed significant differences between two groups. Both AB+ and TB+ sperm cells showed significant increase in group B compared to group A. There was a significant negative correlation between colony number and progressive motility (p=0.01), and sperm viability (p=0.007). In addition, positive correlations were found between colony number and AB+ (p=0.001) and TB+ (p=0.004) as well.
Conclusion: Bacterial contaminations in semen of men from RPL couples had significantly higher levels when compared to fertile controls. Presence of microorganisms in semen may be correlated with irregular sperm parameters and quality.
Key words: Recurrent pregnancy loss, Bacteria, Semen.
P-87
Precedent infertility and attitudes towards preferred Cesarean
Naeimi N.
Department of Biology, Faculty of Science, University of Sistan and Baluchestan, Zahedan, Iran.
Email: nnaeimi@rocketmail.com
Introduction: Prevalence of natural delivery and caesarean section in pregnant women of a country is one of the indicators of health system performance. Currently, we are exposure to increasing elective Cesarean with different reasons and attitudes.
Materials and Methods: In this descriptive-analytical study, 271 pregnant women completed a researcher-made questionnaire including, demographic characteristics (age, precedent of infertility) and 10 attitude questions about the influencing factors on choosing Cesarean, based on five Likert scale which was a score of 10-30 negative attitude (opposite preference for Cesarean) and 31-50 positive attitude (positive preference for Cesarean). Therefore, to analyze the obtained data, Chi-square test was used through applying SPSS19 software.
Results: Overall 74 subjects (28%) had negative attitudes, while 192 subjects (72%) had positive attitudes. In total 7 subject of pregnant women group (2.6%) had a precedent of infertility and 259 pregnant women had not (95.6%). During the study, 5 subjects did not answer questions. In addition, 5 subjects with precedent infertility (73.4%) vs. 69 subjects (26.6%) with no precedent, showed negative attitudes. 2 subjects (26.6%) vs. 190 pregnant women (73.4%) had positive attitude toward preference of Caesarean section. There was a significant relationship between precedent infertility and attitude.
Conclusion: According to the results, women with precedent infertility who are using assisted reproductive techniques, are more aware of advantages and disadvantages of different methods of termination of pregnancy and most of them opposed natural delivery. Therefor giving them information through related groups of women's health, can change their attitudes.
Key words: Precedent infertility, Attitude, Preference cesarean.
P-88
Protective effects of BDNF against oxidative damage and apoptosis in human spermatozoa
Najafi A1, Sobhani AGH1, Amidi F1, Sadighi Gilani MA2, Moawad AR3, Mikaeili S1, Fallah P2, Rezaiian Z2.
- Department of Anatomy, Faculty of Medicine, Tehran University, Tehran, Iran.
- Infertility Center, Shariati Hospital, Tehran University, Tehran, Iran.
- Department of Surgery, Royal Victoria Hospital, McGill University, Quebec, Canada.
- Department of Urology, Shariati Hospital, Tehran University, Tehran, Iran.
Introduction: Oxidative Stress (OS) is a condition when there is an improper balance between the amount of pro-oxidant substances and the amount of seminal plasma antioxidant factors that can be induced by repeated cycles of centrifugation and seminal plasma antioxidants, removal during the sperm preparation in Assisted Reproductive Technology (ART) Reactive Oxygen Species (ROS) can cause decrease of motility, increase of apoptosis and impaired sperm function, and ultimately, influence on fertilization and pregnancy rate. Therefore, currently there is a great interest to the use of antioxidants to prevent ROS generation and ROS-induced apoptosis during the sperm preparation processes. Brain-derived neurotrophic factor (BDNF) is member of neurotrophin family that has anti- oxidant and anti-apoptotic effects on nervous system. Recent researches show that it also plays key role in male and female reproductive system such as spermatogenesis, nuclear and cytoplasmic maturation and embryo development.
Materials and Methods: Liquefied semen samples obtained from normozoospermic men (n=25) after preparation by double wash (400× gr, 5 min) swim-up technique were divided in to two groups of control and treated group (with BDNF). Then motility was evaluated by means of CASA. Reactive oxygen species (ROS) and apoptosis were assessed by flowcytometry.
Results: Addition of BDNF to the sperm media significantly increased mean total motility and progressive motility (75.54±2.63% vs. 66.81±2.48%, p<0.001, and 46.18±2.2 vs. 39.631±1.97%, p<0.002, respectively). Also BDNF treatment caused decrease of H2O2 (50.01±6.08 vs. 38.31±5.250, p<0.02), necrosis (23.26±2.71 vs. 27.52±2.27, p<0.001) and apoptosis (3.26±0.4 vs. 3.76±0.55, p<0.49).
Conclusion: Considering this result, BDNF treatment can be a potential tool against oxidative damage and apoptosis in human spermatozoa.
Key words: Brain-derived neurotrophic factor (BDNF), ROS, Apoptosis, Flowcytometry.
P-89
Iranian couple’s sexual compatibility during the time: A qualitative study
Nekoolaltak M1, Keshavarz Z1, Simbar M2, Nazari AM3, Baghestani AR4.
- Department of Midwifery and Reproductive Health, Faculty of Nursing and Midwifery, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
- Safe Motherhood Research Center, Department of Midwifery and Reproductive Health, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
- Department of Counseling, Faculty of Psychology and Education, Kharazmi University, Tehran, Iran.
- Department of Biostatistics, School of Allied Medical Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Introduction: Since sexual compatibility is facilitators for marital compatibility and the root of many divorces is the sexual incompatibility, and due to the limited data available in this topic, this qualitative study was designed to exploring the changes of Iranian couple’s sexual compatibility during marriage years.
Materials and Methods: In-depth interview and written narrative methods were used. Purposive sampling with maximum variation (age, education, years of marriage, number of children, etc.) was conducted. Ale Yasin Clinic in Tehran and Fatemieh Clinic in Varamin were the setting of research. Times of interviews were almost 40 min. Data reached to saturation after 14 semi-structured depth interviews and 54 written narratives with the 68 married men and women. Conventional content analysis was done in MAXQDA10 software. Lincoln and Guba criteria were used to validate qualitative data.
Results: Emerged categories included: “duration to reach sexual compatibility" and “mainly increasing sexual compatibility during the time" and "rarely decreasing sexual compatibility during the time". Couple’s sexual compatibility increases over time due to “Getting to know each other more", “increasing sexual awareness and experience”, “talking about sex with spouse”, “skill acquisition for mutual pleasure and satisfaction”. Couple’s sexual compatibility may decrease over the time because "children growing and conflict parental role with spousal role" and “financial difficulties”.
Conclusion: With knowing the impact of passing the time on couple’s sexual compatibility many divorces can be prevented by suitable planning and counseling.
Key words: Sexual compatibility, Iranian couples, Qualitative study.
P-90
Effect of non-polar extract of Phaleria macrocarpa (Mahkota Dewa) on spermatogenesis in rats
Parhizkar S1, Aziz Dollah M2, Ferdaus Binti N2.
- Medicinal Plants Research Centre, Yasuj University of Medical Sciences, Yasuj, Iran.
- Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra, Malaysia.
Introduction: Fertility is the natural capability of giving live. In male, fertility requires the production of large numbers of normal and mature spermatozoa by testes through a complex process call spermatogenesis. Evidence shows increase male age is associated with the decline of mature sperm in seminiferous tubule. Aqueous extract of Phaleria macrocarpa (PM) has been shown to improve fertility in rats by increasing the spermatogenesis.
Materials and Methods: Hexane extract of Phaleria Macrocarpa was prepared by extracting grinded dried slices of fruit followed with drying of the extract using rotary evaporator. A total of 30 male rats were divided into 5 different groups and subjected to daily treatments with PM extract at one of the following concentration: 0, 6, 12, 60 mg/kg of hexane extract, or commercial testosterone hormone for seven consecutive weeks. They will be fed with pellet once a day in the morning and drinking water will be given ad libitum. Body weight was measured once a week. On the last day of supplementation period, the rats were sacrificed and the testis was isolated for histological evaluation.
Results: The result showed that no significant effect of non-polar extract of PM on testes size and volume, thickness of seminiferous tubules and spermatogonia cell number. The mean thickness of seminiferous tubules were 62, 68, 62, 60, 60 µm for 60, 12, 6, 0 mg/kg of PM extract and commercial testosterone respectively. The mean for spermatogonia cell count in rats treated with 60, 12, 6 and 0 mg/kg and commercial testosterone were 52.8, 43.8, 47.0, 52.6, 52.0 cells respectively.
Conclusion: The study concludes that the non-polar extract of PM has no effect on the fertility of male.
Key words: Phaleria macrocarpa (PM), Spermatogenesis, Rat.
P-91
The functional SNP analysis of CYP2D6 gene in patients with Polycystic ovary syndrome (PCOS)
Peirosabet M1, 2,Khosravifar M2, Chekini Z2, Rahimi A1, 2, Shiva M3, Afsharian P2.
- Department of Basic Science and Advanced Technologies in Biology, University of Science and Culture, Tehran, Iran.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Endocrinology and Female Infertility at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: PCOS is a common ovulatory disturbance, which influents about 5-10% women of reproductive age. Clomiphene citrate is used for ovulatory induction in PCOS, that is metabolized by Cytochrome P450 2D6 (CYP2D6). More than 120 variable alleles have been reported for CYP2D6 that some of them are poor metabolizer and cause resistance to drugs that are substrates of CYP2D6.
Materials and Methods: Whole blood was collected from drug control, PCOS and fertile women (60 in each group). RFLP method was used to determine CYD2D6*34 (2850C˃T). Patients should have the Rotterdam criteria. Inclusion criteria’s were age under 35 years old, candidates for IUI, not using other drugs and not have ovary cautery.
Results: Genotype distribution of CC, CT and TT in PCOS group was 47.4%, 40.4% and 12.3% respectively (p=0.479), and frequency of these genotypes in drug control group was 36.7%, 50% and 13.3% respectively (p=0.479), which was very close to results in fertile group: 33.3%, 56.7% and 10% respectively. The number of antral follicles (follicle diameter≥15mm) was measured as drug response which was 1.21 (p=0.118) in clomiphene citrate administered PCOS patients (64%) while this was 1.64 (p=0.09) in 36% of patients treated with letrozole (follicle diameter≥15mm). Same results were observed in drug control group: antral follicles were 1.66 in 80% of clomiphene citrate treated whereas it was 1.77 when they received letrozole (20% of patients).
Conclusion: Although genotype frequencies of 2850C˃T polymorphisms were not significant between three groups, we observed cases bearing this polymorphism, showed resistance to clomiphene citrate, in both groups, which is the first time to report.
Key words: CYP2D6, Polycystic ovary syndrome, Clomiphene citrate.
P-92
Does L-carnitine therapy add any extra benefit to standard inguinal varicocelectomy in terms of deoxyribonucleic acid damage or sperm quality factor indices: A randomized study
Pourmand GH1, Movahedin M2, Dehghan S1, Mehrsai A1, Ahmadi A1, Pourhosein M1.
- Tehran University of Medical Sciences, Tehran, Iran.
- Tarbiat Modares University, Tehran, Iran.
Introduction: Varicocelectomy and anti-oxidant therapy are both documented to have positive effect on spermatogenesis and improving semen quality.
Materials and Methods: 100 patients enrolled in this study and were randomly divided into 2 groups (50 patients in each group). In group 1, standard inguinal varicocelectomy and, in group 2, standard inguinal varicocelectomy plus oral antioxidant therapy (oral L-carnitine, 250 mg 3 times a day) were performed for 6 months. For all patients, routine semen analysis and DNA damage test of spermatozoa (by 2 methods of terminal deoxynucleotidyl transferase dUTP nick end labeling and protamine damage assay) were performed at baseline and at 3 and 6 months postoperatively.
Results: In both groups, the improvement in semen analysis parameters and DNA damage was observed, but there was not any statistically significant difference between the 2 groups in these parameters, although the slope of improvement in DNA damage was slightly better in group 2 (that was not statistically significant).
Conclusion: We observed that addition of 750 mg of L-carnitine orally daily to standard inguinal varicocelectomy does not add any extra benefit in terms of improvement in semen analysis parameters or DNA damage.
Key words: DNA damage, Varicocelectomy, L-carnitine therapy.
P-93
Different haplotype frequency in AKAP3 gene in Iranian patients with short tail sperm
Poursafari E1, Mohseni Meybody A2, Sabbaghian M2, Modarresi T2, Hosseini H1, Gourabi H2.
- University of Science and Culture, Tehran, Iran.
- Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: One kind of sperm abnormalities that leads to men infertility is short flagella of sperms. In this defect, fibrous sheath and axoneme are disorganized, the sperms tail is short, the numbers of sperms in the semen fluid reduce and the sperms are immotile. A Kinas anchoring protein 3 (AKAP3) gene encodes a protein that is involved in the fibrous sheath structure, regulation of sperm motility and head-associated functions such as capacitation and the acrosome reaction. AKAP3 interacts with the regulatory subunit of Protein Kinas A via its dimerization/ docking domain. In the present study, 30 patients with short tail sperm defect and 40 males with normal spermogram referred to Royan Institute were enrolled as case and control groups respectively. The genetic variation in exon 5 of AKAP3 gene which encodes the functional domain of this protein was studied.
Materials and Methods: PCR- sequencing was done on extracted DNA from blood samples of control and patient groups.
Results: According to the results, four haplotype polymorphisms 1378 T>C, 1391 C>G, 1437 T>C and 1573 G>A were observed in all samples studied. These polymorphisms were all observed as the mutant alleles. 92% C allele and 8% G allele of 1391 C>G polymorphism has been reported in East Asia. In the present study, 100% G allele was observed in this polymorphism.
Conclusion: This difference in frequency of mutant allele can be due to different ethnic of Asian population and ours. 1391 C>G alternation is located on the outside of the AKAP3 binding domain to AKAP4 and seems that it cannot have a role in the interaction between these two proteins.
Key words: Short tail sperm defect, Fibrous sheath, AKAP3 gene, PKA, Polymorphism.
P-94
Study of genetic alterations of STK11 in patients with polycystic ovary syndrome (PCOS) and their response to ovarian stimulation
Rahimi A1, 2, Khosravifar M2, Peirosabet M1, 2, Chekini Z2, Shiva M3, Afsharian P2.
- Department of Basic Science and Advanced Technologies in Biology, University of Science and Culture, Tehran, Iran.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Endocrinology and Female Infertility at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: PCOS is the main reason of anovulatory caused infertility. Metformin use for PCOS patients to reduces hyperinsulinemia and androgens production through activation of STK11. We aimed to study the associations between c.842C>T and c.996G>A polymorphisms and risk of PCOS susceptibility in women plus the relations of two polymorphisms and response to ovarian stimulation in PCOS patients.
Materials and Methods: Blood samples were collected from patients and drug control group (male infertility) and fertile group (60 in each group). For genotype analysis, we used RFLP method. We used Rotterdam criteria for patient selection. Inclusion criteria's were age below 35 years old, candidates for IUI, not using other drugs and not have ovary cautery. Our data shown that frequency distribution of CT, TT,CC genotypes in exon6, in PCO Patients was 1.7%, 0%, 98.3% respectively (p=0.236), these frequencies in fertile group was 5%, 0%, 95% respectively and in drug control group was 0%, 1.7%, 98.3% respectively (p=0.236). We have not detected c.996 G>A polymorphism in any groups.
Results: The mean of age and BMI were 26.31±3.68 years and 27 kg/m2 in PCOS and control group. Most of PCOS patients (64%) were received clomiphene citrate and 36% used letrozole whereas 80% of drug control was treated by clomiphene citrate and 20% used letrozole. In both groups their responses to drugs was measured by the number of antral follicles (follicle diameter ≥15mm) by ultrasonography that was 1.64 in PCOS, p=0.09 and 1.77 in drug control treated with letrozole that were better than clomiphene citrate (1.21 in PCOS, p=0.118 and1.66 in control).
Conclusion: Our data did not find any significant differences between these polymorphisms and response to ART medicines in PCOS women. According to our study, PCOS patients had higher response to letrozole rather than clomiphene citrate.
Key words: STK11, Polycystic ovary syndrome, Polymorphism.
P-95
Effects of electromagnetic fields of magnetic resonance imaging on male mice fertilization
Rostamzadeh A1, 2, Anjamrooz SH1, Fathi F1, Rezaie MJ1,3.
- Department of Anatomical Sciences and Genetics, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran.
- Brain and Spinal Repair Research Center, Tehran University of Medical Sciences, Tehran, Iran.
- Clinical Infertility Research Center, Kurdistan University of Medical Sciences, Sanandaj, Iran.
Introduction: Currently, the use of electromagnetic waves in medicine, especially in diagnostic devices such as magnetic resonance imaging (MRI) has increased and many of its biological effects have been reported. The aim of this study was to investigate the adverse effects of 1.5T MRI on fertility and reproductive parameters of male mice.
Materials and Methods: 40 NMRI adult male mice were randomly divided into two groups of control and experimental. The mice in the experimental group were exposed to MRI at 1.5T for 36 min once a week for a period of 3 weeks. Then, in the 1st day and 35th day after the final exposure, 10 mice were used for IVF and 10 mice for In vivo studies. MRI effects on testis weight, the duration of pregnancy, the number of newborns, sperm count, and fertility were evaluated. The obtained data were analyzed using ANOVA and Tukey’s tests.
Results: According to the present study, one day after MRI exposure, testis weight, sperm count, and the number of born children were significantly decreased in the experimental group (p<0.05). Moreover, a significant number of the embryos failed to develop to the blastocyst stage. In contrast, 35 days after exposure, no statistically significant difference was found (p>0.05).
Conclusion: Based on the results of the present study, it seems that although the MRI at 1.5T has adverse effects on fertility and reproductive parameters of the adult male mice, these side-effects are reversible.
Key words: Electromagnetic fields, MRI, Sperm, Fertility.
P-96
Interference of histone modification with aberrant expression of HOXA10 gene in eutopic and ectopic endometrial tissues of women with endometriosis during menstrual cycle
Samadieh Y1, Shahhoseini M1, Favaedi R1, Mahdian S3, Ramezanali F2, Ashrafi M2, Afsharian P1, Aflatoonian R2.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Endocrinology and Female Infertility at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: Disruption of the balance of epigenetic networks, which involves DNA and histone modifications, can cause several pathologies, including reproductive disorders such as endometriosis. HOXA10 gene expressed in endometrium plays an important role in uterine receptivity at the time of implantation, uterine organogenesis and functional endometrial differentiation.
Materials and Methods: Epigenetic analysis were assayed by Chromatin Immunoprecipitation (ChIP), using anti- H3K9ac, H3K9Me2, H3K27Me3, H3K4Me3 antibodies and quantitative expression analysis was performed by real-time PCR technique. For this respect, eutopic and ectopic endometrial samples were collected using laparoscopy from 36 women with documented endometriosis, and also endometrial biopsies were obtained from 22 healthy women with male factor problem as a control group. Ethical approval and informed patient consent was gained for the use of tissue samples.
Results: Data showed a harmonious pattern between mRNA expression of HOXA10 and epigenetic state of its promoter region, in the way that, in secretory phase the activating epigenetic marks, H3k9ac and H3k4me3 were higher in ectopic endometrial tissues and H3K9ac itself were lower in eutopic endometrium. In contrast, H3K9me2 and H3k27me3, the epigenetic marks, known to be associated with gene repression, showed a different pattern in which that, H3K9me2 were higher in eutopic endometrium and H3K27me3 were lower in ectopic endometrial tissues.
Conclusion: Our findings suggest epigenetic might be greatly responsible for aberrant expression of HOXA10 gene in eutopic and ectopic endometrial lesions of patients with endometriosis.
Key words: HOXA10 gene, Endometriosis, Menstrual cycle, Histone modifications, Epigenetics.
P-97
Evaluation of action and mechanism of L-Carnitine in improved sperm quality in male infertility
Sheikhpour R.
Yazd Medical Science Branch, Islamic Azad University, Yazd, Iran.
Email: R.sheikhpour@yahoo.com
Introduction: About 15% of married couples are infertile and that approximately 50% of this is due to male factor infertility. A number of drugs have been proposed as being possible causes of male factor infertility. Estudies showed that a diet supplemented with L-carnitine can improve sperm quality in some mammalian species. In humans, 75% of carnitine derives from diet, while 25% is synthesized from lysine and methionine, although the enzyme that catalyses the hydroxylation of the 4-butirrobetain in L-carnitine is present in few tissues.
Materials and Methods: This review was conducted using the following databases: Medline, Pubmed, Scopus and Science direct from 1990-2014 for better understanding of the mechanism and action of L-Carnitine in male infertility.
Results: L-Carnitine can increase the concentration of this component in the epididymal tubules and spermatozoa. This molecule increases their motility and fertilizing abilities. Both free L-carnitine and acetylated L-carnitine can be accumulated in spermatozoa. In sperm cells, L-carnitine transports fatty acid to mitochondria for oxidation and production of energy for epididymal spermatozoa. Also L-Carnitine has antioxidant property; it via deleting excess acyl-CoA (formyl-CoA), due to toxic effect have important role in cellular detoxification. L-carnitine as antiaging protects cellular membranes against oxidative damage. It prevents protein oxidation, pyruvate and lactate oxidative damage. During sperm maturation, a reduction in total lipid content has been seen due to the changing composition of fatty acids in sperm cells. This leads to an increase in the fluidity of spermatozoa’s plasmalemma. Moreover, L-carnitine reduces lipid availability for peroxidation which guards against potential peroxidative damage.
Conclusion: The result of studies showed that addition of L-carnitine to the diet of male infertility is effective in increasing semen quality, but these results need to be confirmed by more clinical trials.
Key words: L-Carnitine, Sperm quality, Male infertility.
P-98
Effects of cell phone radiofrequency electromagnetic waves (RF-EMW) on human spermatozoa
Soltani S1, Khalili MA1, Omidi M1, Nabi A1, Shamsi F2.
- Department of Biology, Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Biostatistics and Epidemiology, College of Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: Cellular phones emit radiofrequency electromagnetic waves (RF-EMW) in the low frequency microwave. The detrimental effects of RF-EMW on the reproductive system and human fertility have been debated in recent years. The goal was to assess the influence of cell phones RF-EMW on different parameters and DNA integrity of human spermatozoa.
Materials and Methods: 50 semen samples were categorized in two groups: a) normospermia (n=24); and b) asthenozoospermia (n=24). After liquefaction, each sample was divided into two aliquots. One aliquot (experimental) was exposed to mobile radiation for 1 hr, and the second aliquot (unexposed) served as control. The sperm parameters and chromatin/ DNA integrity were examined and compared between groups.
Results: Normal samples exposed to RF-EMW showed insignificant differences in sperm motility, viability, and chromatin/DNA integrity. Regarding sperm morphology, normal morphology was significantly decreased in experimental group (p=0.04). Moreover, sperm parameters as well as DNA structure showed no significant differences in astenozoospermic samples.
Conclusion: Data did not show any noticeable impact of RF-EMW on human sperm quality. Further studies should be considered for confirmation of the results.
Key words: Cell phone, Electromagnetic radiation, Sperm parameters, DNA integrity.
P-99
Assessment of genetic variations in intron4 and exon5 of RABL2B gene in Iranian infertile men with immotile short tail sperm defect
Hosseini H1, Sabbaghian M2, Mohseni Meybodi A2, Hosseinifar H2, Poursafari E1, Sadighi Gilani MA2.
- Department of Basic Science and Advanced Technologies in Biology, University of Science and culture, Tehran, Iran.
- Department of Andrology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: The immotile short tail sperm (ISTS) defect, is a syndrome which causes male infertility. Patients with ISTS disorder have immotile short-tailed sperm with disorganized axonem, and a significant decrease in sperm counts too. Numerous proteins are involved in sperm tail formation. One of these proteins is RAB Like 2B (RABL2B), which recently its essential role in sperm intra-flagellar transport and fertility in male mouse has been demonstrated. So its gene, which called RAB Like 2B (RABL2B), is an appropriate candidate gene in human studies. RABL2B protein has 4 GTP binding domains which have important roles in protein function. Exon 5 of RABL2B gene, codes one of these main domains and intron 4 is the location for binding to some important transcription factors. The purpose of this study was to evaluate the genetic variations of exon 5 and intron 4 of RABL2B gene in infertile men with ISTS defect and controls
Materials and Methods: In this study, 30 infertile men with ISTS defect and 30 normozospermic men as controls were recruited. Remarkably it took 2 years to collect patients samples. To study the genetic variations, DNA was extracted from peripheral blood, then PCR sequencing was done.
Results: Sequence analysis results did not identify any mutations or single-nucleotide polymorphisms (SNPs) in exon 5, but an intronic variant (rs:144944885), was found in heterozygote form in one patient. No mutations or SNPs was identified in controls.
Conclusion: Although our data just revealed an intronic variations and no mutations or SNPs was detected in exon 5, due to the high expression of RABL2B gene in testis, and considering the fact that RABL2B is evolutionarily conserved and not many studies have been conducted about the exact role of this gene in human male fertility, evaluation of other exons and regulatory areas of this gene is strongly recommended.
Key words: RABL2B gene, ISTS, Male infertility.
P-100
Localization of septin 14 protein in sperm
Vahabi Barzi N, Hosseinifar H, Nickhah Kalashami Z, Sadighi Gilani MA, Sabbaghian M.
Department of Andrology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: nastaranbarzi@yahoo.com
Introduction: In mammals, 14 septin genes have been identified so far. Disruption of septin functions has been implicated in the pathology of many diseases, including male infertility. Here, we study about one of the new members of the septin family called septin14 that is specially expressed in testis. This gene has two transcripts but only one of them transcript into proteins.
Materials and Methods: In this study, semen obtained from subjects attending Royan Institute that had normal spermogram. After that, immunocytochemistry, which is a common laboratory technique, was used to anatomically localize presence of a specific protein in cells by a specific primary antibody. The primary antibody allowed visualization of the protein under a fluorescence microscope when it was bound by a secondary antibody with a conjugated fluorescence.
Results: The protein expression was detected in the sperm from head to tail and highly localized in the front of the acrosome and the neck.
Conclusion: This is the first report on the localization of septin 14 in sperm. Septin 14 plays an important role in sperm morphology. Regarding the presence of this protein in sperm acrosome and neck, it can be concluded that the probable decrease of septin 14 protein expression in sperm may be associated with the pathogenesis of male infertility.
Key words: Septin 14, Immunocytochemistry, Sperm, Male infertility.
P-101
Molecular studying of sperm surface proteins and their cellular functions
Vaseghi-Dodaran H1, Daghighkia H1, Atshan M2, Jafari S3, Sobhani AGH4.
- Department of Animal Sciences, Faculty of Agriculture, Tabriz University, Tabriz, Iran.
- Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Midwifery and Fertility Health, School of Nursing and Midwifery, Ardabil University of Medical Sciences, Ardabil, Iran.
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Introduction: Fertilization in mammalian is a complicated process with some connections among wide range of glycolipid, glycoprotein and antigenic indexes on sperm and egg surface of every species. We can improve our understanding in fertilization mechanisms and also get some information on molecular or immunological defects responsible for infertility by recognizing the antigens on sperm surface and investigating their role on cell physiological performance. The key challenge is to move from lists of identified proteins to informed understanding of biological function. Studying the sperm surface molecules and investigating their biochemical, biophysical and physiological properties to understand fertilization process.
Materials and Methods: A review of recent bibliography collected from internet database as PubMed, Google scholar, SID by the use of relevant keywords on different markers of cell surface and Sperm cell proteomic published in 2000-2014.
Results: Sperm membrane proteins contain an important part of membrane and are considered as special antigenic index to each species. Every change, increase or decrease in these proteins alters sperm performances and abilities. The important events which occurs during the interaction of sperm and egg and the reactions between them for fertilization relates to identification of these two cells by connecting receptors to ligands which exist on their cell membrane. Discovering the proteins on sperm surface which act as fertility biomarkers and participate in fertilization leads to more apprehension of this cell physiology and performance, so understanding molecular events like sperm acrosome reaction, capacitation and motility.
Conclusion: Sperm surface proteins can use as fertility biomarkers, therefore identification and supplementation these proteins to cryopreservation medium can improve post-thaw parameters of sperm and prevent sperm quality during cryopreservation.
Key words: Proteomics, Sperm, Marker, Fertilization.
P-102
Frequency of chromosome inversion (pericentric and paracentric) in recurrent abortions in patients referred to cytogenetics laboratory of Sarem Hospital
Abdi A1, Bagherizadeh I2, Bahman I2, Shajarehpour L1, Hadipour F3, Hadipour Z3, Shafagh Y4, Behjati F5.
- Azad Islamic University, Tehran, Iran.
- University of Social Welfare and Rehabilitation Seiences, Tehran, Iran.
- University of Ghom, Ghom, Iran.
- School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
- Department of Medical Genetics, Tehran University of Medical Sciences, Tehran, Iran.
Introduction: Recurrent abortion affects almost 15% of diagnosed pregnansis. More than 50% of miscarriages in the 1st trimester are due to chromosome abnormality. The aim of this study is to present the role of chromosomal inversions in recurrent miscarriages in patients referred to the Cytogenetics Laboratory of Sarem Hospital in Tehran. One group of inversions is pericentric inversion of chromosome 9, which is usually regarded as a normal population variant.
Materials and Methods: The samples were studied using high resolution GTG banding technique. For each patient, a minimum of 15 metaphases was examined by light microscopy.
Results: Pericentric inversion around centromere of chromosome 9 was observed in 29 patients (1.37%) and pericentric inversion in hetrochromatin region was found in chromosome 1 in one patient and chromosome Y in another patient, and one patient had pericentric inversion of chromosome 2. Chromosomal inversion involving other autosomal chromosomes included pericentric inversion of chromosomes 1, 5, 11 and 12 (1.9%), and paracentric inversion of chromosomes 3, 6, 7, 8, and 12 (0.23%).
Conclusion: The chromosomal imbalance of gametes may produce spontaneous abortions and malformed offsprings. This suggests that such inversions should not be ignored and they can play an important role in reproduction failure. However, we have shown that the rate of pericentric inversion of chromosome 9 is similar in different referral groups (1-2%) and similar to normal population and thus of no clinical significance.
Key words: Pericentric and paracentric chromosome inversions, Recurrent abortions, inv (9).
P-103
Kit Ligand (KL) promotes the primordial follicle growth in mouse vitrified ovaries
Abdi Sh1, Salehnia M1, Hosseinkhani S2.
- Department of Anatomy, Tarbiat Modares University, Tehran, Iran.
- Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Introduction: Following cryopreservation of ovarian tissue, the primordial follicles are well preserved with minimal damage. An alternative technique to improve the development of follicles within ovarian tissue is ovarian organ culture following cryopreservation. Kit ligand (KL) known as stem cell factor, steel factor or mast cell growth factor is involved in the activation of primordial follicles, oocyte growth and proliferation of granulosa and theca cells.
Materials and Methods: One week old mouse ovaries were collected and divided into vitrified and non-vitrified groups. Then they were cultured in the presence or absence of KL for 7 days. The development of ovarian follicles was evaluated by histology and also the mean area and hormonal level was analyzed during culture period. Apoptosis assessment was done using DNA laddering, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) method and caspase -3/7 activity assay.
Results: The proportion of preantral follicles and the level of hormones were increased in all cultured groups and it was significantly higher in KL treated groups than their control (p<0.001). DNA fragmentation and TUNEL positive signals were seen in vitrified cultured ovaries. The level of caspase -3/7 activity was higher in vitrified cultured ovaries.
Conclusion: KL could improve the development of follicles in vitrified cultured ovaries also it could act as anti-apoptotic factor during culture of vitrified samples. The development potential of follicles in vitrified groups was lower than fresh ovaries.
Key words: In vitro culture, Kit Ligand, Vitrification, Caspase -3/7.
P-104
Association between rs1264457 A/G polymorphism of HLA-E gene and recurrent pregnancy loss
Zarezade Z1, 4, Ashrafzadeh Mehrjardi HR2, 4, Zaimy MA3, Ghasemi N4.
- Department of Medical Genetics, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Biology, Faculty of Science, Azad University, Ashkezar Branch, Yazd, Iran.
- Department of Medical Genetics, Tehran University of Medical Sciences, Yazd, Iran.
- Recurrent Abortion Research Center, Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: Recurrent pregnancy loss (RPL) is defined as two or more consecutive miscarriages before twentieth week of pregnancy. The cause of 50% recurrent miscarriages is unexplained. The goal of this research was to investigate the association between RPL and rs1264457 A/G polymorphism of HLA-E gene.
Materials and Methods: In this case-control study we used Polymerase Chain Reaction Restriction Fragment Length Polymorphism (RFLP-PCR) to determine frequency of this polymorphism in 105 women with RPL in comparison with 109 healthy controls. The collected data were analyzed by the statistical package for the social sciences software (SPSS) and the chi-square test.
Results: The finding showed clear correlation between this polymorphism and RPL. The results will be presented in the congress.
Conclusion: If distinctive data increase frequency of this polymorphism in RPL, it can exponent influence of this polymorphism in generating RPL. We hope result of this investigation to be useful for finding some causes of unexplained recurrent pregnancy loss.
Key words: Recurrent pregnancy loss, HLA-E, Polymorphism.
P-105
Evaluation of the meiotic spindle and zona pellucida after vitrification of mouse MII oocytes
Akyash F, Khalili MA, Safari S, Ashourzadeh S, Agha-Rahimi A, Khoradmehr A.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: fakyash@gmail.com
Introduction: Oocyte vitrification is used as fertility preservation method for young aged females with cancer. The oocyte is one of the biggest mammalian cells with large volume of cytoplasm, compares with other cells. Also, oocyte has less permeability to cryoprotectants (CPAs), since high concentration of CPAs used in vitrification procedure with possible toxic effects. Evaluation of the effect of vitrificaiton on meiotic spindle (MS) and zona pellucida (ZP) of mouse MII oocytes using Polyscope technology was the subject of this study.
Materials and Methods: Ovulation induction performed for 6-8 weeks old NMRI mice by injection of 10 IU PMSG (IP) and 48 hr later with 10 IU HCG. After oviduct removal, COCs were denuded and MII oocytes were retrieved aseptically. The equilibration (7.5% EG, 7.5% DMSO) and vitrification (15% EG, 15% DMSO) solutions were prepared in Hams F10 supplemented with 20% HSA as base medium. For 3 steps of thawing, base medium plus 1, 0.5 and 0.25 M sucrose were used respectively. Assessment of ZP and MS performed at different time intervals after thawing.
Results: After warming, vitrified oocytes showed a significantly fast ZP digestion timing compared to the control group (p<0.05). Presence of MS was detected in 69.23% of vitrified oocytes 1 hr after warming, and presence of MS was showed in 11.11% of vitrified oocytes 2 hr after warming and 26.92% of vitrified- warmed oocytes showed sign of degeneration.
Conclusion: ZP digestion was faster in vitrified group rather than control and recovery of MS structure after vitrification/ warming at 37oC. Vitrification technique still needs more modification before its application in fertility preservation program.
Key words: Oocyte, Meiotic spindle, Zona pellucida, Vitrification.
P-106
Protective effect of vitamin E on testicular germ cells and serum Malondialdehyde concentration in rats fallowing exposure to bisphenol A
Amjadi M, Soleimani Mehranjani M, Naderi Noreini S.
Department of Biology, Faculty of Sciences, Arak University, Arak, Iran.
Email: m.amjadi1369@gmail.com
Introduction: Bisphenol-A (BPA), used in plastic industries, has toxic effects on the reproductive system. It can cause testis injury through the production of Reactive oxygen species (ROS).
Materials and Methods: 24 adult male rats (220±15 gr) were divided into 4 groups (n=6): control, BPA (250 mg/kg/day), Vit E (150 mg/kg/day) and BPA+ Vit E. All groups were orally treated for 56 days. By the end of the treatment, animals were killed, their right testis were taken out, fixed, sectioned, processed and stained with heidenhain azan method. The total number of germ and Sertoli cells was estimated using the optical dissector technique. Serum MDA levels were also measured with the spectrophotometric method. Data were statistically analyzed using one way ANOVA and Tukey's test and means were considered significantly different at p<0.05.
Results: A significant decrease in the total volume of testis, number of long and round spermatids, spermatocytes and Sertoli cells (p<0.05) and a significant increase (p<0.03) in the serum MDA level were found in rats treated by BPA compared to the control group. Histopathology observations revealed morphological changes in BPA-exposed rats including atrophy and vacuolation in the germinal epithelium. The above parameters were compensated to the control level in the BPA+ Vit E group.
Conclusion: Due to the antioxidant activity of vitamin E, it may ameliorate the damaging effects of bisphenol A on spermatogenesis.
Key words: Bisphenol A, Vitamin E, Sertoli and germ cells number, Optical disector.
P-107
Evaluation of follicular genes pattern and growth of preantral follicles after culture in alginate hydrogel following vitrification of the mouse ovarian tissue
Asgari F1, Valojerdi MR1, 2, Ebrahimi B 2.
- Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
- Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: This study was set up to evaluate the effect of ovarian tissue vitrification on the in vitro growth and pattern of follicular genes expression in mouse preantral follicles encapsulated within alginate hydrogel.
Materials and Methods: Ovaries of 12-14 days old female NMRI mice allocated into fresh control and vitrification groups. For cryopreservation, ovaries equilibrated with a solution (ES) that composed of 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 15 min and vitrification was performed by a solution (VS) that composed of 15% EG and 15% DMSO for 30 min then ovaries loaded to nitrogen with needle. Descending concentrations of sucrose (1, 0.5, 0.25 M) used for warming step. After histologic assessment of ovaries, in the next stage, pre-antral follicles was mechanically isolated from control and vitrified ovaries and was cultured for 12 days in 0.7% sodium alginate. Preantral follicles survival rate, growth, antrum formation and relative expression of oocyte- specific genes (Bmp15, Gdf9،Fgf8, Igf1, Kit, Kit ligand) was assessed after 1, 8 and 12 days of culture and finally maturation rate of oocytes was studied.
Results: Preantral follicles in vitrified group showed a lower survival rate on 8 and 12 days of culture (p<0.05) but could retain a comparable morphological appearance, growth and antral formation with the control group. Reduction of Bmp15, Gdf9, Fgf8, Kit, Kit-l showed during 12 days of culture (p<0.05). Although the expression of Gdf9, Kit, Kit-l in vitrification group was more than control group at 1st day of culture but all genes in both groups showed same expression after 12 days of culture (p<0.05).
Conclusion: Although vitrification of ovarian tissue reduces the survival rate, it is a safe method for preservation of preantral follicles and could not modify the relative expression of follicullar genes and oocytes maturation capacity.
Key words: Follicular genes, Preantral follicle, Vitrification of ovarian tissue, Alginate.
P-108
Effects of morphine on sperm parameters, protamine deficiency and DNA integrity in mice
Bahari H1, Talebi AR1, 2, Nahangi H1, Anvari M1, 2, Abbasi Sarcheshmeh A1, Khoradmehr A2, Danafar AH2.
- Department of Biology and Anatomy, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: Morphine as a natural alkaloid (opiate) is the most effective pain-relieving drugs and can be abused because of its high addictive potential. Opiate abuse is considered as one of the problems associated with poor semen production and sperm quality. Therefore, for the first time, this experimental study was carried out to evaluate the effect of intraperitoneal injection of morphine on sperm parameters, protamine deficiency and DNA integrity of spermatozoa aspirated from cauda epididymis of mice.
Materials and Methods: Totally 24 adult male Balb/c mice (8 weeks old. 30g) were equally divided into 3 groups each containing 8 mice. Mice of group 1 served as control fed on basal diet, group 2 received basal diet and normal saline and group 3 received basal diet and morphine (15 mg/kg/daily, intraperitoneal) for 35 days. Finally right tail of epididymis of each mouse was cut and placed in Ham’s F10 for 30 min. Released sperm were used to analyze count, motility, viability (eosin-nigrosin staining), morphology (Papanicolaou), protamine deficiency with chromomycin A3 (CMA3) and apoptosis via TUNEL assay.
Results: In morphine-treated mice a significant decrease was found in sperm viability, normal morphology, count and motility compared to other groups (p<0.05). In relation with protamine deficiency the rates of CMA3-reacted spermatozoa were similar in groups (p>0.05). In addition, in morphine-treated mice there was a significant increase in apoptosis compared to other groups (p<0.05).
Conclusion: The results showed that morphine abuse disturbs sperm parameters and DNA integrity but not protamine content of the sperm nucleus.
Key words: Mice, Morphine, Sperm parameters, Apoptosis, Protamine deficiency.
P-109
Study the protective role of jujube extract on growth index of mice fetuses exposed to Carbamazepine
Doostabadi MR1, Afshar M1, Nahangi H2, Hasanzadeh Taheri MM1, Hassanpour M1.
- Birjand University of Medical Sciences, Birjand, Iran.
- Shahid Sadoghi University of Medical Sciences, Yazd, Iran.
Introduction: Carbamazepine (CBZ) is an anticonvulsant drug that is widely used, primarily to treat seizures. One of the side effects of this medication during pregnancy is the reduced fetal growth. The jujube has been widely used in traditional medicine because of its special compound. The purpose of this study was to evaluate the preventive effects of aqueous extract of jujube on growth index of mice fetuses exposed to Carbamazepine.
Materials and Methods: In this study, 100 Balb/C mice (28-30 gr; 8-9 weeks-old) were divided into eight experimental and two control groups. Experimental groups (I, III, V) and (II, IV, VI) received daily intraperitoneal injections of 50 and 100 mg/kg of CBZ, respectively. Experimental groups (III, IV) and groups (V, VI) were gavaged doses of 200 and 400 mg/kg of aqueous extract of jujube respectively. Groups VII and VIII received only doses of 200 and 400 mg/kg of aqueous extract of jujube respectively. Control groups I and II were gavaged to normal saline and tween respectively. Injections were done on 0-15 gestational days (GD), and aqueous extract was gavaged along with CBZ starting 10 days prior to gestation. On GD 18, fetuses were removed for weighing and crown-rump measuring. Data were analyzed by ANOVA and Tukey tests using SPSS (version 18) at 0.05%.
Results: Average weight and body length of fetuses in experimental groups I and II, that received CBZ, significantly reduced compared with control groups. In the fetuses of experimental groups V (1.20±0.38) and VI (1.21±0.21 gr) that recieved jujube extract (400 mg/day) with CBZ, the mean weight of these fetuses increased meaningfully in comparison with the experimental groups I (0.93±0.19 gr) and II (0.92±0.26 gr) respectively.
Conclusion: This study showed that aqueous extracts of jujube can have preventive effects on reducing effect of CBZ on birth fetal weight.
Key words: Growth index, Jujube, Carbamazepine, Mice.
P-110
Effectiveness of mindfulness-based cognitive therapy on improvement of perceived stress and infertility in infertile women undergoing IVF irrational cognitions
Dortaj A1, Mehdizadeh A1, Dortaj H2.
- Payame Nour University, Dubai, UAE.
- Shahid Sadoughi University of Medical Scienses, Yazd, Iran.
Introduction: Effectiveness of mindfulness-based cognitive therapy on improvement of perceived stress and infertility in infertile women undergoing IVF irrational cognitions.
Materials and Methods: This clinical trial with pre-test post-test was done in 1331 infertile women referred to the Reproductive Health Research Center, Crescent Hospital, UAE who were treated with IVF. In total, 24 infertile women filled an irrational cognitions questionnaire related to childbearing and infertility. They were divided into experimental and control groups. The experimental group received mindfulness-based cognitive therapy for 8 sessions of 2 hr each. The control group did not receive any mental health services. Those two questionnaires were completed before and after the intervention. Data were analyzed by SPSS and p<0.05 was considered significant.
Results: There were no significant differences between the experimental and control groups in terms of reduction of perceived stress of infertility. But, the improvements in the recognition of irrational for childbearing between two groups was significantly different.
Conclusion: Teaching mindfulness-based cognitive therapy on improvement of perceived stress and irrational cognitions related to fertility in infertile women undergoing IVF treatment is effective.
Key words: Perceived stress, Infertility, IVF treatment, Mindfulness-based cognitive therapy, Irrational cognitions of parents.
P-111
Genotype and phenotype frequencies of paraoxonase 1 in fertile and infertile men
Fattahi A1, Tavilani H1, Latifi Z1, Esfahani M2, Khodadadi I2, Karimi J2,Bahrayni E2, Vatannejad A3, Vaisi-Raygani A4, Ghorbani M2.
- Department of Clinical Biochemistry, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
- Department of Clinical Biochemistry, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran.
- Student’s Scientific Research Center, Tehran University of Medical Sciences, Tehran, Iran.
- Fertility and Infertility Research Center, Kermanshah University of Medical Science, Kermanshah, Iran.
Introduction: Paraoxonase1 (PON1) is a glycoprotein associated with high density lipoprotein and has antioxidant activity. The impact of PON1 in various stages of spermatogenesis has also been suggested.
Materials and Methods: Q192R variants of PON1 were determined in 150 fertile and 150 infertile men using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Plasma arylesterase and paraoxonase activities were detected by spectrophotometry and malondialdehyde (MDA) level was measured using thiobarbituric acid.
Results: Our results showed no significant difference in the distribution of PON1 genotypes and alleles between fertile and infertile groups. However morphology and motility of sperm were associated with various genotypes of PON1. The number of fertile males with the BB phenotype (high activity) was significantly higher than that of infertile males, whereas the number of individuals with the AB phenotype (moderate activity) was statistically higher in infertile men compared with the fertile group. Additionally, MDA and arylesterase activity levels were significantly higher in infertile subjects compared with fertile men.
Conclusion: We speculate that the low activity of PON1 can be a risk factor for male infertility probably due to a decrease in antioxidant activity of PON1 and increase in lipid peroxidation.
Key words: Genetic polymorphism, Male infertility, Paraoxonase 1, Reproduction.
P-112
Saccharin consumption increases sperm DNA fragmentation and apoptosis in mice
Rahimipour M, Talebi AR.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: marziehrahimipour@yahoo.com
Introduction: Saccharin is an artificial non-caloric sweetener that used to sweeten products such as drinks, candies, medicines, and toothpaste, but our bodies cannot metabolize it. Sodium saccharin is considered as an important factor in tumor promotion in male rats but not in humans.
Materials and Methods: Totally 14 adult male mice were divided into 2 groups. Group 1 served as control fed on basal diet and group 2 or experimental animals received distilled water containing saccharin (0.2% w/v) for 35 days. After that, the left cauda epididymis of each mouse was cut and placed in Ham’s F10. Swimmed-out spermatozoa were used to analyze count, motility, morphology (Pap-staining) and viability (eosin-Y staining). Sperm DNA integrity, as an indicator of apoptosis, was assessed by sperm chromatin dispersion (SCD) and terminal deoxynucleotidyl transferase (TUNEL) assay.
Results: Following saccharin consumption, we had a reduction in sperm motility with respect to control animals (p=0.000). In addition, the sperm count diminished (17.70±1.11 in controls vs. 12.80±2.79 in case group, p=0.003) and the rate of sperm normal morphology decreased from 77.00±6.40 in control animals into 63.85±6.81 in saccharin-treated mice (p=0.001). Also, we saw a statistically significant increase in rates of sperm DNA damage and apoptosis in experimental group when compared to control one (p=0.001, p=0.002 respectively).
Conclusion: Saccharin consumption may have negative effects on sperm parameters, and increases the rate of sperm DNA fragmentation and apoptosis in mice.
Key words: Sperm, Saccharin, Apoptosis, Mice.
P-113
Increased telomeric repeat containing RNA (TERRA) levels in cumulus cells of infertile polycystic ovary syndrome patients
Ghobadi N1, 2, Shahhoseini M2, Favaedi R2, Hassani F3, Movaghar M3, Karimian L3, Eftekhari Yazdi P3.
- Faculty of Basic Sciences and Advanced Technologies in Biology at University of Science and Culture, Tehran, Iran.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: One of the important reasons of anovulation based infertility is polycystic ovary syndrome (PCOS) which occurs in 5-10% of women in reproductive age. It seems that the length of telomere, TTAGGG tandem repeats, is related to proliferation and differentiation events during follicular development including the mechanisms which regulate successful reproduction. It is widely recognized that bi-directional communications exist between oocytes and the surrounding cumulus cells which are essential for the production of competent oocytes. During the past few years, study on cumulus cells showed that the length of telomere is longer in cumulus cells of mature oocytes than immature ones. Until now, telomeres have been considered to be transcriptionally silent but recent studies have clearly shown that transcription of subtelomere regions produces telomeric repeat containing RNA, named TERRA. In this study, we tried to identify the correlation of TERRA levels with oocytes maturation by evaluating of TERRA transcripts in cumulus cells of PCOS patients.
Materials and Methods: For this respect, cumulus cells were collected from 6 PCOS patients and 9 healthy women with male factor infertility through ICSI/ IVF procedure. TERRA transcripts were measured by using quantitative real- time PCR.
Results: Our data showed that the level of TERRA transcripts was increased significantly in PCOS group vs. control group (p˂0.05).
Conclusion: This finding implies a considerable association between TERRA transcript levels and PCOS, so it can be concluded that any changes in levels of TERRA transcripts can be judged as a potential marker for the quality of oocytes in ART procedure.
Key words: TERRA, Telomere, PCOS, Cumulus cells.
P-114
Epigenetic modification profile of endometrium in endometriosis patients
Hosseini E1, Afsharian P2, Ashrafi M3, 4, Ramazanali F2, Mehraein F1, Shahhoseini M2, Aflatoonian R3.
- Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Endocrinology and Female Infertility at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Obstetrics and Gynecology, Iran University of Medical Sciences, Tehran, Iran.
Introduction: Endometriosis defined as the presence of endometrium-like tissue outside of the uterine cavity, is a common gynecologic disorder. Although endometriosis is a multifactorial disease and the exact etiology is not clearly understood, recently, some evidence suggests that epigenetic is associated with the molecular features of endometriosis. Two main epigenetic regulatory mechanisms, DNA methylation and histone modifications regulate expression of genes and recognize the states of diseases. The aim of this study was to investigate alterations of DNA methylation and histone acetylation and methylation levels in eutopic and ectopic endometrium of endometriosis patients.
Materials and Methods: Informed consents were gained from all patients then eutopic and ectopic endometrium samples (n=5) were collected from endometriosis patients undergoing surgery and biopsy, as well as endometrial tissues from healthy fertile women (n=5) during the proliferative phase of menstrual cycle. Chromatin extracts from samples were prepared following fixation and then shearing into fragments by sonication. Nucleosome ELISA was performed on chromatin extracts, in order to identify global histone H3K9 acetylation/ methylation and DNA methylation, using antibodies against H3K9ac, H3K9me and MeCP2, respectively.
Results: We have identified global histone H3K9 hypermethylation in ectopic and eutopic endometrium, compared with controls. A significant hyperacetylation at histone H3K9 was observed in eutopic samples compared to ectopic and control groups. Furthermore, eutopic endometrial samples were globally DNA hypermethylated in comparison with controls.
Conclusion: These results clearly show an epigenetic switch in endometrial and endometriotic tissue of patients with endometriosis, in the way that aberrant DNA methylation and histone acetylation/ methylation status may play a dynamic role in occurrence of endometriosis and support the opinion that epigenetic abnormalities have causative functions in endometriosis.
Key words: Endometriosis, Endometrium, Epigenetic, Methylation, Acetylation.
P-115
The effects of chronic and acute ethanol administration on sperm chromatin parameters in mice
Hosseini A, Borzouei Z, Faramarzi A, Ghasemi-Esmailabad S, Talebi AR.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: hosseinia30@yahoo.com
Introduction: Sperm chromatin can be damaged by effects of some toxic materials. Among this, ethanol is one of the reproductive toxins that consumes abuse in men and may be associated with poor sperm quality. The purpose of this investigation was to examine the acute and chronic effects of ethanol consumption on sperm parameters and chromatin assay through testing the effects in mice.
Materials and Methods: Animals were randomly divided into 3 groups consisting of 10 mice each. Group 1 and 2 served as alcohol group and were received a daily dose of (3 g/kg body weight as 25%, v/v) ethanol by I.P. for four and eight weeks respectively. Group 3 (control group) was given normal access of food and water. The subdivisions and dosages were based on past works. At the end of treatments, laparotomy was conducted to expose the reproductive system, cauda of epididymis were immediately dissected out for evaluations of sperm parameters and various tests were used to analysis of the chromatin integrity of sperm.
Results: Various tests such as aniline blue (AB), chromomycin A3 (CMA3), toluidine blue (TB), and acridine orange (AO) were used to analysis the sperm quality. We did not find any statistically significant differences in acute and control groups. Although sperm count and motility were increased but viability, normal morphology, DNA and chromatin integrity were unaffected by ethanol in acute group and were identical with control group. Our results demonstrated that count, motility and viability of sperms were dramatically decreased in chronic treated animals. Besides of that, DNA and chromatin integrity were significantly decreased.
Conclusion: Therefore, ethanol abuse can induce abnormality in sperm structure and function, and this may be one possible cause of male infertility.
Key words: Sperm, Ethanol, Chromatin integrity.
P-116
Comparison of oral dydrogesterone with vaginal progesteronefor luteal support in IUI cycles: A randomized clinical trial (RCT)
Khosravi D1, Taheripanah R1, Khosravi R1, Taheripanah A2, Tarighat Monfared V1, Defaee S1, Mehdighalb S1, Hosseini Zijoud SM3.
- Reproductive Health Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
- Department of Molecular and Cellular Sciences, Faculty of Advanced Sciences & Technology, Pharmaceutical Sciences Branch, Islamic Azad University, Tehran, Iran.
- Hamadan University of Medical Sciences, Hamadan, Iran.
Introduction: Progesterone supplementation is the first line of treatment in ovarian stimulation. This study was conduct to compare the effect of oral dydrogestrone with vaginal cyclogest on luteal phase support in intrauterine insemination (IUI) cycles.
Material and methods: This prospective, randomized, double blind study was performed in a local infertility center from May 2013-2014. It consisted of 150 infertile women younger than 35 years old undergoing ovarian stimulation for IUI cycles. They underwent ovarian stimulation with oral dydrogesterone (20 mg) as group A and vaginal cyclogest (400 mg) as group B in preparation for the IUI cycle. Clinical pregnancy and abortion rates, mid luteal progesterone (7 days after IUI) and patients' satisfaction were compared between the two groups.
Results: The mean serum progesterone levels was significantly higher in group A compared to group B (p=0.001). Pregnancy rate in group A was not statistically different from group B (p=0.58). Abortion rate in group B was higher than this rate in group A, but the difference was not statistically significant (p=0.056). Satisfaction rates were significantly higher in group A compare to group B (p<0.001).
Conclusion: We concluded that oral dydrogestrone is as effective as vaginal progesterone for luteal-phase support in woman undergoing IUI cycles. Moreover, the mean serum progesterone levels and satisfaction rates in dydrogestrone group were higher than cyclogest group.
Key words: Dydrogesterone, Cyclogest, Luteal-phase, Infertility.
P-117
The detrimental effects of alcohol on sperm parameters and DNA integrity in diabetic mice
Mangoli E1, Pourentezari M2, Talebi AR1, Rahimipour M3, Taheri F1.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Biology and Anatomical Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Biology and Anatomical Sciences, Tarbiat Modares University, Tehran, Iran.
Introduction: Diabetes mellitus (DM) is a chronic disease that can affect male reproductive function at multiple levels. Ethanol can suppress reproductive function and sexual behavior in laboratory animals and humans. Alcohol also is considered as one of the problems associated with poor semen production and sperm quality. The main goal of this study is to examine the effect of alcohol on sperm parameters and DNA integrity in diabetic mice.
Materials and Methods: Totally 32 adult male Syrian mice (10 weeks old, 35 gr) were divided into 4 groups, mice of group 1 served as control fed on basal diet, group 2 received streptozotocin (STZ) (200 mg/kg, single dose, intra peritoneal) and basal diet, group 3 received alcohol (10 mg/kg, water-soluble) and basal diet and group 4 received streptozotocin and alcohol. After 35 days, the cauda epididymis of each mouse was dissected and placed in 1 mL of pre-warm Ham’s F10 culture medium for 30 min. The swim-out spermatozoa were analyzed for count, motility, morphology and viability. Also the sperm chromatin quality and DNA integrity, was evaluated with Aniline blue (AB), Toluidine blue (TB), Acridine orange (AO) and Chromomycin A3 (CMA3) staining.
Results: In this study all of the sperm parameters were significantly diffrent between groups (p=0.001). Also, regarding the sperm DNA integrity tests, the results from four tests showed significant differences between groups. Also, in diabetes+ alcohol mice, a significant increase was found in mean tests compare to other groups.
Conclusion: Although the DM may have bad effects on sperm fertility potential and DNA integrity, but, on the other hands, consumption of alcohol in diabetic mice, may have detrimental effects on sperm parameters, sperm function and also sperm chromatin condensation in experimentally-induced diabetic mice.
Key words: Mice, Sperm parameters, DNA integrity, Diabetes, Alcohol.
P-118
Protective effect of Silymarin on plasma membrane integrity, acrosome integrity and mitochondrial membrane potential in cadmium-treated mice
Ali Asgarnejad P, Momeni HR.
Department of Biology, Faculty of Sciences, Arak University, Arak, Iran.
Email: asgarnejad3@gmail.com
Introduction: Cadmium as a heavy metal and environmental pollutant is able to exert numerous undesirable effects on human reproduction by inducing oxidative stress. Silymarin, a polyphenolic flavonoid extracted from the seeds of Silybum marianum, has effective antioxidant properties.
Materials and Methods: Adult NMRI mice were divided into four groups: 1) control 2) cadmium chloride (5 mg/kg, sc) 3) Silymarin (100 mg/kg, ip) and 4) Silymarin + cadmium. Treatment period was 24 hours. After treatment mice were dissected and their epididymis was cut into small pieces in HTF medium in order to swim out spermatozoa. The spermatozoa from different groups were used to evaluate sperm parameters. Sperm plasma membrane integrity, acrosome integrity and mitochondrial membrane potential assessed by Hoechst and propidium iodide, coomassie blue and rhodamin staining respectively. Data were analyzed with one way ANOVA and p<0.05 was considered significant.
Results: In cadmium-treated mice, the percentage of intact membrane and acrosome were significantly decreased compared to the control. In addition, mitochondrial membrane potential was significantly decreased in cadmium-treated mice in comparison with the control. In Silymarin + cadmium group, Silymarin could significantly compensate the adverse effects of cadmium compared to cadmium group.
Conclusion: The present study showed that cadmium induces toxic effects on mice sperm plasma membrane integrity, acrosome integrity and mitochondrial membrane potential and Silymarin can compensate the toxic effects of cadmium on these sperm parameters.
Key words: Cadmium, Silymarin, Sperm parameters.
P-119
Evaluating quality of well-being, marital adjustment and sexual dysfunction between infertile women
Mousavi Nik M1, Saberi Zafarghandi MB2, Birashk B3, Assari A4, Eshaghi Farahman SR1.
- Department of Clinical Psychology, Mental Health Research Center, School of Behavioral Science and Mental Health, Iran University of Medical Sciences, Tehran, Iran.
- Department of Psychiatry, Mental Health Research Center, School of Behavioral Science and Mental Health, Iran University of Medical Sciences, Tehran, Iran.
- Department of Psychiatry, Faculty of Clinical Psychology, Mental Health Research Center, School of Behavioral Science and Mental Health, Iran University of Medical Sciences, Tehran, Iran.
- Department of Architecture and Urbanism, Tehran Shargh Branch, University of Payame Noor, Tehran, Iran
Introduction: Infertility is a stressful experience and has a high impact on the infertile women’s psychological status. Infertility and it is treatment create a major and prolonged crisis for the women and create a heavy psychological trauma for the women. The effectiveness of infertility treatment depends on success rate of the treatment facility, the emotional well-being and sexual health of the women seeking treatment.
Materials and Methods: For the propose of the study, 50 fertile and 50 infertile women were selected randomly (n=100) from Midwife IVF and Fertility Research Hospital in Mysore-India. All subjects were assessed using Quality of Well-Being Scale-Self Administered (QWB-SA), Marital Adjustment Test (MAT) and Massachusetts General Hospital Sexual Functioning Questionnaire (MGH) questionnaire were administered.
Results: The results showed significant difference between fertile and infertile women assessment on Quality of Well-Being and Marital Adjustment score. Compare with the fertile women, the patients with infertility had significantly lower scores in the sexual interest and sexual arousal domains and lower frequency of intercourse. The patients with infertility retrospectively reported an orgasm, lubrication and sexual satisfaction score that was similar to that of the fertile women.
Conclusion: Women with a diagnosis of infertility were found to be at higher risk for sexual dysfunction when compare with fertile women. Further infertile women reported poor marital adjustment and quality of well being compare with controls. The results suggest to apply counseling and psychotherapy services in the infertility centers to reduce the psychological problems, increase Quality of Well-Being, Marital Adjustment and identify the factors that can contribute to the development of sexual dysfunction on women facing infertility to help them to enhance fertility chances.
Key words: Quality of well-being, Marital adjustment, Sexual dysfunction, Infertile women.
P-120
Assessment of aerobic bacterial and fungal contaminations in liquid nitrogen tanks
Nabi A, Khalili MA, Torki AR, Fesahat F, Anbari F.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: ali.nabi67@yahoo.com
Introduction: Cryopreservation technologies play an important role in assisted reproduction for both medical and research purposes. But, there are some concerns about the technical and biological safety of this technology because of pathogen transmission during cryopreservation and germplasm banking. The aim of this study was to evaluate the presence of microbial contaminations in liquid nitrogen (LN2) and carrier tanks used in different laboratories of our reproductive science institute.
Materials and Methods: In this descriptive study, different parts of 20 carrier and storage LN2 tanks, such as LN2, vapors, cryocans, and inner surface were qualitatively evaluated for microorganism contaminations through the assessment of bacterial and fungal growth in minimal and selective Petri dishes.
Results: Two fungal (Candida albicans and aspergillus flavus) and two aerobic bacterial microorganisms (Ecoli, Pseudomonas aeruginosa) were the most common findings in bottoms, vapors, and inner surfaces of 2 carriers and 11 storage LN2 tanks. There were Pseudomonas aeruginosa growths in 8 cryocans from contaminated tanks. Also, there were staphylococcus aureus and acinetobacter baumannii contaminations in one storage tank.
Conclusion: Theoretically, LN2 tanks can be considered as a possible source of pathogen transmission. Therefore, performing quality control assays, like microorganism detection tests for LN2 and supplementary equipments should be considered as a routine practice in IVF clinics. Sterilization of tanks should be performed on regular basis.
Key words: Bacterial growth, Contamination, Liquid nitrogen.
P-121
Effect of non-polar extract of Phaleria macrocarpa on sperm quality in adult rats
Aziz Dollah M1, Parhizkar S2, Baharudin N1.
- Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University Putra, Malaysia.
- Medicinal Plants Research Centre, Yasuj University of Medical Sciences, Yasuj, Iran.
Introduction: Diminish production of testosterone hormone could affect sperm quality; hence contribute to the infertility in man.
Materials and Methods: 25 Sprague Dawley adult male rats were randomly divided into five groups. Rats were supplemented orally once a day with 0.25 ml Tween 20 solution containing non-polar hexane extract of PM at one of the following concentrations: 0, 6, 12, and 60 mg/kg for seven weeks. Commercial testosterone hormone was used as positive control. On the last day of supplementation period, all rats were sacrificed and orchidectomy technique was performed to collect semen sample from cauda epididymis of the testis. The sperm count, motility, viability and morphology were determined.
Results: The results showed that the sperm motility was significantly improved by non-polar PM extract (p<0.05) with the value of 66.98% (6 mg/kg), 63.96% (12 mg/kg) and 65.21% (60 mg/kg) compared to 44.08% for 0 mg/kg group. The supplementation of PM extract however did not improved the sperm count, sperm viability and sperm morphology. The sperm count was 852 (0 mg/kg), 918 (6 mg/kg), 906 (12 mg/kg), 1030 (60 mg/kg) and 1027 million cells/ml (commercial testosterone). The sperm viability was 85.90 (0 mg/kg), 85.26 (6 mg/kg), 85.45 (12 mg/kg), 87.88 (60 mg/kg), and 89.32% (commercial testosterone). The sperm morphology was 62.5% (0 mg/kg), 63.7% (6 mg/kg), 72.2% (12 mg/kg), 67.0% (60 mg/kg) and 64.5% (commercial testosterone). The results showed that non-polar PM extract only improved the sperm motility and did not have any effect on the sperm count, morphology and viability.
Conclusion: The study concludes that non-polar extract of PM has no compound that can improve sperm quality except on the motility of the sperm.
Key words: Phaleria macrocarpa, Sperm count, Sperm motility, Sperm viability, Sperm morphology.
P-122
Protective effect of ascorbic acid on testicular tissue in mice exposed with sodium arsenite
Pirozmanesh H, Soleimani Mehranjani M, Naderi Noreini S.
Department of Biology, Faculty of Sciences, Arak University, Arak, Iran.
Email: hp457@yahoo.com
Introduction: Arsenic is one of the major environmental contaminants with carcinogenic and toxic effects. Ascorbic acid as an antioxidant is able to restrict oxidative stress. The aim of this study was to investigate the adverse effect of sodium arsenite on the mice testicular tissue as well as to examine whether vitamin c is able to ameliorate this effect.
Materials and Methods: 24 adult male NMRI mice were randomly allocated into four groups (n=6) including control, sodium arsenite (7 mg/kg/day, orally), ascorbic acid (150 mg/kg/day, orally) and finally sodium arsenite+ ascorbic acid. Mice were treated for 35 days. At the end, mice were sacrificed and their right testis were taken out, fixed, processed and stained with Heidenhain azan method. The total volume of testis, volume of interstitial tissue, volume of seminiferous tubules, diameter and of length of seminiferous tubules, germinal epithelium height and basement membrane thickness were estimated using stereological methods. Data were analyzed using one way ANOVA and Tukey's test and means difference were considered significant at p<0.05.
Results: A significant reduction in total volume of testis, volume of seminiferous tubules and its diameter and germinal epithelium height, was found in sodium arsenite group compared to control group (p<0.04). In ascorbic acid + sodium arsenite group, the above parameters were significantly increased compared with sodium arsenite group (p<0.02). In addition, germinal epithelium height and diameter of seminiferous tubules were increased in mice treated with ascorbic acid alone compared to the control group (p<0.002).
Conclusion: The results indicate that ascorbic acid may be useful in reducing the sodium arsenite- induced toxic effects on testicular tissue.
Key words: Sodium arsenite, Ascorbic acid, Stereology, Testis.
P-123
The comparison of anxiety and depression rate between medical staff of Infertility Centers and Obstetrics and Gynecology Centers of Yazd, Shiraz, Isfahan and Kerman Hospitals
Pourmovahed Z1, Yassini SM1, Khalili MA2, Halvaei I2, Nabi A2, Ghasemi M3, Fesahat F2.
- Research Center of Addiction and Behavioral Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Fertility and Infertility Research Center, Isfahan University of Medical sciences, Isfahan, Iran.
Introduction: Mental health is an important issue in personal, social and occupational function of anyone in the life; moreover, mental problems have many negative consequences on the quality of professional work. Regarding the close and continuous interaction of infertility staff with hopeless infertile couples and in the contrary the atmosphere of happiness especially in Obstetric Wards it make sense that anxiety and depression rates would be different between them.
Materials and Methods: This study was a descriptive-correlation study based on cross-sectional method. 199 individuals who were the staff of Infertility Centers and Obstetrics and Gynecology Wards in four provinces enrolled in this study through stratified sampling. Data collection was done by demographic questionnaire, Spiel Berger and Beck depression inventory tests. Data were analyzed by SPSS software and ANOVA test.
Results: The results showed that there is statistically significant higher rate of anxiety in staff of Obstetrics and Gynecology Wards in Isfahan (54.69±13.58) while there was a higher rate of depression in staff of Infertility Center in Shiraz (14.94±10.87). Overall, correlation between anxiety, depression and work place was statistically significant (p=0.047 and 0.008 respectively). According to ANOVA test, the mean value of anxiety level was higher in the staff of Obstetrics and Gynecology Centers of Isfahan, Shiraz, Shahid Sadoughi and Madar Hospitals in Yazd (p=0.03, 0.006, 0.008, 0.012 respectively) and Infertility Center of Yazd (p=0.00).
Conclusion: Comparison of the mean value of anxiety in staff of Obstetrics and Gynecology Wards and Infertility Centers was not significant in these four centers except for Isfahan Center. As long as we know that infertile couples have little chance for success rate and Obstetrics and Gynecology patients have little risk of failure in treatment it could be mentioned that the anxiety and depression in the staff are not correlated with the client illness.
Key words: Anxiety, Depression, Infertility centers, Obstetrics and gynecology centers.
P-124
Developmental competence of immature oocytes aspirated from ovarian antral follicles as a method for fertility preservation
Safian F1, Khalili MA2, Karimi Zarchi M3, Mohsenzadeh M1, Ashourzadeh S1, 4.
- Department of Biology and Anatomical Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Obstetrics and Gynecology, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Afzalipoor Clinical Center for Infertility, Afzalipoor Hospital, Kerman University of Medical Sciences, Kerman, Iran.
Introduction: Advances in the treatment of cancerous women in reproductive age have markedly been increased recently. Also, in vitro maturation (IVM) of immature oocytes collected from ovary has been proposed for fertility preservation. In addition, the morphology of oocytes post IVM is one of the factors determining its developmental competence. By using the non-invasive PolScope system, the meiotic spindle and zona pellucida (ZP) can be assessed in living oocytes.
Materials and Methods: The ovarian cortex from 26 cancer patients (21-45 years old), were obtained directly from collaborating hospitals, and transported to the IVF center on ice. 61 immature oocytes were aspirated, of which 18 (29.5%) were degenerated and discarded. The remaining 43 (70.5%) healthy oocytes were cultured for 48 hr IVM culture media. The rate of maturity was assessed and the ZP birefringence and meiosis spindle (MS) were imaged with Polscope technology.
Results: 43 immature oocytes underwent IVM technology, of which 30.2% reached viable metaphase II (MII) oocytes. The ovarian tissues of 9 (34.6%) women were lacking oocytes at any stage. There was a positive correlation between the recovered number of oocytes and the ovarian volume. During polarized light microscopy examination, MS could be visualized only in one of the MII oocytes, but high ZP birefringences were observed in most of the oocytes post IVM (61.5%).
Conclusion: Oocytes maturation post IVM from unstimulated ovaries showed a good developmental competence in cancerous patients. Further studies should be performed to advance the oocyte maturation program, such as co-culture system, for fertility preservation.
Key words: Fertility preservation, Ovarian tissue, IVM, Polscope.
P-125
Effect of Heracleum persicumm oil and alcoholic extracts on sperm parameters and chromatin quality in mice
Taghizabet N1, Mangoli E2, Anbari F2, Talebi AR2, Masoodi A1.
- Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: Seminal plasma from infertile men has lower antioxidant levels than that of fertile men, particularly of patients whose semen have poor sperm motility. Evaluation of the importance and the effects of plant derived drugs on fertility of laboratory animals have long been recognized. Antioxidant activity has been reported from Heracleum persicum (Golpar).
Materials and Methods: 80 adult male mice (10 weeks old, 35 gr) were divided to 3 groups: group1 received hydroalcoholic extract (1000 mg/kg, ip), group 2 received oil extract (200 ml/kg, ip) and group 3 serving as the sham control group that received water. After 35 days, the cauda epididymis of each mouse was dissected and placed in 1 mL of pre-warm Ham’s F10 culture medium for 30 min. The swim-out spermatozoa were analyzed for count, motility, morphology and viability. The sperm chromatin quality and DNA integrity, was evaluated with Aniline blue (AB), Toluidine blue (TB), Acridine orange (AO) and Chromomycin A3 (CMA3) staining.
Results: In sperm analysis, progressive and non-progressive motility were significantly differences between groups. Regard to sperm chromatin quality, the results of TB and AO tests showed statically significant differences between groups, but in AB and CMA3 staining, we didn’t see any differences between them. So we can say that the Heracleum persicum extracts doesn’t have any detrimental effects on histone-protamines replacement during the testicular phase of sperm chromatin packaging.
Conclusion: According to our results, Heracleum persicum extracts as an antioxidant although improved sperm motility but may influence the male fertility potential via affecting DNA/ chromatin quality in mice.
Key words: Heracleum persicum extracts, Sperm, Mice, Chromatin.
P-126
Effect of vitrification on high magnification morphology, chromatin condensation, and fertility potential of human spermatozoa
Taherzadeh S2, Khalili MA1, 2, Anvari M1, 2, Ghazali Sh1, Agha-Rahimi A1, Anbari F1, Ghasemi-Esmailabad S1.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Anatomy, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: Sperm vitrification is a technique of ice- and CPA- free cryopreservation by direct plunging of a sperm suspension into liquid nitrogen (LN). Sperm characteristics of motility and morphology are important for normal spermatozoa-oocyte interaction. However, defects on chromatin condensation can cause vacuolization on sperm morphology. Motile sperm organelle morphology examination (MSOME) is an unstained real time high magnification that analysis viable sperm morphology. This study investigated the influence of vitrification on human sperm structure by MSOME technique, and fertility potential by zona binding assay (ZBA) and chromatin condensation by toluidine blue (TB) and aniline blue (AB) assessment.
Materials and Methods: 30 normozoospermic ejaculates were prepared by swim up technique, and supernatants were divided into two parts: fresh and vitrified groups. For vitrification, sperm suspension was mixed with equal volume (1:1) of Hams F10 +5% HSA +0.5 M sucrose. Then, 30 µl sperm suspension was dropped into LN. Warming was performed by quick submerging spheres into pre-warmed 5ml Hams F10 with 5% HSA at 37˚C. Sperm motility, stained morphology, MSOME and ZBA were evaluated for each sample. Three classes were considered for MSOME analysis: high quality sperm with a score of 4-6 (Class 1); medium-quality sperm with a score of 1-3 (Class 2); low-quality sperm with a score of 0 (Class 3). 2×10⁶ spermatozoa in each 25µl droplet containing 4 Oocytes were performed for ZBA. In addition, samples were fixed for TB and AB assessments.
Results: Cryopreservation significantly reduced both progressive motility and normal morphology of spermatozoa. There was no significant differences between the rates of MSOME in class 1 (14.93±14.66 vs. 13.56±11.34), class 2 (53.53±13.99 vs. 55.63±12.16), class 3 (31±20.81 vs. 30.80±16.03) pre and post vitrification, respectively. However, vitrification reduced the fertility potential of spermatozoa from normozoospermic samples (13.40±22.73 vs. 9.00±13.87) and chromatin condensation (TB: 60.32±16.60 vs. 57.28±17.19) (AB: 38.32±8.14 vs. 34.35±6.87).
Conclusion: Vitrification had adverse effects on sperm parameters of motility and morphology. However, this technique did not increase the rate of vacuolization of sperm head or severe alteration in fertility potential. Since, the majority of human spermatozoa contained vacuolization in head region, it is highly recommended to use MSOME technology for assessment of sperm fine morphology during clinical microinjection procedure.
Key words: Sperm vitrification, Motile sperm organelle morphology examination (MSOME), Zona binding assay (ZBA).
P-127
Vitamin C attenuates detrimental effects of diabetes mellitus on sperm parameters, chromatin quality and rate of apoptosis in mice
Talebi AR1, Mangoli E1, Nahangi H2, Anvari M2, Pourentezari M2, Halvaei I1.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Biology and Anatomical Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: Diabetes Mellitus (DM) may affect male reproductive functions at multiple levels. It is shown that intake of antioxidants such as vitamin C and E can reinforce the stability of testicular blood barrier and protect sperm DNA from oxidative stress.The main goal was to examine the protective effects of vitamin C on sperm parameters, sperm chromatin condensation and apoptosis in experimentally-induced diabetic mice.
Materials and Methods: 28 adult Syrian mice were divided into 4 groups. In group 1, the mice were diabetic that received a single dose of Streptozocin (STZ) (200 mg/kg) intra-peritoneally (ip). Group 2 included diabetic mice that received vitamin C (10 mg/kg/daily, ip). Mice in group 3 received vitamin C and group 4 was considered as control. After 35 days, sperm analysis was done accordingly. To asses sperm chromatin and DNA quality, we used aniline blue (AB), toluidine blue (TB), chromomycin A3 (CMA3), acridine orange (AO) and terminal transferase mediated deoxyuridine triphosphate biotin end labeling (TUNEL) tests.
Results: All of the sperm parameters (count, motility, morphology and viability) had significant reduction in diabetic mice but, the data showed a significant increase in all of the sperm parameters in diabetic + vitamin C when compared with diabetic and control animals (p<0.05). There were significant differences (p<0.001) between groups regarding TB staining (48.8±5.92 vs. 34.3±4.13), AO test (35.9±6.11 vs. 20.8±2.89) and TUNEL test (39.42±7.18 vs. 22.00±3.65) in diabetic and diabetic + vitamin C groups, respectively. Nevertheless, in CMA3 and AB staining assays, there were not any significant differences between different groups.
Conclusion: Vitamin C, as a potent antioxidant, can attenuate detrimental effects of diabetes mellitus on the sperm parameters, chromatin quality and apoptosis in an experimental model.
Key words: Sperm, Diabetes mellitus, Mice, Vitamin C, Chromatin, Apoptosis.
P-128
DNA fragmentation in mammalian spermatozoa and its relationship with male infertility
Vaseghi-Dodaran H1, Daghighkia H1, Atshan M2, Sobhani A3, Sobhani AGH4.
- Department of Animal Sciences, Faculty of Agriculture, Tabriz University, Tabriz, Iran.
- Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Animal Sciences, Faculty of Agriculture, Mohaghegh Ardabili University, Ardabil, Iran.
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Introduction: It has been suggested that altered nuclear chromatin structure or damaged DNA in spermatozoa is implicated as a possible cause of increased infertility in males. Since the first reports on sperm DNA integrity, this subject has become the focus of numerous studies. Up to 8% of infertile men have been shown to have high levels of sperm DNA fragmentation despite a normal semen analysis. Currently, there are eight major tests of sperm DNA fragmentation, including the comet assay, sperm chromatin structure assay (SCSA), acridine orange test (AOT), tritium-labeled 3H-actinomycin D (3H-AMD) incorporation assay, terminal TdT-mediated dUTP-nick-end labeling (TUNEL) assay, in-situ nick translation (ISNT) assay, DNA breakage detection-fluorescence in-situ hybridizations (DBD-FISH) assay and sperm chromatin dispersion (SCD) test. New studies suggest that sperm with certain levels of DNA fragmentation serve as a strong predictor of reduced male fertility. Diagnosis of the fertilizing ability of a semen sample is important for consistently high reproductive efficiency. This paper aimed at investigating the potential use of sperm DNA fragmentation (SDF) to improve the routine screening of male infertility.
Materials and Methods: This article discusses some of the current techniques employed for evaluating chromatin structure or DNA damage in spermatozoa. A review of recent bibliography was carried out in PubMed, Google scholar and SID by the use of relevant keywords, in order to evaluate the possible correlation between the conventional seminal parameters and sperm DNA fragmentation assessment as diagnostic tools in male infertility evaluation.
Results: A negative correlation was found between sperm characteristics and the proportion of sperm showing DNA fragmentation. For fragmentation >30%, a significant decrease of the fertilization rate was observed.
Conclusion: The proportion of sperm with DNA fragmentation appears to be potentially useful as a predictor of infertility. Sperm DNA fragmentation is a parameter worth integrating in routine clinical practice. However, additional large scale studies based on the optimization of sperm DNA integrity are needed.
Keywords: Spermatozoa, Male, DNA damage, Infertility.
P-129
Establishment of spermatogonial stem cell line in mouse testicular culture
Azizi H1, Amirian M2, Skutella Th2.
- Department of Biotechnology, Amol University of Special Modern Technologies, Amol, Iran.
- Institute for Anatomy and Cell Biology, Medical Faculty, University of Heidelberg, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany.
Introduction: In the seminiferous tubules of the testis spermatogonial stem cells (SSCs), Sertoli cells and differentiating cells during spermatogonesis are present. Materials and Methods: Here we report the establishment of a new VASA/ DAZL/ PLZF/-, Sox9/ Nanog/ GFRa1 low and KLF4, SOX2, VIMENTIN, N-MYC, OCT4+ cell line both from neonate and old mice, which could be expanded on SNL feeders for more than a year.
Results: Unlike undifferentiated SSCs they have a smaller nuclear/cytoplasm ratio in EM. FACS analysis showed the expression of CD49, CD29, CD9, GFRa1 and E-CAD. In Fluidigm analysis the cells expressed pluripotency markers OCT4, NANOG, SOX2 and KLF4 but only partially expressed the typical germ cell profile of SSCs. One month after transplantation in busulphan treated NOD SCID mice we observed localization of GFP labelled cells in the basal compartment of the seminiferous tubule.
Conclusion: These more differentiated SSCs could provide an ideal cell system for studying both pluripotency and in vitro differentiation of SSCs to sperm and also provide a new strategy for isolation of SSCs from neonate and old mice by morphology based selection.
Key words: Germ cells, Spermatogonial stem cells, Testicular culture.
P-130
The association between TNF-alpha -308 polymorphism and susceptibility to spermatogenic failure
Khademi Bami M1, Dehghan Tezerjani M2, Vahidi Mehrjardi MY2, Ashrafzadeh Mehrjardi HR1, Ghasemi-Esmailabad S2, Montazeri F2, SabbaghNejad Yazd S2, Shadram Y2, Golzade M2, Karimi R2, Sheikhha MH2, Kalantar SM2.
- Department of Biology, Islamic Azad University, Ashkezar Branch, Yazd, Iran.
- Research and Clinical Center for Infertility, Shahid Sadoughi Medical Sciences University, Yazd, Iran.
Introduction: Approximately half of infertility reasons are related to male factors among which genetic etiology is the main cause. Signaling molecules can regulate spermatogenesis during regulation of germ cells maturation. Tumor necrosis factor-α (TNF-α) is one of the prominent cytokines that supports signaling molecules and it can affect spermatogenesis.
Materials and Methods: This case-control study includes 50 azoospermic men who referred to Yazd Research and Clinical Center for Infertility and 50 healthy controls. After sperm analysis, DNA was extracted and Restriction Fragment Length Polymorphic-Polymerase Chain Reaction (RFLP-PCR) was carried out for TNF alpha -308 polymorphism.
Results: The frequencies of A allele and G allele were 58% and 42% in azoospermic group, and 34% and 67% among controls, respectively. Among azoospermic patients, 42% presented AA homozygous genotype, 32% AG heterozygous genotype, and 26% GG homozygous genotype. In the control group, 20% presented AA homozygous genotype, 28% AG heterozygous genotype, and 52% GG homozygous genotype. According to our findings, A allele (p=0.001, OR=2.681) and AA genotype (p=0.030, OR=2.89) have a positive association with spermatogenic failure in the cases.
Conclusion: Our data suggest that the polymorphism might be associated with the risk of spernatogenic failure in Iranian azoospermic and it can be used as an infertility marker for screening as well as further treatment.
Key words: Azoospermic, Cytokines, TNF-α, Polymorphism, RFLP-PCR.
P-131
The role of FSH receptor gene alterations in poor ovarian response to gonadotropins
Masoudi N1, Zari Moradi S1, Mohseni Meybodi A1, Mansouri Z1, Totonchi M1, Mirghavam-Aldin N2, Ashrafi M2, Gourabi H1.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Endocrinology and Female Infertility at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: Poor ovarian response is an infertility disorder in which women’s ovaries don’t have proper response to gonadotropins. Follicle stimulating hormone (FSH) has a critical role in the maturation of the ovarian follicles from the antral to the graffian stage. FSH will start a signaling cascade in the granulosa cells after sitting on its receptor (FSHR). Alteration of this receptor may change follicle maturation and therefore result in improper response to gonadotropins. We investigated the association of FSH receptor gene alteration in ovarian response of poor responder patients.
Materials and Methods: The presence of P.Ala307Thr, P.Ser680Ala, P.Ala665Thr and Mut.Val341Ala were analyzed in a case control study. 70 Iranian poor responder patients were selected as the case group. 60 Iranian fertile women were enrolled as the control group. The patients DNA were extracted from their peripheral blood and amplified by relevant primers. For determining allelic variant status all PCR products were analyzed by Sequencing.
Results: The results showed that the homozygous Ser680 and Ala307 variants seem not to be significantly associated with poor response to gonadotropins. The FSHR P. Ala665Thr genotype frequency was similar in all patients and controls. The number of oocytes retrieved was comparable between patients with different FSHR genotype.
Conclusion: Although data are accumulating with evidence suggesting that the ovarian response to gonadotropins may be mediated by different genetic alterations, the optimal biomarkers and the efficacy of the tests still remain to be evaluated.
Key words: Female infertility, Poor ovarian response, Follicle stimulating hormone receptor (FSHR).
P-132
A review of studies about vascular endothelial growth factor gene polymorphisms as genetic cause of infertility
Nabat E1, Farahzadi R2.
- Center of Excellence for Biodiversity, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran.
- Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Introduction: Infertility is defined as an inability of couples to achieve pregnancy after one year of regular, non-contraception intercourse. Diverse factors such as environmental, immunological, endocrine and genetic predispositions are thought to be involved in the development of the infertility. Infertility may be due to male or female factors or both. Causes of female infertility may include ovulation disorders, uterine abnormalities and fallopian tube blockage. Recurrent miscarriage (RM) and endometriosis are the common cause of infertility in female. Genetic factors can play an important role in susceptibility to RM and endometriosis, such as polymorphisms in vascular endothelial growth factor gene (VEGF). Recently researchers have found that endothelial dysfunction and placental ischemia/hypoxia are important factors in miscarriage. VEGF plays a pivotal role in the formation of blood supply of the fetus through the placenta and development of endometriosis. Many recent studies have indicated that a reduced VEGF serum concentration was associated with increased risk of RM and endometriosis. Therefore, the aim of this study was to investigate the association of VEGF polymorphisms such as +405 G/C, -460 C/T in 5’-UTR, +936 C/T in 3’-UTR and -1154 G/A in promoter region, with RM and endometriosis.
Materials and Methods: This review is focused on the most recent laboratory and clinical findings to investigate the association of VEGF polymorphisms with RM and endometriosis.
Results: -1154A and +93 6T alleles are increased in RM, while only in the study of Hsieh et al +405 C/C and -460 T/T genotypes are highest in patients suffering from endometriosis.
Conclusion: According to many functional studies, A allele of -1154G/A, T allele of +936C/T, C/C genotype of +405 G/C and T/T genotype of -460 C/T are associated with reduced VEGF transcriptional levels. These could lead to inadequate angiogenesis thus, lower endometrial vascularization, impaired placentation and subsequent predispose to RM and risk of endometriosis.
Key words: Infertility, Endometriosis, Recurrent miscarriage, Polymorphism, VEGF.
P-133
Association study of GSTO1 gene polymorphism (E155del) among patients with recurrent miscarriage from NorthWest of Iran
Yasari S1, Bonyadi M1, Taghavi S2.
- Center of Excellence for Biodiversity, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran.
- Maternal Fetal Medicine Unit, Department of Obstetrics and Gynecology, Alzahra Hospital, Tabriz University of Medical Sciences, Tabriz, Iran.
Introduction: Recurrent miscarriage (RM) is the occurrence of three or more consecutive pregnancies that end in miscarriage of the fetus before viability. About 1-5% of couples trying to conceive are affected by recurrent miscarriage. RM is considered as a multi-factorial disease and nearly half of RM cases cannot currently be explained clinically. Several studies have indicated that genetic polymorphisms could be factors for causing susceptibility of RM with unknown etiology. GSTO1 protein has a crucial role in detoxification metabolism and it is believed that GSTO1 gene could be involved in RM. In this study the possible association of GSTO1 gene polymorphism [E155del (rs11509437)] with recurrent miscarriage in NorthWest of Iran was evaluated.
Materials and Methods: In this case-control study, forty-eight patients with a history of RM and forty-eight referent women, who had at least two live births without other pregnancy complications, were included. Glutathione S-transferase Omega 1 (GSTO1) gene polymorphism (E155del) was screened using polymerase chain reaction with confronting two-pair primers (PCR-CTPP). Subsequently, PCR products were electrophoresed in 2% agarose gel and visualized by ethidium bromide staining.
Results: This study revealed that there was a significant association with the allelic frequency of del155 between the patient and control groups (p=0.04). However, analysis of genotype’s frequencies between patient and control showed no significant association (p=0.14).
Conclusion: Although some studies have indicated that the genetic polymorphisms of detoxification genes, encoding related enzymes, could be involved in RM diseases, current study showed the weak association of GSTO1 gene polymorphism (E155del) with susceptibility to develop RM in this population.
Key words: NorthWest of Iran, Recurrent miscarriage, GSTO1 gene, E155del polymorphism, Cellular detoxification.
P-134
Effects of in vitro maturation (IVM) on epigenetic changes in oocytes and early embryos
Allahveisi A1, Rezaei MJ2, Nikkhoo B3, Yousefian E4.
- Department of Reproductive Biology, Infertility Center of Beassat Hospital, Kurdistan University of Medical Sciences, Sanandaj, Iran.
- Department of Histology and Embryology, Infertility Center of Beassat Hospital, Kurdistan University of Medical Sciences, Sanandaj, Iran.
- Department of Pathology, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran.
- Department of Anatomical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
Introduction: In vitro maturation (IVM) of oocyte is an effective technique for avoiding ovarian hyperstimulation syndrome in patients with polycystic ovaries (PCOS) during in vitro fertilization (IVF). Pervious study indicated that more than 1000 children have been born from IVM technique, especially in patient with PCOS. However, IVM remains questions even today. Epigenetic reprogramming occurs in the periods of gametogenesis and embryogenesis, which regulates the gene activity without alteration of DNA sequences. This review discuss about effects of in vitro maturation (IVM) on epigenetic changes in oocytes and early embryos.
Material and methods: This article presents result of a systematic review about effects of in vitro maturation (IVM) on epigenetic changes in oocytes and early embryos.
Results: Different studies showed that IVM down regulated the protein expression of enzymes controlling histone acetylation such as histone acetyltransferase GCN5 (GCN5) and histone deacetylase 1 (HDAC1), as well as their common target, acetyl-histone H3 (Ac-H3), in metaphase II (MII) oocytes and two-cell embryos. The significantly decreased HDAC1 mRNA levels in oocytes and early embryos from IVM strongly suggested that IVM technique down regulated the transcription of HDAC1 gene in oocytes before and after fertilization.
Conclusion: Our studies indicated that IVM could affect the protein and gene expression level related to histone acetylation in oocytes and early embryos. Therefore, the impacts of IVM on epigenetic changes may be reasons for the lower rates of fertilization and early embryos in IVM.
Key words: IVM, Oocyte, Epigenetic.
P-135
Association of 14-bp insertion/deletion polymorphism of HLA-G gene with idiopathic recurrent miscarriage
Arjmand F.
Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: fateme.arjmand@yahoo.com
Introduction: HLA-G is supposed to play a pivotal role in tolerance of the semi-allogeneic graft in pregnancy by inhibiting the cytotoxic functions of T- and NK cells. A 14-bp insertion/deletion polymorphism in exon-8 has a possible role in HLA-G expression.
Materials and Methods: Genomic DNA from 200 recurrent miscarriage (RM) patients and 200 normal fertile control individuals were isolated using the routine salting out method. Exon-8 of HLA-G gene of the two groups were amplified using polymerase chain reaction and analyzed by electrophoresis on 10% non-denaturing polyacrylamide gel electrophoresis containing ethidium bromide and visualized under ultraviolet light. Statistical analysis used SPSS 19 software. HLA-G allele frequencies and genotypes in RM women and the fertile control group were compared using a Chi-square test.
Results: There was a difference in allelic frequencies of 14-bp insertion polymorphism between fertile controls and RM patients; frequency of +14/-14 bp heterozygotes was significantly increased in RM patients as compared with fertile controls.
Conclusion: A 14-bp insertion/deletion polymorphism in exon 8 plays a possible role in HLA-G expression in certain cases of recurrent miscarriage. However, additional studies are needed in this regard.
Key words: 14-bp insertion, Deletion polymorphism, HLA-G, Recurrent miscarriage.
P-136
Mutation analysis of exone 2 of Tnp2 gene in varicocele patients
Danafar AH1, Heidari MM2, Khatami M2.
- Department of Biology, Ashkezar Branch, Islamic Azad University, Ashkezar, Iran.
- Department of Biology, Faculty of Science, Yazd University, Yazd, Iran.
Introduction: Varicocele is one of the main causes of male infertility that caused by inflammation of the veins of the pampiniform plexus. Several studies have given great evidences that revealed the relationship between sperm DNA damage and varicocele. Because of the crucial role of Transition Nuclear Proteins (TNPs) and Protamines in sperm DNA condensation and integrity, the mutations in these genes can increase the risk of sperm DNA damage and infertility in varicocele condition.
Materials and Methods: DNA was extracted from total blood of 78 infertile patients with varicocele and 75 fertile control men for PCR amplification and SSCP analysis. DNA from samples with altered band pattern in the SSCP was then sequenced to search for mutations.
Results: The results of sequencing showed one variant at position IVS1-26G>C (rs8043625) in the intronic region of this gene. Comparison of the genotypes between cases and controls showed significant differences in frequencies of GG and CC (p=0.002, p=0.01), but not in GC genotype of this polymorphism (p=0.41). Also it was found that varicocele risk in men who have the CC and GC genotypes is respectively 3.07 and 1.37 fold higher than those who don't have these genotypes (OR=3.07, OR=1.37).
Conclusion: High conservation of this SNP position during evolution can represent the effects of this nucleotide in some important processes associated with the expression of this gene like mRNA splicing; but the exact mechanism is not clear.
Key words: Varicocele, Infertility, Tnp2, SSCP.
P-137
Assessing the polymorphism frequency rate of insulin receptor gene in PCOS
Khazamipour A1, Nejatizade A1, Kalantar SM2.
- Department of Genetic, Faculty of Science, Hormozgan University, Bandar Abbas, Iran.
- Research and Clinical Center for Infertility, Shahaid Sadoughi University of Medical Scinces, Yazd, Iran.
Introduction: Polycystic ovarian syndrome (PCOS) is a common endocrine disorder with prevalence of 5-10% globally among women of reproductive age. PCOS is being characterized by chronic anovulation, polycystic ovaries, hyperandrogenism, hirsutism, overweight, insulin resistance and infertility. It is well-known that PCOS is a complex trait like type-2 diabetes where both genetic and environmental factors play a crucial role in pathogenesis of the disease. Some type-2 diabetes susceptibility genes including those for insulin secretion and action such as insulin receptor showed considerable contribution to genetic predisposition of PCOS. Since insulin receptor gene seems to be a strong candidate gene to PCOS, we aimed to investigate the role of insulin receptor gene polymorphism (T/C) located in a transcription enhancer element of the gene to the disease susceptibility.
Materials and Methods: We carried out a cross-sectional case-control study. Using simple random sampling 90 healthy women were selected. All 90 patients fulfilled the 2003 Rotterdam criteria of PCOS. The subjects were genotyped for insulin receptor gene polymorphism (T/C) using PCR-Sequencing. Differences in genotype distributions between case and control subjects were examined via a chi-square test.
Results: In the present study 180 women between 18-40 years old age were assessed. Clinical, biochemical and metabolic characters of women were compared. Among them, obesity (Waist round more than 85 centimeters), hirsutism and irregular menarche (monthly cycles more than 35 days), LH/FSH >2, BMI>25, (mean: 28.19) in the PCOS group were significantly higher than control group (p<0.05). There was no significant associated between two groups for diabetes mellitus type II, hypertension (>140/90 mmHg) and hormonal level (TSH >5 and prolactin >29.2). Similarly in PCOS and control group, 95% of women had TT genotype and 2.2% had CC genotype. Frequency of C allele in PCOS and control group were 3.3% and 2.2% respectively. Frequency of TC genotype in PCOS and control group were 2.2% and 4.4% respectively. Noted polymorphism had no significant difference between PCOS and control women (p=0.99).
Conclusion: Although PCOS showed high prevalence among women in Bandar Abbas, Iran, insulin receptor gene polymorphism (T/C) didnot show significant influence on susceptibility to PCOS.
Key words: Polymorphism, Insulin receptor gene, Polycystic ovarian syndrome.
P-138
Association of SNP rs.2414096 of CYP19 gene with polycystic ovarian syndrome in Iranian women
Mehdizadeh A, Kalantar SM, Sheikhha MH.
Department of Genetics, International Campus, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: anahita.mz@gmail.com
Introduction: Polycystic ovarian syndrome (PCOS) is the most common endocrinologic disorder of women in their reproductive age. Major characteristics of PCOS are clinical and/ or biochemical hyperandrogenism, ovulatory dysfunction and polycystic ovaries. The etiology of PCOS is unknown, however, it has been suggested that genetic factors play major role in development of PCOS. Of those the ones implicating in metabolism of androgens are important as they are impaired in PCOS. CYP19 gene encodes aromatase which has a crucial role in androgen synthesis and variants of this gene might implicate in the pathogenesis of PCOS. Investigations have indicated the role of single nucleotide polymorphism of CYP19 in hyperandrogenism and PCOS in some racial groups. There is no data on the variants of this gene in PCOS population in Iran.
Materials and Methods: In a case-control study 70 PCOS women and 70 normal controls were selected. Following informed consent, 5 ml blood was taken from each woman of which genomic DNA was extracted by salting out method. Then a set of PCR reactions for CYP19 gene was carried out using specific primers for SNP rs.2414096 followed by a subsequent enzyme digest (RFLP) with HSP92II.
Results: Genotype frequencies of SNP rs.2414096 in PCOS women were as follows: AA (14.4%), AG (44.3%) and GG (41.4%) while in normal group, these genotypes were 24.3%, 52.9% and 22.9%, respectively. While allele frequencies in PCOS group were 49.3% for A and 50.7% for G, normal group had a different percentage of A (36.4%) and G (63.6%). The calculations for both genotype and allele frequencies showed statistical significance.
Conclusion: Variants of SNP rs.2414096 in CYP19 could play a role in development of PCOS in Iranian women.
Key words: Polycystic ovarian syndrome, CYP19, Single nucleotide polymorphism.
P-139
Exposure to tertiary-butyl hydroperoxide (TBHP)- down regulates the expression of Ddx3y in mature mice testis
Mokhtari P, Fatemi N, Golkar Narenji A, Mohseni Meybodi A, Gourabi H.
Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email:gourabi@royaninstitute.org
Introduction: Approximately 40% of infertility is associated with male factors. Two major causative factors of male infertility are oxidative stress (OS) and genetic factors. OS damages the sperm plasma membrane, the genome integrity and alter the expression profile of genes involved in spermatogenesis. Ddx3y gene is one of the important azoospermia factor (AZF) genes in Y chromosome. AZF deletion causes a severe block in spermatogenesis which affects the proliferation of spermatogonia, and consequently male infertility. The expression profile of Ddx3y gene was evaluated in testis tissues of Balb/c mice after OS induction.
Materials and Methods: A model of oxidative stress in adult male Balb/c mice testis by injection of the 1:10 concentration of tertiary-butyl hydroperoxide (TBHP) was created. Case group included treated mice by TBHP for 2 weeks and control group treated only by injection of dH2O. Induced ROS levels in testes tissue samples of all mice were measured by flow-cytometry. Consequently the expression of Ddx3y gene was quantitatively measured in samples of both groups by real-time PCR.
Results: According to flow-cytometry results, an increase of oxidative stress in TBHP treated mice in comparison to control group was observed. The gene expression of Ddx3y in testis was significantly down regulated in OS-exposed and ROS induced mice.
Conclusion: Our results indicated that Ddx3y may be a major target gene of OS and the down regulated expression of Ddx3y can be closely related to male reproductive toxicity induced by TBHP.
Key words: Male infertility, Oxidative stress, TBHP, Ddx3y.
P-140
Association of +331G/A polymorphism and differential expression of PR-B isoform in endometriosis
Mousazadeh S1, Afsharian P1, Shahhoseini B1, Aflatoonian R2.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Endocrinology and Female Infertility at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: It is well accepted that endometriosis is a progesterone resistance disease. The effects of progesterone are mediated by its two progesterone receptor (PR) isoforms, namely PR-A and PR-B. These isoforms are functionally different. The most widely studied polymorphism in the promoter region of PGR is the G to A substitution at position +331 (rs10895068). Recently, many studies have investigated the role of +331G/A polymorphism in the etiology of various types of cancers.
Materials and Methods: Blood samples were recruited from 98 women undergoing laparoscopy for endometriosis and 102 healthy fertile women at Royan Institute, Tehran, Iran in 2013-2014. After DNA extraction, allele and genotype frequencies were determined by PCR-RFLP. Then, RNA was extracted from selected eutopic tissue samples of endometriosis patients. Analysis of PR-B mRNA expression was performed using Real-time PCR.
Results:Our data showed the frequency distribution of GG, G/A and AA genotypes in +331G/A polymorphism was 98.04%, 1.96% and 0.0% in patients and 97.96%, 2.04% and 0.0% in control groups respectively (p=0.968). Although our data didn’t show any significant association with +331G/A in our groups, however, we were able to demonstrate higher expression level of PR-B in patients with G/A compared to patients with GG genotypes.
Conclusion: Many studies have been shown that the expression level of PR-B reduced severely during endometriosis, which can affect the function of progesterone. Our findings support this observation, patients with G/A genotypes have high expression level of PR-B compared to patients with GG genotypes. Therefore, +331G/A have been found to lead increased transcriptional activity of PR-B in patients by favoring G/A or AA genotypes that may be able to influence the function of progesterone and reduce the susceptibility and symptoms of endometriosis.
Key words: Endometriosis, PGR, Polymorphism.
P-141
Association between GSTT1 null mutation and endometriosis in an Iranian population
Rastegar M1, Seifati SM1, Sheikhha MH2.
- Department of Biology, Islamic Azad University, Ashkezar Branch, Yazd, Iran.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: Endometriosis is a common gynecological disease that involves growth of endometrial tissue outside its normal location. It seems that genetic factors have an important role in the pathophysiology of endometriosis. In addition, there are reports suggesting a role for environmental pollutants in developing endometriosis. GSTTl is one of the phase II enzymes involved in detoxification of some environmental carcinogens.
Materials and Methods: Genomic DNA from 95 patients with endometriosis and 141 healthy controls was collected. GSTT1 null genotyping was performed using polymerase chain reaction (PCR) method. Results: Frequency of GSTT1 null deletion in both patients and controls was 22%, which shows no significant difference between two groups (p=0.983).
Conclusion: These results suggest that GSTT1 null mutation is not associated with endometriosis in our population.
Key words: Endometriosis, Glutathione s-transfers, GSTT1.
P-142
The low frequency of breast and ovary cancer protective allele (D302H) of CASP8 gene among Iranian patients
Rasti A1, Farashahi Yazd E2, Shiryazdi SM3, Nematzade H4, Rastegar M1, Sheikhha MH1.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Genetic, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
- Department of Surgery, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
- Hematology, Oncology and Genetic Research Center, Shahid Sadoughi Hospital, Yazd, Iran.
Introduction: Suppression of apoptosis is one of the major mechanisms underlying the origin and progression of cancer. The CASP8 plays a key role in the initiation of apoptosis. Previous case-control studies have indicated that the D302H genotype of CASP8 is associated with a reduced risk of breast and ovarian cancer. However, the frequency of this polymorphism in the Iranian people has not been reported yet. Therefore, this polymorphism was genotyped among patients with breast cancer and healthy women.
Materials and Methods: PCR-RFLP technique was used to genotyping of CASP8 D302H polymorphism among 100 female patients with breast cancer and 100 healthy women in this case-control study. Fisher's exact test and SPSS software were used to analyze the data.
Results: The mean age of patients and healthy groups was 51.37±1.27 and 52.57±1.21 years respectively. The frequencies of the GG and GC genotypes in case group were 91% and 9%, respectively, and among the control group, the frequencies were 97% and 3%, respectively )p=0.2). CC genotype was not found in any of the groups. Furthermore, the C allele frequency was 4.5% among the cases and 1.5% among the control (p=0.14). There was no significant difference between cases and controls. In total, the frequency of the C allele was observed in about 3% of people in our study (breast cancer patients and healthy women).
Conclusion: Previous case-control studies have indicated that the D302H genotype of CASP8 was associated with a reduced risk of breast and ovary cancer. In the present study the frequency of allele C was detected 3%. Indeed, the frequency of C allele in Iranian study population is less than one-fourth the frequency of this allele in Caucasian populations (13.29%). According to the low frequency of this allele in Iranian population and the protective effect of allele C, one of the conclusions that can be raised is that the Iranian population is more prone to breast and ovarian cancer than Caucasian populations in relation to this polymorphism. Therefore further studies would be needed with appropriate sample size to investigate this matter.
Key words: CASP8 D302H, Polymorphism, Apoptosis, Breast cancer.
P-143
“Cell-free fetal DNA” a novel promising biomarker for prenatal diagnosis and complicated pregnancies
Sadeghi F1, Khalaj kondori M1, Sadeghi M2.
- Department of Biology, Faculty of Natural Science, University of Tabriz, Tabriz, Iran
- Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
Introduction: Cell free fetal DNA (cff DNA) is a novel promising molecular biomarker that has been applied in various aspects of obstetrical research, notably in prenatal diagnosis and complicated pregnancies.
Materials and Methods: Data base was browsed using key words “cell-free fetal DNA” and “noninvasive prenatal diagnosis” for obtaining related reports. Articles were screened for relatedness and the recent progresses in the field.
Results: Cell-free fetal nucleic acids can be detected in the maternal circulation during pregnancy, potentially offering an excellent method for early non-invasive prenatal diagnosis (NIPD) of the genetic status of a fetus. Using molecular techniques, fetal DNA and RNA can be detected from 5 weeks gestation and are rapidly cleared from the circulation following birth. Cell-free fetal DNA comprises only 3-6% of the total circulating cell-free DNA, therefore diagnoses are primarily limited to those caused by paternally inherited sequences as well as conditions that can be inferred by the unique gene expression patterns in the fetus and placenta. Broadly, the potential applications of this technology fall into two categories: first, high genetic risk families with inheritable monogenic diseases, including sex determination in cases at risk of X-linked diseases and detection of specific paternally inherited single gene disorders; and second, routine antenatal care offered to all pregnant women, including prenatal screening/diagnosis for aneuploidy, particularly Down syndrome (DS), and diagnosis of Rhesus factor status in RhD negative women. Already sex determination and Rhesus factor diagnosis are nearing translation into clinical practice for high-risk individuals.
Conclusion: In conclusion, the discovery of Cell free fetal DNA (cff DNA) has revolutionized the field of non-invasive prenatal diagnosis (NIPD) and has opened a new avenue in the field of obstetrical research and may in future form part of national antenatal screening programmes for DS and other common genetic disorders.
Key words: Cell free fetal DNA, Noninvasive prenatal testing (NIPT), Prenatal diagnosis, Pregnancy complications.
P-144
Identification of a time window for spontaneous establishment of pluripotency in mouse spermatogonial stem cells in vitro
Azizi H1, Conrad S2, Hajizadeh Moghaddam A1, Baharvand H3, Skutella T4.
- Department of Biotechnology, Amol University of Special Modern Technologies, Amol, Iran.
- Institute of Anatomy, University of Tübingen, Tübingen, Germany.
- Department of Stem Cells and Developmental Biology at the Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
- Institute for Anatomy and Cell Biology, Medical Faculty, University of Heidelberg, Im Neuenheimer Feld, Heidelberg, Germany.
Introduction: Although testis-derived embryonic stem cell-like (ES-like) cells have been obtained in several studies, the time window for the shift to pluripotency is not clear yet.
Materials and Methods: Here we describe, that only during a special time window (41 until 125 days) after initiation of germ line stem cell (GSCs) cultures from neonate and adult promoter-reporter Oct4-GFP transgenic mouse the spontaneous appearance of germline-derived pluripotent stem (gPS) cells from both neonate and adult GSCs occurred.
Results: The isolated and long-term cultured (more than one year) GSCs which were isolated by a morphology based selection procedure expressed germ cells markers and exhibited a similar morphology with a high nucleus/cytoplasm ratio in comparison to undifferentiated SSCs (spermatogonial stem cells) in vivo. The generated gPS cells expressed pluripotency marker, in-vitro differentiated into all three germ lineages, formed complex teratoma after transplantation in SCID mice and produced chimeric mice.
Conclusion: Although the exact mechanism of the development of gPS cells from GSCs is still unclear, this new information could provide an ideal strategy for scheduling natural conversion mechanisms of ES-like cells from mouse testis.
Key words: Germ cells, Spermatogonial stem cells, Pluripotency.
P-145
Association between MspI polymorphism of CYP1A1 gene and endometriosis in an Iranian population
Babaki A, Seifati SM.
Department of Biology, Ashkezar Branch, Islamic Azad University, Yazd, Iran.
Email: babakiaz@yahoo.com
Introduction: Endometriosis is defined as the presence of endometrial tissue outside the uterine cavity. It is associated with dyspareunia, dysmenorrhea, pelvic pain and infertility and about 3-10% of women suffer from it in reproductive age. It seems that genetic and environmental factors play a role in the development of endometriosis. The relationship between genetic polymorphisms and endometriosis has been investigated in several genetic studies. CYP1A1 gene is a member of the cytochrome P450 gene family. It is involved in estrogen metabolism and in detoxification.
Materials and Methods: In a case-control study, DNA extraction was performed from the blood specimens of 93 cases of endometriosis and 139 healthy controls. The polymorphism was determined by PCR-based restriction fragment length polymorphism (RFLP) method.
Results: There was no significant difference in frequency of TT, TC, and CC genotypes of MspI polymorphism of CYP1A1 gene between case and control groups (p=0.961).
Conclusion: Results of this study revealed no association between the MspI polymorphism of the CYP1A1 gene and susceptibility to endometriosis in the studied population.
Key words: Endometriosis, MspI polymorphism, CYP1A1 gene.
P-146
Preimplantation genetic screening: Which stage? Which technique?
Bazrgar M.
Department of Genetics at Reproductive Biomedicine Research Centre, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: bazrgar.masood76@gmail.com
Introduction: Preimplantation genetic screening (PGS) techniques have a wide range of resolution, complexity and costs. Additionally each of biopsy stages has advantages and disadvantages.
Materials and Methods: Recent publications certainly systematic reviews and randomized clinical trials were considered in details.
Results: Polar body biopsy (PB) is the least invasive however provides the least capture of abnormalities. PB focuses on female meiotic errors, fails to identify approximately 40% of aneuploidies from postzygotic and paternal origins. PB is suitable in countries with legal restrictions for embryo biopsy. Cleavage stage biopsy (CB) of few cells (1 or 2) does not affect differentiation potential, however removal of 2 cells can result in an impaired implantation potential while mosaicism is not detectable in one cell biopsy. In trophectoderm biopsy (TB) several cells are available for analyses. In day 5 TB when the genetic results cannot be obtained within 24 hours, cryopreservation should be considered. The least and the most aneuploidy rates has been reported for TBs and CBs, respectively (1 versus more than 3). TB increases absolute implantation rate about 20% in comparison with CB. Blastocoel fluid aspiration is a new source of DNA for PGS with few publications. FISH-based PGS is low resolution due to limitation in number of both chromosomes and probes per chromosome which could be tested. Therefore it has not been effective in improvement of clinical outcomes while PGS methods with ability for detection of all 24 chromosomes (PGS-24) result in increased implantation and pregnancy rates and diminished effect of advanced maternal age. Concerning PGS-24 methods, digital PCR is mostly applied for PBs; real time qPCR is only applicable to TBs and limited by number of samples, two on each 384-well plate. Array CGH is robust and scalable, turnaround time as short as 12 hours, decreased cost per sample as increased case numbers. SNP arrays and NGS-based methods for copy number analysis are likely to be the most accurate and informative but complex and high-cost equipment. Low pass NGS-based PGS is moving to be cost effective in addition to performance same as array CGH.
Conclusion: Clearly, the largest reported increases in implantation and live birth rates to date have been with TB. TB is a good choice for good-prognosis patients particularly for those wishing to have elective single-embryo transfer. However, the cleavage-stage embryos of some poor-prognosis patients may implant and develop in utero but not develop to the blastocyst stage in vitro. At the moment, array CGH seems to be the most applicable method for PGS however future would be with higher resolution methods following decrease of related costs, hence ethical concerns on designer babies will need more serious considerations.
Key words: PGS, Biopsy, Stage, Technique.
P-147
Expression of CYP19A1 in patients with endometriosis and normal endometrium during the menstrual cycle
Khateri E1, 2, Shahhoseini M2, Mahdian S3, Aflatoonian R3, Afsharian P2.
- Department of Molecular and Cellular Science, Faculty of Advanced Sciences and Technology, Islamic Azad University, Tehran, Iran.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, Tehran, Iran.
- Department of Endocrinology and Female Infertility at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, Tehran, Iran
Introduction: Endometriosis is an estrogen-dependent inflammatory disease defined by the growth of endometrial tissues outside of the uterus. Epidemiological and clinical studies show that estrogen is essential for the growth of endometriosis. Molecular studies have revealed the presence of aromatase P450, the key enzyme in the biosynthesis of ovarian estradiol, inside the endometriotic tissues, indicating local synthesis of estradiol. So, it is proposed that the enzyme aromatase P450, which is coded by CYP19A1 gene, will be expressed aberrantly in endometriosis.
Materials and Methods: To evaluate the expression profile of CYP19A1 gene in women with endometriosis during menstrual cycle, ectopic endometriotic lesions and eutopic endometrium samples were collected using laparoscopy from 20 women with endometriosis and endometrial biopsies were obtained from 10 healthy fertile women as a control group. For this respect, ethical approval and informed patient consent was gained for the use of tissue samples. Quantitative expression analysis was performed using real-time PCR technique.
Results: Data showed that mRNA expression of CYP19A1 was significantly higher in ectopic endometrium of patients with endometriosis, in both proliferative and secretory phase, in comparison with eutopic and control group. CYP19A1 expression in eutopic endometrium was significantly higher in comparison to control group in proliferative phase.
Conclusion: Higher expression of CYP19A1 mRNA in the endometriotic tissues, may contribute to the etiology and progression of endometriosis and it is involved in disease pathogenesis.
Key words: Endometriosis, CYP19A1, Menstrual cycle.
P-148
A headache in prenatal diagnosis: A case of vanished twin or hermaphroditism?
Mousavi F1, Mohammad Khani N2, Bagherizadeh E1, Hadipour F3, Hadipour Z3, Shafeghati Y4, Behjati F5.
- Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran.
- Islamic Azad University, Karaj Branch, Karaj, Iran.
- Qom University, Qom, Iran.
- School of Medicine, Tehran University of Medical Sciences,Tehran, Iran.
- Department of Medical Genetics, Tehran University of Medical Sciences,Tehran, Iran.
Introduction: The finding of a mixture of both 46,XX and 46,XY cells in amniotic fluid culture has been frequently described. In the great majority of cases, the finding is followed by the birth of a normal male infant, leading to a consensus that the finding is the result of contamination with maternal cells in a normal male fetus. There are, however, several other possible explanations including the presence of cells from an undiagnosed twin pregnancy, cross contamination in the laboratory, the presence of cells from a “vanished” male twin, and true fetal chimerism. An accurate obstetric history and thorough sonography can be of great value in correct assessment of such situations.
Materials and Methods: A 29 Years-old woman was referred for genetic counseling. Her gestational age was 16 weeks and maternal biochemical serum screening test indicated high risk for Down Syndrome. She underwent amniocentesis for chromosome study using standard high resolution GTG banding technique.
Results: Karyotype result was 46,XY and sonography revealed a female fetus with normal internal genital organs. A normal baby girl was borned with 46,XX karyotype. Upon reviewing of amnio GTG slides both 46,XY and 46,XX cell lines were detected. The patient admitted that she had experienced a miscarriage and heavy bleeding early in pregnancy. The sonography at the time of amniocentesis had shown a shrinking cyst next to the fetus. These findings strengthened the vanished or resorbed twin as the reason for such a finding.
Conclusion: The fetus was most likely a non-identical twin. Most of grown cells were from the vanished male fetus which led to misdiagnosis. Thorough genetic counseling with the view of obstetric history is of great value in such situations.
Key words: Amniocentesis, Resorbed twin, Hermaphroditism, Genetic counseling.
P-149
+49A/G CTLA-4 gene polymorphism in NorthWest Iranian population with recurrent pregnancy loss
Parsa S, Jabbarpour bonyadi M, Zeinal zadeh N, Taghavi S.
University of Tabriz, Tabriz, Iran.
Email: sara.parsa6928@gmail.com
Introduction: Recurrent pregnancy loss is defined as three or more pregnancy loss before 20th gestational weeks. There are several factors which involved in pregnancy loss including anatomical, hormonal, immunological and infectious factors. Immunological factors are important due to the interaction between mother and fetus. In spite of allogeneic proteins encoded by paternal genes, it is clear that a series of regulatory mechanisms should exist in maternal immune system not to reject fetus during pregnancy. T regulatory cells which are one of the important component of humeral immune response plays role in the fetu-maternal interface. One of the regulatory ways for these cells is mediated by antigen independent co-stimulatory signals. Interaction of B7/ CTLA-4 is one of these signals. Cytotoxic T Lymphocyte Antigen 4(CTLA-4) is a glycoprotein transmembrane molecule which down regulate the activation and proliferation of T cells in a competitive interaction with CD28 to bind to B7.
Materials and Methods: We have studies 120 cases; patient group consist of 60 women with the experience of two or more pregnancy loss and control group consist of 60 women with at least two live births without any previous history of pregnancy loss and autoimmune diseases. Genomic DNA was extracted from whole blood using standard protocols. The CTLA-4 +49 A/G were detected using polymerase chain reaction- restriction fragment length polymorphisms assay.
Results: In this study CTLA-4 +49 A/G polymorphisms showed no significant differences between patients and controls. Polymorphic allele G showed the frequency of 39.16% among patients whereas its frequency for controls was 35.83%. The age of patient varied from 19 years to 39 years.
Conclusion: The results of the present study suggest that the CTLA-4 do not have association with recurrent miscarriage in NorthWest Iranian population.
Key words: Recurrent pregnancy loss, CTLA-4, Immunological factors.
P-150
Investigation of mitochondrial Gln tRNA molecular alterations in idiopathic repeated pregnancy loss
Vahidi Mehrjardi MY, Seyedhasani SM
Shahid Sadoghi University of Medical Sciences, Yazd, Iran.
Email: mmvahidi@gmail.com
Introduction: Recurrent pregnancy loss (RPL) is a critical medical problem in about 0.5-2% of women. The molecular genetics background for spontaneous abortion is being increasingly understood, and some polymorphisms associated with it have been reported
Materials and Methods: The nucleotide variations of threonine and proline were investigated in 96 women with idiopathic repeated pregnancy loss. The related mitochondrial area was amplified using a polymerase chain reaction (PCR). The PCR products were demonstrated by 2% agarose gel electrophoresis, and all the positive samples were purified and verified by an automated DNA sequencing method.
Results: The sequence analysis revealed 2 mutations in tRNA Gln . These mutations were 4343 in 1 cases and 4336 in 1 cases .
Conclusion: These tRNAs mutations can alter their steady state level and affect the structure of tRNAs. It results in protein synthesis defects and, in turn, mitochondrial dysfunction. The mutations of these genes may help in the assessment of RPL. Further study of an expanded series of these tRNA mutants is recommended to describe their etiologic role in idiopathic RPL.
Key words: tRNA, Mitochondrial mutation, Repeated pregnancy lo.
P-151
A novel mutation in morquio syndrome
Yavari M1, Asadollahi S2, Hasheminasab SH2, Harazi F3.
- Department of Biology, Faculty of Sciences, Yazd University, Yazd, Iran.
- Department of Biology, Faculty of Sciences, Isfahan University, Isfahan, Iran.
- Department of Biology, Faculty of Sciences, Ashkezar University, Ashkezar, Yazd, Iran.
Introduction: The Mucopolysaccharidoses (MPS) are a group of inherited metabolic disorders caused by a deficiency or malfunctioning of lysosomal enzymes which are needed to break down complex carbohydrates known as mucopolysaccharides or glycosaminoglycans (GAGs). Accumulation of GAGs causes a cascade of events leading to the progressive damage of cells, tissue and organs. Morquio disease or Mucopolysaccharidosis Type IV (MPS IV) belongs to this group and has two sub-types, A and B. Type A is also known as Morquio A, GALNS deficiency, N-acetylgalactosamine-6-sulfate sulfatase deficiency or more simply MPS IVA. This lysosomal enzyme involved in the catabolism of keratan and chondroitin sulfate. Patients who inherit two mutated GALNS gene alleles have a decreased ability to degrade the glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate, thereby causing GAG accumulation within lysosomes and consequently pleiotropic disease. The objective was to report the results of clinical characteristics, enzyme activity determination and mutation analysis of GALNS gene in an Iranian patient with mucopolysaccharidosis (MPS) type IVA (Morquio A disease).
Materials and Methods: The 10 years old Iranian boy with MPSIV was firstly diagnosed by urin GALNS determination who w:as char:actraised by multiple skeletal abnormalities and dwarfism, kyphosis, hypermobility joints, difficultly in walking to 8 years old and now he can’t walk. But his intelligence was normal. We investigated for all coding exons and adjacent intron regions of GALNS gene by PCR sequencing method.
Results: We performed urin test (MS/MS) from patient. In this test total GAGS was high and MPS was likely. So lysosomal enzymes in dried blood were checked and MPS IVA was diagnosed. Two heterozygous missens mutation as c.135 G>T (S>I) and c.510 C>A (P>H), and a homozygous mutation as c.181 A>G (y>C) in GALNS gene were detected in this patient. The mutation c.181 (y>C) is a novel variant which is not reported yet.With the method of gene analysis of new variant, the mutation c.181 (y>C) was considered to be a pathogenic mutation.
Conclusion: The MPS IVA patient showed severe multiple skeletal deformities, normal intelligence, muscle weakness, short stature, who carries homozygous mutations c.181 A>G (y>C). The bioinformatics analysis in POLYPHEN predicted this mutation as being probably pathogenic.
Key words: Mucopolysaccharidoses (MPS), Morquio disease, GALNS gene.
P-152
A newborn with ambiguous genitalia and a complex X;Y rearrangement
Dehghani MR1, Hashemian Z2, Zuffardi O3.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of medical Sciences, Yazd, Iran.
- Shahid Sadoughi University of medical Sciences, Yazd, Iran.
- University of Pavia, Pavia, Italy.
Introduction: In most mammals, sex is determined at the beginning of gestation by the constitution of the sex chromosomes, XY in males and XX in females.
Materials and Methods: Array Comparative Genomic Hybridization (Array-CGH) analysis was performed by using oligonucleotide aCGH platforms (180K SurePrint G3 Human Kit, Agilent Technologies, Santa Clara, CA), as reported elsewhere. Changes in DNA copy number at a specific locus were observed as the deviation of the log2 ratio value from 0 of at least three consecutive probes, by using Genomic Workbench v. 5.0.14 software (Agilent, ADM-2 algorithm with a threshold of 5). Oligomer positions refer to the Human Genome GRCh37 (hg19) assembly.
Results: Array-CGH revealed an unbalanced rearrangement resulting in the deletion of the distal Xp and the duplication of the proximal Xp contiguous region with presence of the Y chromosome from Ypter to Yq11. Fluorescent in situ hybridization (FISH) showed that this portion of the Y was translocated to the tip of the abnormal X and that the duplicated portion of chromosome X was inverted. Altogether, the abnormal chromosome was a dicentric one with the centromere of the Y chromosome apparently inactivated.
Conclusion: The presence within the translocated Y chromosome of the SRY gene explains the devolopment of testes although it is not clear the reason for the genitalia ambiguity.
Key words: Ambiguous genitalia, 46, XX testicular DSD, Inverted duplication and Xp terminal Deletion (Invdup del), Rearrangement, Array Comparative Genomic Hybridization, FISH.
P-153
Effect of genetic variation of beta defensin 126 on ICSI and IVF outcome in unexplained infertile men
Abedini M, Mohseni Meybodi A, Sabbaghian M.
Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: maria.abedini@gmail.com
Introduction: Despite improved methods for evaluation of sperm quality, infertility remains unexplained in about 20% of affected couples. During sperm maturation, a Cysteine-rich secretory glycoprotein α-defensin 126 secreted by the epididymal epithelium adsorbed to the entire sperm surface. It remains on the sperm until sperm become capacitated in the female reproductive tract. Its removal from over the head of sperm is required for sperm zona recognition. A cytosine dinucleotide deletion in the open reading frame of second exon of DEFB126 gene generates an abnormal mRNA. Men homozygous for this mutation have reduced chances of successful fertilization.
Materials and Methods: Genomic DNA from the peripheral blood of 92 male partners of unexplained couples who underwent ICSI (n=74) and IVF (n=18), were extracted. PCR was performed and molecular genotyping for the DEFB126 variant was done by single strand conformational polymorphism (SSCP), tetra PCR and DNA sequencing. ELISA and immunocytochemistry by indirect immunofluorescence antibody performed for the assessment of this protein expression on sperm cells.
Results: In our study this allele frequency in Iranian men was 0.54. Statistical analysis shows, no significant differences were found between homozygote mutation and wild type carriers in fertilization rates, implantation rates and clinical pregnancy of IVF and ICSI. Our results by ELISA and immunocytochemistry showed that the protein expression was less in men with del/del genotype in comparing to other genotypes (p<0.005).
Conclusion: Although previous studies found that DEFB126 variation would affect sperm function and male fertility rate, in the present study, no significant differences were found between homozygote mutation and wild type carriers in fertilization rates, implantation rates and clinical pregnancy. Further confirmation in a larger scale study is needed.
Key words: Unexplained male infertility, Deletion, DEFB126, IVF, ICSI.
P-154
Association of TNFR1 36 A/G polymorphism with azoospermia in Iranian infertile males
Ashrafzadeh Mehrjardi HR1, 4, Dehghan Tezerjani M2, Khademi M1, Ghasemi-Esmailabad S2, Mossadegh AM3, Ghasemi N4.
- Department of Biology, Faculty of Science, Islamic Azad University, Ashkezar Branch, Yazd, Iran.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Pharmacy, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Recurrent Abortion Research Center, Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: Infertility is the failure to achieve a pregnancy after one year of regular sexual intercourse without contraception. Half of the cases of infertility are due to male factors and nearly 60-75% of reasons of male infertility are unknown, due to unknown mechanisms of molecular defect. Tumor Necrosis Factor α (TNF-α) is a multi-functional cytokine that regulates cellular processes related to spermatogenesis. Tumor Necrosis Factor Receptor 1 (TNFR1) mediate TNF-α activity and changes in its structure can affect TNF-α activity. Studies show that variation in the TNFR1 gene may be associated with male infertility.
Materials and Methods: This case-control study includes 108 azoospermic men and 119 healthy controls. We investigated the association of TNFR1 36 A/G in the population with idiopathic azoospermia who referred to Yazd Institute of Reproduction Sciences. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR- RFLP) method was used to genotype the single nucleotide polymorphism in the both groups. PCR fragments digested by enzymes Mspa1I, and were separated by gel electrophoresis and then the frequency of A→G substitution in azoospermic males and fertile men were counted.
Results: According to our study, G/G genotype in the control group has higher frequency among men with azoospermia [p=0.01; OR=2.29 (1.248-4.229)]. Our findings also show that the allele frequency of G allele in azoospermic men is significantly different in comparison with control group [p<0.001; OR=2.302 (1.580-3.55)].
Conclusion: It seems that the G/G genotype and G allele are significantly associated with increased risk of idiopathic azoospermia.
Key words: Polymorphism, Male infertility, Sperm, Cytokines, Tumor necrosis factor alpha.
P-155
Epigenetic evaluation of histone methylation on HOXA1-5 genes in cumulus cells of polycystic ovary syndrome patients
Asiabi Kohneh Shahri P1, 2, Shahhoseini M2, Favaedi R2, Hassani F3, Movaghar B3, Karimian L3, Eftekhari Yazdi P3.
- Faculty of Basic Science and Advanced Technologies at University of Science and Culture, ACECR, Tehran, Iran.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: Polycystic ovary syndrome (PCOS) is the major cause of an ovulatory infertility with uncertain etiology. Epigenetics and environment play critical roles in PCOS. Of important genes correlated with human reproductive system disorders are HomeoboxA (HOXA) cluster genes. Regarding the epigenetic role of histone modifications in regulation of gene expression, lysine (K) methylation of histone3 (H3K9me), as a repressive epigenetic mark, on the promoter regions of HOXA1-5 genes were evaluated in this study. For this respect, cumulus cells (CCs) which have critical roles during folliculogenesis, oocyte maturation, ovulation and fertilization, were aimed to monitor expression profile and epigenetic alterations of HOXA1-Poster Presentations
P-1
Fertility preservation in cancer patients
Abtahi NS, Ebrahimi B, Rezazadeh Valojerdi M, Fathi R.
Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: b.ebrahimi@royaninstitute.org
Introduction: Fertility preservation before gonadotoxic treatments is necessary in cancer patients. Ovarian tissue cryopreservation is the only available option for prepubertal girls and women who cannot delay cancer treatment or when ovarian stimulation is contraindicated.
Materials and Methods: Human ovarian tissue biopsies were obtained from six women (29-40 years old) who were candidates for oophorectomy for benign gynecologic conditions. Their medulla were removed, cortical parts were thinned and then cut into 10×5×1 mm strips. Cortical strips were vitrified in two steps by using vitrification solutions including; V1 [HTCM as base medium (BM)+ 7.5% DMSO+ 7.5% EG+ 20% HAS] and V2 (BM+ 15% DMSO+ 15% EG+ 0.25M sucrose+ 20% HSA) for 15 and 10 min respectively. Morphology of ovarian tissue and enclosed follicles were compared in control and vitrification groups by histological assessment.
Results: Ovarian stromal integrity, granulosa cells distribution, follicular population and morphology were well preserved in vitrified-warmed strips as fresh one. Oocyte degeneration, vacuolization and granulation were rarely observed in vitrified-warmed ovarian tissues. Primordial follicles were more distributed in cortical region in both groups. The mean percentage of intact follicles (primordial, primary and pre antral) are higher in control groups compared to vitrification one but the difference was not significant.
Conclusion: Accordingly, used vitrification method could be suitable for human ovarian tissue preservation in cancer patients.
Key words: Human, Fertility preservation, Vitrification, Ovarian tissue.
P-2
Effects of sesame-supplemented diet on the histology of adult rat reproductive system
Amini Mahabadi J1, Hassani Bafrani H1, Nikzad H1, Taherian AA2, Seyed Hoseini ES1.
- Gametogenesis Research Center, Faculty of Sciences, Kashan University of Medical Sciences, Kashan, Iran.
- Anatomical Sciences Research Center, Faculty of Sciences, Kashan University of Medical Science, Kashan, Iran.
Introduction: Studies show that antioxidants are important in improving male infertility. There are several antioxidant compounds in sesame seeds.
Materials and Methods: This experimental study was carried out on 30 adults Wistar rat (200 gr). Rats were randomly divided into experimental and control groups. The control group received standard diet and experimental group received a diet containing 70% standard diet and 30% sesame seed for 12 weeks. At the end of the study, body weight, testis weight and volume were measured and histology of testis, epididymis and prostate were evaluated. Serum FSH, LH and Testosterone levels were measured as well. SPSS software was used to calculate t-test and p<0.05 was considered as significant.
Results: The measured sperm count and motility, the number of epithelial cells and lumen diameter were increased significantly in the experimental group (p<0.0001). The number of spermatogonial cells, primary spermatocytes, spermatid and spermatozoa were increased significantly in the experimental group compared to the control group (p<0.0001). The epididymal diameter, lumen diameter and epithelial thickness did not change significantly. Fibromascular and epithelium diameter of seminal vesicle in treatment group were very significant differences and volume density epithelium significantly increased in treatment group compared to control group but volume density, Fibromascular and lumen significantly decreased in treatment group compared to control group. LH concentration increased significantly in the experimental group (p<0.03).
Conclusion: This is the first study, which evaluated the histology effect of sesame seed on reproductive system of adult Wistar rat. These results highly suggest that sesame seed can improve male reproductive parameters.
Key words: Rat, Male Sex hormones, Sesame seed, Testis, Prostate, Epididymis.
P-3
The effects of sperm preparation media on motility, viability and DNA integrity of human spermatozoa
Anbari F1, Halvaei I1, Nabi A1, Ghazali Sh1, Khalili MA1, Johansson L2.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Origio A/s, Knadrupvej 2, DK-2760 Malov, Denmark.
Introduction: The main goal was to compare the effects of three different sperm preparation media on sperm motility, viability and DNA integrity of semen samples from normozoospermic men.
Materials and Methods: A total of 15 normozoo spermic males were included in the study. The semen analysis was performed in accordance with the WHO guidelines (2010). After semen analysis, each sample was divided into three aliquots and swim-up performed with three different sperm preparation media (Sperm Preparation Media, Origio, Denmark, Ham’s F10, Biochrome, Berlin, Germany, and VitaSpermTM, Innovative Biotech, Iran). Sperm motility, viability and DNA fragmentation,were evaluated at 0, 1, 2, and 24 hr after swim-up.
Results: There were no significant differences, at any time intervals, in the totalsperm motility between the different sperm preparation media. However, the rate of progressive motility was significantly higher in spermatozoa prepared by the media from Origio in comparison to VitaSperm TM (68.3±11.5 vs. 58±9.8, p=0.03), whereas no significant difference was found against Ham’s F10. No significant differences in sperm viability were seen between the media products .One hour after swim-up therate of sperm DNA fragmentation was also significantly lower inthe medium from Origio versus VitaSpermTM (p=0.02).
Conclusion: The type of medium for preparation of semen samples from normozoospermic men significantly affects the performance of spermatozoain assisted conception.
Key words: Sperm preparation media, DNA fragmentation (SCD), Viability, Motility.
P-4
Investigating the underlying factors of preterm delivery prevalence preceded by preeclampsia among pregnant visitors of Shariati Hospital, Bandar-Abbas, Iran: 2011-2012
Aslavi N1, Fallahi S2, Nowroozi S1, Najafian A3, Ghanbarnejad A1.
- Student Research Committee, Hormozgan University of Medical Sciences, Bandar Abbas, Iran.
- Department of Molecular Medicine, Hormozgan University of Medical Sciences, Bandar Abbas, Iran.
- Department of Fertility and Infertility, Hormozgan University of Medical Sciences, Bandar Abbas, Iran.
Introduction: Along with blood loss and infection, disorders pertaining to blood pressure are the three deadly factors in preterm childbirth morbidities and disabilities. In other countries the prevalence rate of this crisis is maximally 10%, while in Iran, it is 7%.
Materials and Methods: The present descriptive/ analytic research is a cross-sectional retrospective study. It was carried out on 863 pregnant women (167 had a preterm delivery due to pre-eclampsia and 696 were diagnosed with preterm delivery due to other reasons). The data were collected via a checklist about mother’s age, blood group, pre-eclampsia history, systemic disease, preterm delivery pain, etc. SPSS 16 analyzed the data. T-test and Chi-squared tests were used too. Significance level was set at p<0.05.
Results: Participants with a preterm delivery after pre-eclampsia were found to have a higher average age than peers afflicted with preterm delivery for other reasons. Most of the women afflicted with pre-eclampsia were of the B+ blood type. Significant differences were observed in these variables: pre-eclampsia history, preterm delivery pain, preterm rupture in fetal membrane, vaginal bleeding, intrauterine growth restriction, mother’s systemic diseases, blood group, history of intrauterine death and amniotic fluid disorders.
Conclusion: There is an increase in the probability of preterm childbirth due to pre-eclampsia with intrauterine growth restriction, history of intrauterine death and mother’s background diseases. Timely medical and preventive attempts and instructions for pregnant mothers on pre-natal healthcare can help to decrease irrevocable a consequence that endanger mothers and baby’s health.
Key words: Pre-eclampsia, Premature childbirth, Disease prevalence, Bandar-Abbas.
P-5
Infertility and quality of life in women with polycystic ovary syndrome
Bahri Khomami M1, Ramezani Tehrani F1, Azizi F2.
- Reproductive Endocrinology Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
- Endocrine Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Introduction: Polycystic ovary syndrome (PCOS), the most common endocrine disorder, may cause infertility among affected women. Infertility is a life crisis and blamed for psychosocial distress and poor quality of life (QoL). Ethnicity and socio-cultural factors may have an impact on this negative influence on QoL.
Materials and Methods: The study procedure was completed by 796 women with polycystic ovary syndrome, aged 15-49 years. A reliable validated Persian version of the health related quality of life questionnaire (HRQoL) for polycystic ovary syndrome patients (PCOSQ) was filled for each participant. They were subdivided into 2 groups according to their fertility status: fertile and infertile. Using linear regression the association between infertility and health related quality of life in women with PCOS was assessed.
Results: Out of all participants 120 (15.1%) and 482 (60.6%) women were infertile and fertile, respectively. Infertility was associated with lower HRQoL score before and after adjustment for age, body mass index and other perceived PCOS symptoms (CI 95%: -16.76, -3.81; p=0.002 and CI 95%: -15.02, -3.67; p=0.001). Women who had longer duration of infertility, had got better scores.
Conclusion: We found that infertility causes poor quality of life in PCOS women. Care providers should pay attention to the effect of each PCOS symptoms on patients emotional health along with physical health and plan to treat them accordingly.
Key words: Quality of life, Health-related quality of life, Infertility, Polycystic ovary syndrome.
P-6
The effect of hydrostatic pressure on parthenogenetic activation of mouse oocytes
Bashiri Z, Azadbakht M.
Departeman of Biology, Faculty of Sciences, Razi University, Kermanshah, Iran.
Email: zbashiri88@gmail.com
Background: Parthenogenetic activation of mammalian oocytes using artificial stimuli is commonly used in various reproductive biotechniques. Hydrostatic pressure can act as a mechanical stimulator that rearranges egg contents. In this study, we investigated the effect of hydrostatic pressure on parthenogenetic activation of MП oocytes derived from superovulation and matured oocytes in vitro.
Materials and Methods: In experiment 1, immature oocytes were dissected from ovary of female NMRI mouse (8-week-old) and transferred to α-MEM medium for in vitro maturation. After 24 hr, MП oocytes were transferred to T6 medium. In experiment 2, mice were superovulated by injections of 10 IU of PMSG and 10 IU of HCG 48 hr apart. MП oocytes were collected 12 hr after HCG injection and transferred to T6 medium. Then oocytes from two groups (experiment 1, 2) were divided into experiment and control groups. Oocytes of experiment group were subjected to 20 mmHg pressure for 10, 20, 30 min (treatments I, П, III). Oocytes without exposure to pressure were considered as control. Oocytes were cultured for 72 hr and embryo development was assessed.
Results: In experiment 1, cleavage rate in treatments I, П, III and control was 21.42%, 15.71%, 8.52%, 5.71% respectively. The best cleavage rate were associated with treatment I which were significantly different with treatment III and control group (p<0.05). In experiment 2, cleavage rate in treatments I, П, III and control was 22.91%, 53.12%, 29.15% and 9.34% respectively. Oocyte activation rate in experiment group was higher than control group. The highest cleavage rate associated with treatment П which were significantly different with treatments I, III and control (p<0.05).
Conclusion: Exposure of MП oocytes derived from superovulation to hydrostatic pressure, on account of cumulus cells presence around oocyte, could improve embryonic development and by affecting on calcium channels, probably leading to increase rate of cleavage in the mouse oocyte.
Key words: Parthenogenetic activation, Hydrostatic pressure, Oocyte, Mouse.
P-7
Evaluation the knowledge and awareness of nulliparous women from adverse outcome of delayed childbearing
Behboudi-Gandevani S.
Reproductive Endocrinology Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Email: s.behboudi@hotmail.com
Introduction: Recently the numbers of women who delay childbearing in older age were rapidly increased. The aim of this study was to evaluate the nulliparous women’s knowledge and awareness from adverse outcome of delayed childbearing.
Materials and Methods: In this cross-sectional survey study, a total of 700 healthy nulliparous women 35 years and older that had self-choice delayed pregnancy were selected by cluster sampling in Tehran. To evaluate the knowledge and awareness of these women, a standard questionnaire about adverse pregnancy outcomes in older age were used. The questionnaire consists of three parts: demographic characteristics, assessment of maternal adverse outcomes (10 questions) and to assessment of fetal-neonatal adverse outcomes in maternal older age (6 questions). Data were analysis by SPSS software and the significance level was set at p˂0.05.
Results: The psychometric properties of questionnaire including forward and backward translation, face and content validity and reliability with cronbach's-alpha: 0.078 were done. The result of the study showed that women’s awareness about the risks of delayed childbearing including gestational diabetes, congenital anomaly, Down syndrome, caesarean section, low birth weight, stillbirth and long-term health problems like learning difficulties among neonate varies between 40%-72%. In this respect, most of the women were aware about the increased risk of infertility in older age (82.9%), but a little were aware about the possibility of a twin or multiple pregnancy and gestational hypertension in older ages of mother (35.2%). Awareness of delayed childbearing risks were independently related to the women’s educational levels (OR: 1.87; 95% CI: 1.25-2.78) and family income (OR: 3.12, 95% CI: 0.01-5.12).
Conclusion: It seems that women are largely unaware about the adverse consequences of pregnancy in older age among mother and child. Women’s should be more inform about these risks by health care professional.
Key words: Awareness, Delayed childbearing, Adverse outcome.
P-8
Study the protective role of jujube extract on teratogenic effects of Carbamazepine in Balb/c mice embryos
Doostabadi MR, Hasanzadeh Taheri MM, Afshar M, Hami J, Hosseiny M.
Birjand University of Medical Sciences, Birjand, Iran.
Email: doostabadim@yahoo.com
Introduction: Carbamazepine (CBZ) is an anticonvulsant medication that is consumed during pregnancy and can produce congenital anomalies including; neural tube defects, cardiac, skeletal and craniofacial abnormalities.
Materials and Methods: In this experimental study, 100 pregnant mice of Balb/c 25±3 gr body weight randomly divided into 8 experimental groups (E) and 2 control groups (C) (n=10). Experimental groups (E1, E5 and E6) and (E2, E7 and E8) received daily intraperitoneal injection (IP) of 50 and 100 mg/kg/ of CBZ respectively from gestational day zero (GD 0) to GD 15 and also groups (E5, E7) and (E6, E8) in addition to medicine, received jujube extract (AJE) at doses of 200 or 400 mg/kg/ from ten days prior to gestation, to GD 15. E3 and E4 groups gavaged only by 200 and 400 mg/kg of AJE respectively. Two control groups (C1, C2) received normal saline or tween -20. Dams underwent cesarean section on GD 18 and fetuses harvested from uterine. First, absorbed embryos were counted. Thereafter, morphological studies were done on the offspring by stereomicroscope. All malformed fetuses were stained with Alizarin red S and alcian blue for detection of skeletal anomalies. Data were analyzed by ANOVA, Tukey and c2 tests and using SPSS software version 18 and p<0.05 were considered significant.
Results: The findings of this study showed that administration of CBZ purely in pregnant mice induced various anomalies in their fetuses such as; limb defects, deformities of vertebral column, craniofacial malformations and etc in experimental groups so that their differences were significant when compared with control groups. But these anomalies decreased significantly in those experimental groups which received CBZ and AJE synchronously when compared with experimental groups that received CBZ only.
Conclusion: According to our findings it can be concluded that although administration of CBZ can induce several malformations in fetuses of pregnant mice, but consumption of AJE synchronously with CBZ, can prevent teratogenicity of CBZ. Therefore, probably AJE can play a protective role against CBZ induced anomalies.
Key words: Teratogenic, Jujube, Carbamazepine.
P-9
Depression and clinical markers in polycystic ovary syndrome
Enjezab B1, Ghadiri anari A2, Eftekhar M3, Mohajeri M1.
- Department of Midwifery, Faculty of Nursing and Midwifery, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Internal Medicine, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
Introduction: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among reproductive-age women. Previous studies have raised conflicting results of depressive disorders in PCOS compared with healthy people.
Materials and Methods: In this cross-sectional analytic study, 62 patients with PCOS diagnosed by Rotterdam criteria and 61 women without PCOS were selected after an initial survey conducted to determine demographic characteristic, systolic and diastolic blood pressure and reproductive status, the Beck depression questionnaire short form was used to assess depression in both groups. All women were introduced to determine fasting insulin and blood sugar and testosterone.
Results: The mean age, BMI, systolic and diastolic blood pressure of PCOS and Non PCOS was respectively (29.96±6.85, 29.49±7.44 years), (29.15±6.56, 25.65±5.84), (117.42±10.17, 117.18±66.57) and (75.41±9.27, 76.42±6.56) respectively that only mean of BMI was different (p˂0.05). The average Beck score in patients was 7.47±5.54 and in the control group was 7.57±5.79, (p˂0.05), which was not statistically significant. 37.1% of the patient group and 36.1% of the control group have degree of moderate to severe depressed mood (p˃0.05).
Conclusion: Despite non significant difference in prevalence of depression in both groups, due to the high prevalence of depression in both groups may be there is other reasons for depression in this population that overcomes on the psychological effects of this disease.
Key words: Depression, Polycystic ovary syndrome, BMI.
P-10
Comparison of metabolic syndrome in postmenopausal women with natural or surgical menopause: Community based cohort study
Farahmand M, Ramezani Tehrani F, Azizi F.
- Reproductive Endocrinology Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
- Endocrine Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Introduction: The ovaries produce female sex hormones that after natural menopause gradually and following surgical menopause abruptly end.
Materials and Methods: The study subjects were selected from 5019 women, 59-35 years old, which were participated in the Tehran Lipid and Glucose Study. They include 357 natural menopauses and 63 surgical menopauses during the follow-up that was 10 years. Data were collected by questionnaires in this project.
Results: Changes in metabolic and biochemical profiles of these two groups of women during follow-up were compared with each other. The findings of our study showed that the incidences of metabolic syndrome in surgical menopause and natural menopause subjects were 14.5 and 8.9% respectively. Mean serum low density lipoprotein in surgically menopausal women was significantly lower than natural menopause subjects. Also, mean systolic blood pressure in naturally menopausal women was significantly higher than surgical menopause women.
Conclusion: It seems the metabolic disorders associated with menopause differ in surgical and natural menopause women. The metabolic complications should be considered following menopause.
Key words: Surgical menopause, Natural menopause, Metabolic syndrome.
P-11
Medical staffs’ viewpoints on “ART nursing” working at Reproductive Institutes in Iran
Faramarzi A, Khalili MA, Borzouei Z, Hosseini A, Rahiminia T.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: faramarzi_90@yahoo.com
Introduction: As, assisted reproductive techniques (ART) advances, multifaceted changes are noted in the roles, responsibilities, and commitments of each members of the infertility team. The aim was to determine the level of knowledge and attitude of medical staff working at reproductive institutes about role of nursing in ART program.
Materials and Methods: In this descriptive cross-sectional study, knowledge and attitudes of 199 Bio-medical staffs working at reproductive institutes were investigated through a questionnaire including 20 questions. All participants based on work experience, education and communication with patients were classified. Chi-square test was used for data analysis and p<0.05 was considered as statistically significant.
Results: There was no significant relationship between educational levels and viewpoints on ART nursing. But, significant relationship between work experience, kind of relation to the patients and viewpoints on ART nursing were found. 52.5% of participants were familiar with ART nursing. By increasing experience, knowledge and attitudes of Bio- medical staffs enhanced. Laboratory technician, embryologist, ART Laboratory technician and nurses were more familiar with ART nursing; but, Urologists were less familiar with ART nursing. Over 97% of participants requested offering a master degree of ART nursing in Iran (p<0.05).
Conclusion: There is a need for nursing professionals to assume an ongoing, visionary, scientific, and academic approach to advancement. It seems that appropriate continuing education and opportunities are essential to support this group of nursing professionals.
Key words: Viewpoint, Nurse, ART, Medical staffs.
P-12
The effect of microtubule stabilizer pretreatment in IVM program
Fesahat F1, Khalili MA1, Khoradmehr A1, Anbari F1, Bazrgar M2, Taheri F1.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Genetics, Royan Institute for Reproductive Biomedicine, Tehran, Iran.
Introduction: It has been reported that in vitro maturation (IVM) increases the risk of abnormal spindles and chromosome configurations of oocytes compared with oocytes matured in vivo. This is one possible explanation for the reduced developmental potential of IVM oocytes compared with those matured in vivo. Paclitaxel is known to stabilize the microtubules that constitute the spindle. The aim was to investigate whether pretreatment by a microtubule stabilizer, paclitaxel, would improve IVF outcomes in IVM program.
Materials and Methods: In this ongoing experimental study, 75 GV oocytes were retrieved from 5 mice primed with rec-FSH for 48 hr. The immature oocytes were cultured 24-48 hr for IVM. After identifying mature oocytes by the presence of a first polar body extrusion under a stereomicroscope, Normal morphologically MII oocytes were divided in control and 5 experiment groups. Experiment groups were incubated in presence of 1μM paclitaxel for different times (30 min/group 1,1 hr/group 2,2 hr/group 3,3 hr/group 4,4 hr/ group 5 and group 6 as control) prior to IVF. Standard IVF program was performed for the 60 MII oocytes after pretreatment with paclitaxel and embryo development was followed until blastocyst formation.
Results: Total maturation rate of GV oocytes was 80% and fertilization rates of MII oocytes were 85% in groups 6, 83% in groups 1, 2 and 3 and 74% and 65% in groups 4, 5 respectively. Blastocyst formation rates of embryos were 95% in group 1, 2 and 6, 60% in group 3 and 40% in groups 4 and 5. The final results, which include probable relationship between paclitaxel pretreatment and the aforementioned variables, will be presented.
Conclusion: The preliminary results showed by performing critical concentration with optimized timing of microtubule stabilization procedure IVM, MII oocytes could efficiently develop to the blastocyst stage. Paclitaxel may cause cell toxicity with irreversible harm on normal fertilization and embryo development in higher concentrations.
Key words: Paclitaxel, IVM, IVF outcomes.
P-13
The effect of ovarian drilling on poly cystic ovary syndrome (PCOS) women
Ghasemi-Esmailabad S, Eftekhar M, Pourmasumi S, Rajabi Firoozabadi Sh, Sabeti P, Heidarian Meimandi H.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: Qasemisaeed@yahoo.com
Introduction: One of the most common causes of female infertility factors is ovulation disorder. Poly cystic ovary syndrome (PCOS) has the highest prevalence rate about 5-10% among women. There are different diagnostic methods and treatments for PCOS syndrome, including medical and surgical treatments. Laparoscopic surgical treatment is for the patients resistant to medical treatment, and could be done with ovarian drilling at some points.
Materials and Methods: In this retrospective study we evaluated 289 patients. All patients had proven clomiphene resistant PCOS with ultrasound and hormonal tests. Laparoscopic ovarian cautery was performed for all these women. Age, duration of infertility and assisted reproductive techniques in each patient were asked.
Results: Of the 289 patients, we access to the information of 135 patients. The mean age and the duration of infertility were 26.90 and 4.83 years respectively. Among these, 63 patients (46.6%) had not pregnancy and 72 patients (53.33) were pregnant, 52 patients (72.22%) were naturally pregnant and 20 patients (27.77%) used assisted reproductive techniques.
Conclusion: PCOS patients who resistant to clomiphene could be treated with ovarian drilling.
Key words: Polycystic ovary, Clomiphene, Ovarian drilling.
P-14
Comparison of two different embryo loading techniques for embryo transfer in IVF/ET cycles
Halvaei I, Mangoli E, Omidi M, Khalili MA.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: ihalvaei@gmail.com
Introduction: Embryo loading (EL) is one of main steps in embryo transfer (ET) technique that plays an important role in IVF success. This study was aimed to compare the effect of two different techniques for EL on rates of pregnancy and delivery in IVF/ET cycles.
Materials and Methods: In total 195 fresh ET and 171 frozen-thawed ET were included in this retrospective study in which 76 and 119 cycles in fresh ET and 76 and 95 cycles in frozen-thawed ET were placed in two groups of A and B, respectively. Embryo catheter loading techniques were divided into two groups of A and B. In group A, the whole catheter was flushed with a 1-ml air-tight syringe with Ham’s F10 medium. Then the embryos were drawn into ET catheter between two air brackets. In group B, 70 µl airs was held in the syringe and the catheter was flushed by Ham’s F10 medium. Then the media, air, embryos, and air were respectively drawn into catheter. The main outcome measure of the study was delivery rate.
Results: The groups were matched for number of fertilized oocytes, etiology of infertility, source of sperm, type of stimulation protocol, percent of conventional IVF or ICSI, high quality embryos, type of embryo loading catheter, fresh ET and ease of transfer. In fresh ET cycles, delivery rate showed an increasing trend in group B compared to group A (21% vs. 11.8%, respectively, p=0.1). In frozen-thawed ET cycles, the rate of delivery rate was also higher in group B compared to group A (16.8% vs. 13.1%, respectively), but the difference was not significant (p=0.5).
Conclusion: It seems different embryo catheter loading has no effect on delivery rate.
Key words: Embryo loading technique, Embryo transfer, Pregnancy.
P-15
Application of sonography in infertility treatment cycles: instructions for midwives and nurses
Irani Sh, Ahmadi F, Javam M.
Department of Reproductive Imaging at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: Irani_shohreh@yahoo.com
Introduction: Ultrasound has a pivotal role in imaging modality in the study of the female pelvis, and provides fundamental information in detecting and characterizing pelvic masses of uterine, ovarian, or adnexal origin.
Materials and Methods: A narrative review was performed within articles published at PubMed, Elsevier, SID and original text books to reach the aim.
Results: Every patient in the process of infertility treatment needs to go through four steps of sonography to roll out disorders and malformations in ovaries, uterus and tubes then to opt for the best treatment method and to follow up the results. Infertility Workup: 1) Base sonography is applied in examination of the following organs: -For overall evaluation of the pelvis and determining any pathologic condition in uterus such as polyps, fibroms, uterus malformations. -Ovarians are examined for ovarian cysts and Poly Cystic Ovary Syndrome (PCOS). 2) Pre-treatment sonography is applied in checking the result of operative surgeries- like Hysteroscopy, Laparoscopy, Myomectomy. 3) Monitoring sonography is done at different stages of treatment cycle, especially at ovulation stimulation period, in order to investigate ovarians and in picking up the eggs as well as endometrial thickness in response to drugs. 4) Post-Assisted reproductive techniques (ART) cycles is done for confirmation of pregnancy (gestational sac, fetal heart), and the side effects of treatment cycles such as ovarian hyper stimulation syndrome (OHSS), pregnancy complications such as ectopic pregnancy (EP) and mole.
Conclusion: Ultrasound has a key role in diagnosis and proposing of different options of treatment regimes, and the post-treatment follow-ups. It is important to know the application of the aforementioned four steps to improve the accuracy and efficiency of infertility treatment.
Key words: Ultrasonography, ART cycle, Infertility.
P-16
Evaluation of anxiety, depression and risk factor associated with them in women after tubal ligation
Jahanian Sadatmahalleh Sh1, Ziaei S1, Kazemnejad A2, Mohamadi E3.
- Department of Reproductive Health and Midwifery, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
- Department of Biostatistics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
- Department of Nursing, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Introduction: Tubal ligation is recommendable for women completed their family. The existence of anxiety and depression following this procedure has been the subject of debate for decades.
Materials and Methods: A historical cohort study was carried out on 200 subjects with tubal ligation and on 200 subjects using condom as contraceptive method. The two groups were matched in demographic and personal characteristics. Data collection tool was a questionnaire including questions regarding demographic and obstetrical characteristics. Anxiety and depressive symptoms were evaluated by the Hospital Anxiety and Depression Scale (HADS). All statistical analyses were carried out using software package used for statistical analysis (SPSS) version 20 (SPCC Inc., Chicago, IL, USA). Student’s t-test and chi-square test were carried out to reveal the statistical differences between the groups. Multiple linear regression was done to build a prediction model in anxiety and depression.
Results: The mean (SD) duration of tubal ligation was 4.1 (1.6) years. The overall prevalence of anxiety and depression in the two groups was 81.5% and 48.5% respectively (p<0.0001). Multiple linear regression analysis revealed a significant association between low education (p<0.0001), post-sterilization regret (p=0.03) and no consultation prior tubal ligation (p=0.03) with risk for anxiety and depression in women who have undergone tubal ligation.
Conclusion: Termination of fertility with tubal ligation may be a risk factor for anxiety and depression. We found significant differences in anxiety and depression between women with and without tubal ligation. Therefore, women should be informed by the health providers regarding the advantages and disadvantages of tubal ligation before the procedures.
Key words: Historical cohort study, Tubal Ligation, Anxiety, Depression.
P-17
Polymorphism in CGA affects the function of miR-1302 and increases the risk of men infertility
Jamalvandi M, Motevali bashi M.
Department of Biology, Faculty of Sciences, Esfahan University, Esfahan, Iran.
Email: mina.jamalvandi@yahoo.com
Introduction: Infertility occurs in 10-15% of couples worldwide and close to half of it is caused by male factors. Despite decades of efforts to clarify mechanism of male infertility, most of cases are still idiopathic. Many factors such as genetics and sexual problems can affect infertility. Among these problems, genetic disorders are the most common factors. A study has shown that one of genes that can affect male infertility is CGA. This gene is involved in miotic. CGA, α subunit of glycoprotein hormones, is the main part of thyrotropin glycoprotein hormone (pituitary TSH), lutropin (LH), follitropin (FSH), and Chorionic gonadotropin (human placental gonadotropin, hCG) that has essential role in development and function of tyroid and gonads. CGA gene is located on 6q14-q21. Rs6631 in CGA has strong association with men infertility. Studies have shown that miR-1302 can negatively regulate CGA and substitution of T with A may interfere this process. This miRNA can band with rs6631-A more strongly than rs6631-T.
Materials and Methods: Tetraprimer technique is an appropriate way to study this polymorphism; because it is faster and cheaper than ordinary PCR. Also laboratories with low equipment can use this method.
Results: Primers designed by the use of Primer1 and then checked by Oligo7 software. By the use of these primers and tetraprimer technique, this polymorphism can be studied for the first time in Iran.
Conclusion: SNP in genes specially the ones that are target of miRNAs, can have important role in complex disease such as infertility. In this study, miR-1302 negatively regulates CGA, and the substitution of T by A at rs6631 within the binding site disrupts its regulation. It is not exactly clear that what is the real role of this polymorphism in idiopathic male infertility; but it has been suggested that the variant allele of rs6631 may elevate the risk of idiopathic male infertility through up-regulation of the expression of CGA. It has been showed that α-subunit, that is produced by CGA, can has growth factor activity and can induce the differentiation of lactotrope and secretion of RPL. Because CGA and PRL play an important role in the development of gonads, the abnormal hormone level may elevate the risk of male infertility.
Key words: Men infertility, miRNA, SNP, CGA.
P-18
Evaluation of testosterone levels after GnRH agonist administration in the adult male rat
Khadivi B1, Peirouvi T1, Zirak Javanmard M2, Rasmi Y3, Mohammadbeigi H1.
- Department of Histology, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran.
- Department of Anatomy, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran.
- Department of Clinical Biochemistry, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran.
Introduction: Gonadotropin-releasing hormone (GnRH) is secreted from the hypothalamus in a pulsatile manner and stimulates the biosynthesis and release of gonadotropins, LH and FSH, via GnRH receptor located in gonadotropin cells. These gonadotropins regulate various gonadal functions such as gametogenesis and steroidogenesis.
Materials and Methods: 24 male adult Wistar rats were divided into three groups. In the first study group, 300 µg/kg buserelin, in the second study group, 600 µg/kg buserelin and in the control group, salin was injected subcutaneous for 5 days. Thirty day after the first injection, blood samples were collected from the heart, centrifuged and plasma was isolated from blood. Plasma testosterone level was measured by ELISA.
Results: The findings reveal no significant differences in plasma level of testosterone in the first study group (2.71±0.71) and second study group (2.12±1.74) compared with control group (3.14±1.20) (p>0.05).
Conclusion: Short-term administration of buserelin has no effect on the plasma testosterone level.
Key words: GnRH, Buserelin, Testosterone, Adult rat, ELISA.
P-19
Apoptotic cells and loss of follicle development were resulted after administration of Nano dioxide titanium on immature mouse ovary
Khoradmehr A1, Danafar AH1, 2, Hosseini M3, Halvaei I1, Golzadeh J1, Akyash F1, Anvari M1.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Young Researchers Club, Islamic Azad University, Yazd, Iran.
- Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran.
Introduction: Titanium dioxide (TiO2) is used as an antimicrobial and whitening agent in food and products such as chewing gums, candies, toothpastes, lip balms, shampoos, deodorants and sunscreens. Previous studies showed that ovary might be one of the target organs that TiO2 nanoparticles induced genotoxicity and cytotoxicity in ovary cells and exposure to TiO2 can effect on follicle development and fertility with changes the levels of sex hormones. The aim of the present study was to evaluate ovarian dysfunction by detect of follicle development.
Materials and Methods: 40 immature Balb/C female mice (4week ages) randomly divided into five groups. First, second and third groups were administrated by oral gavage 2.5, 5 and 10 mg/kg TiO2 for thirty days respectively. Forth group was gavaged normal saline as placebo and in control group we didn’t gavage anything. Finally female mice were dislocated and ovary was fixed in bouin's fluid. Ovaries were dehydrated with ethanol series, embedded in paraffin and then serially sectioned at 5μm thickness. Hematoxylin and Eosin used to evaluate primordial, primary, preantral and antral follicles by light microscope. TUNEL assay was used to detect cell apoptosis. Statistical significance was analyzed by Kruskal-Wallis test and the level of significance was determined to be at p≤0.05.
Results: According to data, primordial, primary, preantral and antral follicles were significantly decreased in third group in comparison with the controls and shams (p≤0.05) and also, there was increasing trend in primary and preantral follicles in first and second groups compared to control group during TiO2 exposure. Although, significant reduction of apoptotic cells was observed in third group compared to control and sham groups (p≤0.05).
Conclusion: Furthermore, there is little knowledge about physicochemical characteristics of TiO2 nano materials; our findings suggested that TiO2 exposure can defect follicle development and perhaps fertility potential in young mice.
Key words: Titanium dioxide, Ovary, Follicle development, TUNEL.
P-20
Correlation between 25-OH vitamin D in follicular fluid and implantation rate in infertile women undergo IVF/ICSI
Latifi Z1, Farzadi L2, Khayatzadeh Bidgoli H2, Fattahi A1, Nouri M2.
- Department of Clinical Biochemistry, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
- Women’s Reproductive Health Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Introduction: Vitamin D in combination with its receptors can be involved in implantation by regulating gene expression, endometrium immune response and stimulation of endometrium decidualization. So, it seems that the amount of vitamin D in follicular fluids (FF) may has an association with ART success.
Materials and Methods: In a prospective study, 80 infertile female candidates for IVF/ ICSI were enrolled. Blood samples (on the day of human chorionic gonadotropin) and follicular fluids were taken, and then levels of serum estradiol and follicular fluids 25-OH vitamin D were measured. Also clinical characteristics of patients (duration of infertility, causes of infertility, and menstrual status), number and quality of oocytes, number of fertilized oocytes, estradiol levels, and clinical pregnancy were evaluated.
Results: Concentration of FF 25-OH vitamin D in pregnant women was significantly higher than non-pregnant women (p=0.007) but there were no significant differences in age, body mass index (BMI), duration of infertility, menstrual status, number of oocytes, oocytes quality, number of fertilized oocytes and serum estradiol levels between the two groups (p>0.05). Statistically positive correlation was found between 25-OH vitamin D levels with patient age and implantation rate (r=0.264, p=0.018 and r=0.301, p=0.007 respectively).
Conclusion: The obtained results suggest that vitamin D without affecting the number and quality of oocytes can independently improve implantation rate and IVF outcome.
Key words: Embryo implantation, Vitamin D, IVF outcome.
P-21
The protective effect of N-acetyl-L-cysteine on spermatogenesis and sperm characteristics in mice following exposure to Para-Nonylphenol
Malmir M, Soleimani Mehranjani M, Naderi Noreini S.
Faculty of Science, Arak Univercity of Medical Scinecs, Arak, Iran.
Email: mehhdi@yahoo.com
Introduction: Para-Nonylphenol (p-NP) is an environmental contaminant with wide industrial applications which causes oxidative stress in different organs such as the reproductive system. The purpose of this study was to investigate the protective effect of N-acetyl-L-cysteine (NAC), as a powerful antioxidant, on spermatogenesis indices, sperm parameters, chromatin quality and tail length following treatment with para-Nonylphenol in adult mice.
Materials and Methods: 24 adult male NMRI mice (32±4 gr) were divided randomly into 4 groups (n=6), control, NAC (150 mg/kg/day), p-NP (250 mg/kg/day) and p-NP+NAC, and they were treated orally for 35 days. By the end of the treatment, mice were weighed and sacrificed, Their right testis was also weighed then the left caudal epididymis was cut in Ham's F10. Released spermatozoa were used to analyze the motility, viability and the abnormalities of the sperm. The sperm tail length was estimated by stereological methods. Sperm chromatin quality was also assessed by nuclear staining using acridine orange and aniline blue dyes.In continue, the right testis were taken out, fixed, sectioned, processed and stained using heidenhain azan method. Spermatogenesis indices including the tubular differentiation index (TDI), Sertoli cell index (SCI) spermatogenesis index (SI), meiotic index (MI) and repopulation index (RI) were studied. Data were statistically analyzed using one way ANOVA and means were considered significantly different at p<0.05.
Results: A significant decrease in the spermatogenesis indices, sperm motility, viability, sperm tail length and the number of sperms with normal morphology was observed in the p-NP group compared with the control (p<0.002), while no significant change was found in the sperm chromatin quality. The above parameters significantly increased in the p-NP+NAC group compared to the p-NP treated ones (p<0.01).
Conclusion: N-acetylc-L-ysteine, as an antioxidant, can prevent the adverse effects of para-Nonylphenol exposure on spermatogenesis indexes and sperm parameters in mice.
Key words: Para-Nonylphenol, N-acetyl-L-cysteine, Spermatogenesis indices, Sperm parameters.
P-22
The effect of vitamin C on human vitrified sperm parameters in normozoospermic men: raw semen and washed semen
Mangoli E1, Anvari M2. Talebi AR3, Taheri F1, Rahiminia T1, Vatanparast M1.
- Research and Clinical Center For Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Biology and Anatomical Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Andrology, Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: Vitamin C is a common component in seminal fluid. It can improve sperm parameters as an antioxidant. The objective was to evaluate the effect of Vitamin C on human vitrified sperm parameters in normozospermia men.
Materials and Methods: Semen samples were collected from 40 normozoospermic samples and divided into 4 groups. Group 1, raw semen was vitrified. Group 2, semen processed by swim up method and then vitrified. Group 3, vitamin C (600 µm) was added to raw semen and then vitrified. Group 4, vitamin C (600 µm) was added to prepared spermatozoa and then vitrified. The semen analysis was performed according to WHO criteria before and after vitrification.
Results: In this study data showed that progressive motility and immotile spermatozoa after vitrification were significantly different between groups (p=0.014, 0.005 respectively). All groups were compared, and the results revealed that progressive motility and immotile spermatozoa after vitrification were statistically significant in washed semen+vitamin C group compared with wash semen and raw semen groups (p=0.05, 0.01 for progressive motility and p=0.04, 0.007 for immotile spermatozoa respectively).
Conclusion: Adding vitamin C, as an antioxidant, to washed semen has shown beneficial effect on sperm motility after vitrification.
Key words: Sperm parameters, Vitamin C, Vitrification.
P-23
Review of fertility diet and its effect on infertility among women from the viewpoint of Iranian traditional medicine in comparison with modern medicine
Tork Zahrani Sh1, Mirahi A1, Tansaz M2, Alaeddini F3, Hekmatdoost A4.
- School of Nursing and Midwifery, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
- School of Traditional Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
- Ministry of Health, Tehran, Iran.
- Institute and Faculty of Nutrition Sciences and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Introduction: In the recent years the infertility diagnosis and treatment methods have undergone a radical change and development but still, the success rate of modern treatments like in vitro fertilization (IVF) even in the best centers is between 30-40%.
Materials and Methods: The available texts on the Iranian traditional medicine were reviewed. Besides, the relationship between fertility diet factors and infertility among women was investigated through reviewing databases on the world-wide web.
Results: Iranian traditional medicine investigates the relationship between nutrition and infertility from a perspective postulating that complying with fertility diet principles helps maintain healthiness and improve fertility. From this view, body members have their special temperaments; if balanced, body members function normally. Any kind of abnormal temperament happened to the semen or uterus results in infertility or hardship in fertility. These are seen as disorders in sanguine, choleric, melancholic, and phlegmatic temperaments. It should be noted that the bad temperaments are mainly caused by not following the health and fertility diet.
Conclusion: The different viewpoint of the Iranian traditional medicine towards the relationship between infertility and nutrition and its focus on the nature of food and nature of a person could be of interest to researchers in the field and if approved, help to prevent infertility or treat infertile couples.
Key words: Infertility, Iranian traditional medicine, Infertility diet.
P-24
The in vitro fertilization outcome and luteal phase GnRH antagonist administration
Eftekhar M1, Miraj S1, 2.
- Department of Obstetrics and Gynecology, Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Obstetrics and Gynecology, School of Medicine, Shahrekord University of Medical Sciences, Shahrekord, Iran.
Introduction: Genital tissues (ovary, endometrium and placenta) express GnRH receptors. GnRH plays essential roles in embryo implantation, invasion of trophoblastic tissue and steroid synthesis in the placenta. In IVF-ICSI cycles, the use of GnRH antagonists is limited to the last days of ovulation. The aim of this study was to evaluate the effects of GnRH antagonist at pharmacological doses given in the early implantation period on pregnancy.
Materials and Methods: This retrospective study was performed in Yazd Research and Clinical Center for Infertility, 2014-2015. Women under 40 years old, with >20 follicles (>11mm) and risk of OHSS were included. Participants with history of endometriosis, hysteroscopy and history RIF were excluded. The treatment for all of participants was antagonist protocol. Twenty seven patients did not receive Cetrotide in luteal phase, and 67 patients received Cetrotide. Pregnancy outcomes were assessed based on chemical and clinical pregnancy.
Results: Totally 94 patients were included. The mean age of participants was 28.40±4.25 years old. There were no differences in ART and demographic characteristics (p>0.05). The most frequent causes of infertility were: male factor (45.2%) and polycystic ovary syndrome (19.4%). The pregnancy outcome was not significantly different between Cetrotide and non Cetrotide group (p=0.224).
Conclusion: The present study proposed that luteal phase GnRH antagonist administration does not influence the chance of successful pregnancy outcome. The incidence of chemical and clinical pregnancy in two groups was not significantly different.
Key words: GnRH antagonist, Pregnancy outcome, IVF.
P-25
Hazardous effect of acrylamide on development landmarks in rat offspring and the role of Glycyrrhiza Glabra
Mirgalili A1, Yadegari M1, Talebi AR2, Dortaj H1.
- Department of Biology and Anatomy, Faculty of Medical Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Research and Clinical Center for Infertility, Faculty of Medical Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: Acrylamide (AA) is a chemical substance used mainly in certain industrial process. Monomer of Acrylamide formed in food stuffs containing carbohydrates during high temperature cooking.
Materials and Methods: 24 pregnant rats were selected for this study. The pups of these mothers divided into four groups: group A:(control group); group B: (ACR administration 10 mg/kg/day orally); group C: (GG administration 150 mg/kg/day orally); group D: (ACR+GG). At day 21 the pups were evaluated for developmental study. The results were analyzed by SPSS software (15) and p<0.05 was significant.
Results: The result demonstrate that eye opening and fur development appeared occurred slightly later in AA administration group.
Conclusion: These results showed that AA treatments induce delay in developing offspring and GG as an antioxidant reduce these changes.
Key words: Acrylamide, Glycyrrhiza glabra, Eye opening, Fur appearance, Offspring.
P-26
The effect of long-term exercise via TNF-α level on the type-1 diabetic rats in testis tissues
Moghaddami Z1, Sheikhzadeh Hesari F1, Banan khojasteh SM1, Hemmati AR2, Dastranj A3.
- Department of Animal Biology‚ Faculty of Natural Sciences‚ University of Tabriz‚Tabriz‚ Iran.
- Department of Anatomical and Histologyical, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
- Department of Pathology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz‚ Iran.
Introduction: Approximately 90% of diabetes patients often show sexual abnormalities and impotence infertility. Damages caused by hyperglycemia and oxidative conditions result in inflammatory mediators increasing such as cytokines in tissues of sex organs. The TNF-α is the first locally produced cytokines in chronic inflammation pathway. TNF-α level in diabetics blood is higher than normal people that probably histological studies of TNF-α could determine source of this increasing. Also exercise can reduce complications of diabetes especially in tissue by reducing of oxidative elements and establishing of Hypoglycemia.
Materials and Methods: In this study, 40 male rats weighing 200-250 gr were used and randomly were classified into four groups; each group consist 10 rats (control, diabetic, healthy with period of 60-day exercise, diabetic with period of 60-day exercise). Treadmill exercise daily was 1 hr with 22 m/min speed. Diabetes was induced by injection intraperitoneal of streptozotocin with the amount of 60 mg/kg. After expelling of the testis under general anesthesia, samples were homogenized and TNF-α protein levels were measured according to ELISA kit (special rat TNF-α).
Results: The level of TNF-α showed no significant changes among diabetic groups in testicular tissue compare to control groups. Also, exercise didn’t have any negative effects on the level of TNF-α in exercise control group compare with control group. Therefore, we didn’t show any significant change of TNF-α level between our groups.
Conclusion: In this study, the period of disease time may be is not enough to determine the level of TNF-α via exercise treatment in testis tissues. It seems that duration of diabetic developing, severity and type of exercise could be many important reasons to change inflammatory mediators.
Key words: Cytokine, Hyperglycemia, ELISA Kit, Sexual dysfunction.
P-27
Evaluation of progesterone and estradiol levels after GnRH agonist administration in the adult female rat
Mohammadbeigi H1, Peirouvi T1, Rasmi Y2.
- Department of Histology, Faculty of Sciences, Urmia University of Medical Sciences, Urmia, Iran.
- Department of Clinical Biochemistry, Faculty ofSciences, Urmia University of Medical Sciences, Urmia, Iran.
Introduction: Gonadotropin-releasing hormone (GnRH) is secreted from the hypothalamus in a pulsatile manner and stimulates the synthesis and release of gonadotropins, LH and FSH, via GnRH receptor located in adenohypophysis. These gonadotropins, regulate various gonadal functions such as folliculogenesis and steroidogenesis. Various data are reported of GnRH agonists effects on ovary steroidogenesis.
Materials and Methods: 24 female adult Wistar rats were divided into three groups. In the first study group, 300 µg/kg buserelin, in the second study group, 600 µg/kg buserelin and in the control group, the same amount of salin was injected subcutaneous for 5 days. 30 days after the first injection, blood samples were collected from the heart, centrifuged and plasma was isolated from blood. Plasma esteradiol and progesterone levels were measured by ELISA.
Results: The findings reveal a significant incereas in plasma level of estradiol in the second study group (165±104.89 pg/ml) in comparison with control group (55.47±50.32 pg/ml) and in the second study group compared with the first study group (47.28±32.95 pg/ml) (p<0.05). But no significant differences were observed in plasma level of progesterone in the three groups (p>0.05).
Conclusion: Short-term administration of high doses buserelin increase plasma estradiol level, while has no effect on the plasma progesterone level.
Key words: Buserelin, Progesterone, Estradiol, Adult rat.
P-28
A comparative study on the effect of face-to-face or group education during the pregnancy period on sexual function of the couples under coverage of selected clinics in Isfahan in 2013
Mohammadi M1, Khani MB1, Bahadoran P2.
- Department of Nursing and Midwifery, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Nursing and Midwifery, Isfahan University of Medical Sciences, Isfahan, Iran.
Introduction: Pregnancy can conflict with sexual function that can be affected by physical and psychological changes during Pregnancy.
Materials and Methods: In this quasi-experimental pre-post-test study, 64 couples with pregnant women were selected and randomized in two groups in Isfahan. The data were collected via the triangulation of FSFI, BFSI and demographic characteristics questionnaires. SPSS -18 was used to analyze the data by descriptive and inferential statistics.
Results: No significant difference was found in the demographic characteristics between two groups. Education was effective on sexual function in two groups of women (p<0.001), but no significant difference was found between two groups (p=0.61). Also education was effective on sexual function of the men in both groups (p<0.001), and there was a significant difference between the two groups (p=0.003). Meanwhile, there was no significant difference between couples regarding the education (p=0.104).
Conclusion: The results of the study showed that type of education plays a role in improvement of sexual function in pregnancy. In addition, sex education is effective on prevention of sexual disorders in pregnancy. Therefore, having a special approach toward sex education classes during pregnancy is inevitable for the health providers, particularly midwifery professionals.
Key words: Group education, Face to face education, Sexual function, Pregnancy, Iran.
P-29
Assisted reproductive technology outcomes in couples with hepatitis virus infection
Mohsenzadeh M, Omidi M, Nabi A, Khalili MA.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, iran.
Email: mohsenzade@sums.ac.ir
Introduction: Currently, many hepatitis positive infertile couples attend assisted reproductive technology (ART) program, so there is concern as this viral infection may be deleterious to human fertility. Some have suggested that hepatitis virus have an adverse effect on pregnancy outcomes.
Materials and Methods: The study was conducted in two groups of patients: A) seropositive for hepatitis B virus (HBV) and hepatitis C virus (HCV) (n=46); and B) patients lacking these viruses as a control group (n=32). Patients included couples seeking ART from January 2010 to October 2014. ART cycle characteristics and clinical outcomes were assessed between two groups.
Results: Demographic characteristics and age were similar between two groups. The mean±SD age was 30.19±5.54 and 34.31±6.86 years in female and male, respectively. Regarding to oocyte number and embryo score, there was no significant differences between groups A and B. The rate of pregnancy was, however, significantly higher in group B in comparison with A (p=0.03).
Conclusion: Although, the data on embryo formation was similar in both groups of infertiles, but the pregnancy outcomes were noticeably reduced in infertile patients with hepatitis virus infections.
Key words: Hepatitis virus infection, ART, Pregnancy outcome.
P-30
The influence of the Corpus Luteum (CL) on hormonal and biochemical metabolites composition of follicular fluid from different sized follicles and their relationship to serum concentrations in dairy cows
Moradi kor N.
Research Center of Physiology, Faculty of Medicine, Semnan University of Medical Sciences, Semnan, Iran.
Email: moradikor@semums.ac.ir
Introduction: Metabolic changes in blood serum may be rejected in the biochemical composition of follicular fuid (FF) and could indirectly influence oocyte quality. In addition, the levels of hormonal and biochemical metabolites in the FF were related to follicular size and interestingly to the presence or absence of a corpus luteum. The purpose of this study were to examine the influence of the corpus luteum on hormonal and biochemical metabolites composition of follicular fluid (FF) harvested from different sized follicles and their relationship to blood serum in dairy cows.
Materials and Methods: Ovaries were recovered from 30 female adult cows (Holstein Friesian) 4-7 years of age with clinically normal reproductive tracts after slaughtering. Blood samples were collected from the jugular vein before slaughter from each cow. The stage of the cycle in the cows slaughtered was diestrus determined post mortem. The ovaries collected per cow were classified with corpus luteum (CL+) and without corpus luteum (CL-). Visible follicles on the surface of the ovaries were classified, based on their diameter, into (I) small (3-5 mm), (II) medium (6-9 mm) and (III) large (10-20 mm) categories. Follicular fluid was aspirated from different sized follicles in CL+ and CL- ovaries. Serum and FF samples were analyzed for hormones (estradiol-17β, progesterone, testosterone, T3 and T4) and biochemical metabolites (glucose, cholesterol, triglyceride, total protein, albumin and globulin).
Results: Results showed that the FF concentration of estradiol-17β, progesterone and testosterone in different size follicles categories (small, medium and large follicles in CL+ and CL- ovaries) were significantly higher (p˃0.05) when compared with the serum. The FF concentration of estradiol-17β, testosterone, glucose and cholesterol in same follicle size categories of CL- ovaries were significantly higher (p˃0.05) when compared with CL+ ovaries. In the present study, the serum concentration of glucose, cholesterol and triglyceride were significantly higher (p˃0.05) when compared with the fuid from different sized follicles categories in CL+ and CL- ovaries. The differences between follicle size categories in CL+ ovaries were only significant for concentration of estradiol-17β and in CL- ovaries were significant for concentrations of estradiol-17β, glucose and triglyceride.
Conclusion: According to the results of the present study, the levels of the biochemical metabolites in the FF were related to follicular size and interestingly to the presence or absence of corpus luteum.
Key words: Dairy cow, Follicular fluid, Corpus luteum, Hormone, Metabolite.
P-31
Health related quality of life in women with polycystic ovary syndrome
Moridi M1, Rahebi SM1, Seyf Zadeh Z2.
- Guilan University of Medical Sciences, Rasht, Iran.
- Alzahra Hospital, Rasht, Iran.
Introduction: Polycystic ovary syndrome (PCOS) is associated with symptoms that affect psychological wellbeing and health-related quality of life (HRQoL).
Materials and Methods: This was a retrospective case-control study which was conducted on 116 women from April-August 2014 in Rasht, Iran. Cases were 60 newly diagnosed women with PCOS, according to the Rotterdam criteria while the controls were 60 healthy women and groups were matched on sociodemographics characteristics. Data needed to determine health-related quality of life were collected via convenience sampling using modified PCOS quality-of-life questionnaire (MPCOSQ) that contains 6 subscales: emotional disturbances, hirsutism, infertility, weight, menstrual and acne. Data collected were analyzed using statistical T-test, chi square and Kolmogorov-Smirnov.
Results: Finding demonstrated that groups were matched in age, body mass index, marital status, occupation, educational status and socioeconomic status. The mean HRQoL score was significantly lower among women with PCOS (20.96±7.4) than among controls (29.8±4.5) (p<0.001). The mean scores of all subscales in cases (32.01±13.56 for emotional disturbances, 23.34±9.35 for hirsutism, 12.02±6.6 for infertility, 21.25±10.16 for weight, 16.89±6.71 for menstrual and 20.27±7.25 for acne) were significantly lower than controls (47.53±8.28 for emotional disturbances, 31.81±6.68 for hirsutism, 25.83±4.19 for infertility, 20.23±7.70 for weight, 21.15±7.27 for menstrual and 24.26±6.17 for acne) (p<0.001).
Conclusion: According to the results of this study, the average of HRQoL and all the six subscales of MPCOSQ in the case group was significantly lower than in the control group. The psychological implications of PCOS are easily underestimated and have been largely ignored. Clinicians has a pivotal role in recognizing these concerns and implementing therapy to improve quality of life in women with PCOS.
Key words: Polycystic ovarian syndrom, Quality of life, Infertility.
P-32
Counseling during pregnancy and women with precedent infertility
Naeimi N.
Department of Biology, Faculty of Science, University of Sistan and Baluchestan, Zahedan, Iran.
Email: nnaeimi@rocketmail.com
Introduction: Delivery is physiological phenomenon and among the beautiful events in the life of every woman and yet stressful reality. In the present era considering the increasing of caesarean sections, it require using consulting services for informed, correct decisions.
Materials and Methods: In this descriptive-analytical study, 271 pregnant women, completed a researcher-made questionnaire including, demographic characteristics (age and precedent of infertility) and kind and rates of counseling. To analyze the obtained data, chi-square test was used through applying SPSS19 software.
Results: Our results showed that 7 pregnant women (2.6%) had a precedent of infertility while 259 pregnant women had not (95.6%). In total 53 (19.2%) subject did consulting during pregnancy and 213 (80.8%) did not. From these, 6 women with precedent infertility (85.7%) and 47 (14.3%) without, perform counseling during pregnancy. There was a significant relationship between counseling during pregnancy and fertility (p<0.05).
Conclusion: The results showed that women with precedent infertility because of the importance of pregnancy and because of the history of information which were obtained during the course of treatment were more acquainted with the role of consultant, and therefor used consultant services mostly during pregnancy. Ferthermore, considering to the high prevalence of elective cesarean, counseling during pregnancy can informe the pregnant women about the advantage of normal delivery, causing the reduction in cesarean section prevalance.
Key words: Counseling, Precedent infertility, Pregnancy termination.
P-33
Overweight and obesity of patients: Blind spot in assisted reproductive technology treatment
Naghibzadeh M.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: naghib330@yahoo.com
Introduction: The effects of lifestyle on health were investigated via several studies. But, the impacts of overweight and obesity on fertility and reproductive system are poorly understood. Overweight and obesity may reduce fertility rate via reducing the IVF success rate in both women and men undergoing treatment via assisted reproductive technology (ART). Improve lifestyle, including physical exercise and weight loss program, may improve fertility and ART outcome.
Materials and Methods: The results of the related epidemiological and randomised control trials (RTCs) sited on Pubmed and ISI database were selected as references.
Results: The results indicated that the patients with a BMI above 30 have about 68% less chance to have a live birth following their first ART cycle compared with women with a BMI less than 30. Also, overweight and obesity of the patients were associated with both lower implantation and more risk for cycle cancellation. A correlation between obesity and the risk of spontaneous abortion (22% increased risk) was observed in obese women compared with normal weight women. The body composition, specific fat mass, was adversely associated with the success of the IVF procedure. Finally, some studies indicate that weight loss is associated with a higher spontaneous pregnancy rate.
Conclusion: Lifestyle behaviours may have a significant impact on pregnancy rates in women with infertility problems. The results support the clinical recommendation of advising overweight and/ or obese women to lose weight prior to ART. Nevertheless, it seems that the prospective randomized controlled trials are required to prove efficient evidence-based guidelines for weight loss interventions in overweight and/ or obese women before the ART procedure.
Key words: Lifestyle, Obesity, Fertility, ART.
P-34
Role of nitric oxide in the developmental process of ovarian follicles in pregnant rats
Naseri Sh, Khanehzad M, Noori Moogehi SMH.
Department of Histology, Faculty of Medicine, Tehran University of Medical Sciences (TUMS), Tehran, Iran.
Email: naserishafighe@yahoo.com
Introduction: Nitric Oxide (NO)- as one of the smallest active products, involved in control of a number of physiological processes including cell growth, apoptosis, regulation of blood pressure, defense mechanism and especially in the reproductive process.
Materials and Methods: We used 40 Wistar pregnant rats between 200 and 250 grams in weight and aged eight weeks. Based on observation of vaginal plug, pregnant mice were divided into five groups. The first group was received 2 mg/kg normal saline and the others were received respectively 200 mg/kg L-Arginine, 20 mg/kg L-NAME and a mixture of the same doses of L-Arginine and L-NAME on 3, 4 and 5th gestational days via intraperitoneal. The control group did not received any injection. Ovaries were removed on 18th gestational days, and after fixation and tissue preparation via staining by the routine H&E method, studied by Light microscopy.
Results: Comparing the groups using ANOVA, there was a significant difference in reducing the number of primary follicles and increasing atretic follicles in the L-Arginine group in comparision with the other groups. The histological changes were also observed in the L-Arginine group.
Conclusion: The study results showed that nitric oxide during pregnancy has damaging effects on ovaries and is recommended to be used with caution during pregnancy.
Key words: Ovarian follicles, Nitric oxide, L-Arginine, L-NAME.
P-35
Comparison of ovarian stimulation protocols base on AMH level in patients undergoing intracytoplasmic sperm injection
Nikmard F1, Hosseini E1, Bakhtiyari M1, Ashrafi M2, Aflatoonian A3, Aflatoonian B4, Aflatoonian R2.
- Anatomy Department, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
- Department of Endocrinology and Female Infertility at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Obstetrics and Gynecology, Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Yazd Centre for Stem Cell Biology, Research and Clinical Center for Infertility, Shahid Sadoughi University for Medical Sciences, Yazd, Iran.
Introduction: Controlled ovarian hyperstimulation (COH) plays important role in reproductive medicine because selection of appropriate ovarian stimulation strategy can improve assisted reproductive technology outcomes. Antimullerian hormone (AMH) is a predictor of ovarian response, which can help to select the best treatment strategies in women undergoing agonist and antagonist protocols to optimize safety and clinical pregnancy rates.
Materials and Methods: This study is a retrospective study conducted in a private assisted reproductive unite and a total of 243 patients with tubal factor infertility are selected.
Results: In both of GnRH agonist and antagonist protocol, with increasing of AMH level, oocyte and good embryo number is increased. On the other hand in AMH˂1.1, ˃ 2.8 ng/ml levels, GnRH agonist leads to higher oocyte and embryo number that is significant. On the contrary, pregnancy rate with increasing of AMH level is not increased and the highest rate of pregnancy is observed in AMH 1.1-2.8 ng/ml levels that it observed with antagonist protocol.
Conclusion: Based on AMH levels we can predict assisted reproductive outcomes. In three range of AMH levels, GnRH agonist protocol can lead to better results. In the women with poor prognosis and low and high levels of AMH, it should focus on improving results with increasing of endometrial receptivity or embryo quality.
Key words: Antimullerian hormone, Ovarian response, ovarian stimulation protocols.
P-36
Prenatal exposure to a single dose of testosterone leads to appearance of polycystic ovary syndrome in female rat’s offspring in adulthood
Noroozzadeh M1, Ramezani Tehrani F1, Zahediasl S2, Piryaei A3, Azizi F2.
- Reproductive Endocrinology Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
- Endocrine Physiology Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
- Department of Biology and Anatomical Sciences, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Introduction: Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women, affecting 8-12% of reproductive-aged women. PCOS is associated with reproductive and metabolic disorders, including infertility, hyperandrogenism, luteinizing hormone hypersecretion, polycystic ovaries, insulin resistance and dyslipidaemia, with an increased risk of cardiovascular disease and type 2 diabetes in those affected. PCOS is known as a genetic disease. Beside genetic factors, environmental factors may contribute to appearance of this syndrome.
Materials and Methods: Pregnant rats were divided into two groups, experimental and control groups (n=10 in each group). Experimental group were subcutaneously injected with 5 mg free testosterone on gestational day 20 and control rats received only solvent at the same time. The female offspring of these mothers were examined for the functioning of their reproductive system in adulthood.
Results: Levels of testosterone and luteinizing hormone and the luteinizing hormone/follicle-stimulating hormone ratio were increased in prenatally androgenized offspring compared with control rats (p<0.05). The numbers of preantral and antral follicles in the ovaries of prenatally androgenized offspring were also increased compared with control rats (p=0.07 and p<0.01, respectively). The number of corpora lutea was decreased in prenatally androgenized offspring compared with control rats. Cystic follicles were observed in the ovaries of prenatally androgenized offspring.
Conclusion: Prenatal exposure to a single dose of testosterone during the critical period of fetal development leads to appearance of PCOS phenotype in female rats with minimal morphological disorders in their reproductive system in adulthood. Production of a functional rat model that resembles many features of PCOS may contribute to a better understanding of this syndrome.
Key words: Fetal life, Testosterone, Polycystic ovary syndrome, Rat.
P-37
Advanced paternal age does not influence the outcomes of ART cycles
Omidi M, Halvaei I, Khalili MA, Ghazali Sh, Tahajjodi S, Sabeti P.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: omidi.marjan@ymail.com
Introduction: The reports in assisted reproductive technology (ART) cycles noted that the fertilization rates were significantly decreased in men over 50 years of age. However, data have shown that there was no adequate substantiation to prove the effects of paternal age on fertility outcomes.
Materials and Methods: In a retrospective study, the outcome of intracytoplasmic sperm injection (ICSI) in ART cycles in two groups of patients was studied. Patients regarding etiology of infertility were divided into two groups: A) male factor infertility (n=47), and B) female factor infertility (n=16). Sperm parameters, ART cycle characteristics and clinical outcomes were assessed between two groups.
Results: There were no significant differences for rates of sperm count and morphology between two groups, but rate of spermatozoa with progressive motility were higher in group B in comparison with A (p=0.002). The rates of high quality embryos, pregnancy and live birth showed no significant differences between the groups (p=0.4, p=0.4, p=0.2, respectively).
Conclusion: It appears that the paternal age has no detrimental effect on rates of pregnancy and live birth in ART program.
Key words: Paternal age, ART, Live birth.
P-38
Effect of non-polar extract of Phaleria macrocarpa (Mahkota dewa) on serum testosterone level and libido behavior in male rats
Aziz Dollah M1, Parhizkar S2, Ferdaus Binti N1.
- Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University Putra, Malaysia.
- Medicinal Plants Research Centre, Yasuj University of Medical Sciences, Yasuj, Iran.
Introduction: Phaleria macrocarpa (PM) has been claimed to overcome male fertility problems related to reduction of testosterone level.
Materials and Methods: Non-polar hexane extract of PM was prepared using soxhlet extraction technique, dried to powder using rotary evaporator and been kept for later use. In total 30 male adult Spraque Dawley rats weight about 250 gr were divided into five groups. For libido study, 30 female rats weight about 250 gr each were used for the purpose of mounting latency and mounting frequency study. The male rats were kept in individual cage while for female rats, there were five rats for one cage. Three groups of rats were given three different concentration of PM non-polar extract [High dose (60 mg/kg), Medium dose (12 mg/kg) and Low dose (6 mg/kg)]. One group was given tween 20 solution (negative control) and another group was given commercial testosterone hormone (positive control) orally for seven weeks. At the end of experiment period each male rat was introduced to one female rat to determine the libido behavior. Blood sample was then collected using cardiac puncture and the testosterone level was determined by radioimmunoassay kit (TESTO-CTK P3093).
Results: Administration of non-polar hexane extract of PM showed no significant changed for the serum testosterone level and mounting latency of the rats. While, there was a significant change (p<0.05) in the mounting frequency in different groups; 6, 14, 14, 4 and 7 times for the negative control, low dose, medium dose, high dose, and positive control respectively.
Conclusion: PM non-polar hexane extract did not show a potential value as an alternative way to improve the sexual strength. However it showed a potential for the improvement of the mounting frequency.
Key words: Phaleria macrocarpa (PM), Testosterone level, Libido behavior.
P-39
Effect of vitamin C on sperm parameters and serum malondialdehyde levels in mice fallowing treatment with sodium arsenite
Pirozmanesh H, Soleimani Mehranjani M, Naderi Noreini S.
Faculty of Science, Arak University of Medical ASciences, Arak, Iran.
Email: hp457@yahoo.com
Introduction: Arsenic as an environmental toxicant is able to exert malformation in male reproductive system by inducing oxidative stress. Ascorbic acid with potent antioxidant property is able to restrict oxidative stress.
Materials and Methods: In this experimental study, 24 NMRI mice were divided into four groups (n=6): control, sodium arsenite (7 mg/kg), ascorbic acid (150 mg/kg) and ascorbic acid + sodium arsenite. Oral treatment was performed for five weeks. At the end, left cauda epididymis was removed and used to analyze sperm viability, morphology, motility and sperm tail length. Serum MDA levels, as lipid peroxidation index, was measured by spectrophotometeric method. Data were analyzed using one way ANOVA and means difference was considered significant at p<0.05.
Results: The mean of sperm viability, morphology, motility and sperm tail length significantly decreased in sodium arsenite group compared to control (p<0.04). A significant increase in the serum MDA levels was found in sodium arsenite group compared to control (p<0.05). In ascorbic acid+ sodium arsenite group, a significantly reversed in adverse effect of sodium arsenite on these parameters was observed when compared with sodium arsenite group (p<0.02). In addition sperm viability, motility and sperm tail length was significantly increased in the mice treated with ascorbic acid alone in comparing to the control ones (p<0.001). Also, a significant reduction in MDA levels was found in ascorbic acid group compared with control group (p<0.001).
Conclusion: Ascorbic acid could compensate the adverse effects of sodium arsenite on viability, morphology, motility, sperm tail length and serum MDA levels in adult mice. Hence, this study suggests that ascorbic acid may improve the male fertility by improving sperm parameters.
Key words: Sodium arsenite, Ascorbic acid, Viability, Motility, Sperm tail length.
P-40
The predictors of spiritual growth and interpersonal relations in infertile couples referring to the Infertility Center, Al-zahra Hospital, Tabriz, Iran.
Rahimi M, Sehhatie Shafa'I F, Mirghaforvand M.
Tabriz University of Medical Sciences, Tabriz, Iran.
Email: marieh.rahimi@yahoo.com
Introduction: In all cultures, involuntary childlessness is recognized as a crisis that has the potential to threaten the stability of individuals, relationships, and communities. Every society has culturally approved solutions to infertility involving, either alone or together with alterations of social relationships (e.g., divorce or adoption), spiritual intercession (e.g., prayer or pilgrimage to spiritually powerful sites), or medical interventions.
Materials and Methods: This study is a descriptive cross-sectional study on 322 infertile couples referring to the Infertility Center at Al-zahra Hospital in Tabriz. Samples were selected through simple random sampling method. Questionnaires used in the study were demographic data questionnaire and Lifestyle Profile II (HPLP-II) questionnaire with 52 questions. The multivariate linear regression analysis method was used for defining the social-individual predictors of Spiritual Growth and Interpersonal relations. Data were analyzed by SPSS Win/13.
Results: The findings showed that the mean (SD) score of Spiritual Growth and Interpersonal relations in couples was 2.6 (0.5) out of 1-3 grade range. The variable of job, family member, history of contraception, reason of infertility, and education level were the predictors of Spiritual Growth and Interpersonal relations.
Conclusion: According to the results of the study, the mean score of Spiritual Growth and Interpersonal relations in infertile couples is average, therefore we should focus on predictive variables for recovering the infertile couples’ Health-promoting behaviors status.
Key words: Health-promoting behaviors, Spiritual Growth, Interpersonal relations.
P-41
Comparing in-vitro maturation of oocytes in presence of mature or immature cumulus cells in mice
Rahiminia T, Faramarzi A, Khoradmehr A, Khalili MA.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: tahere_1980@yahoo.com
Introduction: Cumulus cells (CCs) are somatic cells which are coupled with oocyte during maturation. During process of maturation, the secretions by CCs influence oocyte to maintain the functional competence.
Materials and Methods: Superovulated mice were killed and ampulla was ruptured to release cumulus oocyte complex (COCs) in IVM culture media. COCs were incubated for 30 min before removing oocytes. Then, denuded mature oocytes were removed and remaining mature CCs were collected for culturing germinal vesicles (GVs) for exp II. After dissecting ovaries, denuded GVs were considered as control group and put in IVM medium. In exp I, collected GVs with intact immature CCs were put in IVM medium and observed for maturation after 24 and 48 hr. In exp II, GVs were cultured with mature CCs and maturation was checked as mentioned for exp I group.
Results: The rate of maturation was (77.36±14.4) in control group. In exp I and exp II, the rate of maturation was (91.32±22.5) and (63.33±7.4) respectively. Difference in maturation rate was significant between two groups of exp I and exp II (p=0.04) at 24 hr. However, maturation rate did not increase after 24 hr. The average of maturation rate in metaphase I (MI) oocytes was as low as about 15% in different groups at 24 hr and did not increase by 48 hr. Degeneration rate was increased from time 0-48 hr in exp II more than this rate in the other two groups.
Conclusion: Presence of mature CC did not improve maturation of GVs comparing to exp I with immature CCs, or control group as conventional IVM.
Key words: In vitro maturation, Cumulus cells, Mice.
P-42
Investigating the effect of culture pH, on Chinese hamster ovary (CHO) cells growth and recombinant human chorionic gonadotropin (hCG), expression
Rahnama M, Sanati MH, Amiri Yekta A, Gourabi H.
Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: mhsanati@yahoo.com
Introduction: The demand for production of recombinant therapeutic proteins by mammalian cell lines, such as Chinese hamster ovary (CHO) cells, continues to grow. Significant achievements in process optimization including development of cell culture strategies for large-scale, cost effective production have been made. One of the key parameters, that has effect on mammalian cell growth, metabolism and productivity, is pH. Human chorionic gonadotropin (hCG), is a member of pituitary glycoprotein hormone family, which has an important role in regulating human reproductive functions and is widely used in assisted reproductive technologies (ART).
Materials and Methods: Equal number of rCHO cells were cultured in four DMEM media with different pH ranging from 6.8-7.7 (6.8, 7, 7.3 and 7.7). The cells were harvested at confluence and viable cell concentration was determined by trypan blue exclusion. The rhCG production was assessed using SDS-PAGE, Bradford and Western blotting techniques. ImageJ software was used for quantitating Western blotting results.
Results: Results demonstrated that maximum viable cell concentration was at pH=7. Bradford assay showed that total protein concentration reached its maximum level at pH=7 and its minimum at pH=6.8. hCG expression was confirmed by SDS-PAGE and western blotting methods. According to ImageJ analysis, maximum hCG expression level was found at pH=7, and minimun expression at pH=7.7 but no significant difference was found at pH=7.7 and pH=6.8. Overall, pH=7 was assessed as optimum pH for culturing rCHO cells.
Conclusion: Several strategies including culture condition optimization, have developed to improve productivity of a recombinant cell line. Findings suggest that optimizing simple parameters such as culture pH, had the potential to increase the viability and productivity of recombinant CHO cells.
Key words: Chinese hamster ovary (CHO) cell, Optimization, pH, Human chorionic gonadotropin (hCG), Assisted reproductive technology (ART).
P-43
Noscapine induced cell death in human endometriotic epithelial cells
Rashidi Z1, Khazaei M2, Khazaei MR2.
- Department of Anatomy, Faculty of Medicin, Tehran University of Medical Sciences, Tehran, Iran.
- Department of Anatomy, Faculty of Medicin, Kermanshah University of Medical Sciences, Kermanshah, Iran.
Introduction: Endometriosis is the presence of a functional endometrial tissue outside the uterine cavity and defines as cancer like model. Noscapine is a safe cough suppressant which has been introduced as cancer suppressor.
Materials and Methods: Human endometriotic biopsies (n=7) were digested by enzymatic method (collagenase, 2 mg/ml). Epithelial glands were collected by sequential filtration through nylon meshes (70 and 40 µm). The cells were divided to five groups: control and 10, 25, 50 and 100µM concentration of noscapine and cultured for 24, 48 and 72 hr.Viability of cells was assessed by MTT assay; cell morphological analyses with Acridine orange (AO)-ethidium bromide (EB) double staining also cell death was assessed by TUNEL assay.
Results: Viability of endometrial epithelial cells were decreased in 10, 25, 50 and 100 µM noscapine concentration (84.1%, 80.8%, 72.7%, 67.1%) compared to control group (91.0%) in 48 hr respectively. Cell death increased in high concentrations noscapine and were increased TUNEL positive cells in 10, 25, 50 and 100 µm noscapine concentration (9.04%, 13.07%, 15.15% and 17.82%) respectively compared to control group (4.96%) in 48 hr (p<0.05).
Conclusion: Noscapineas inducer cell death decreased endometriotic epithelial cells viability and decreased apoptotic index in dose dependent manner. It can suggest for endometriosis treatment.
Key words: Apoptosis, Epithelial Cell, Endometriosis, Noscapine.
P-44
The effect of chronic prenatal stress on insulin secretion from Langerhans isolated islets in male offspring rats
Rostamkhani F1, Zardooz H2, Shirvani H3.
- Young Researchers and Elite Club, Yadegar-e-Imam Khomeini (RAH) Branch, Islamic Azad University, Tehran, Iran.
- Neuroscience Research Center and Department of Physiology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
- Exercise Physiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Introduction: A large number of studies have reported associations between prenatal stress and offspring lifetime consequences. Chronic gestational stress alters maternal glucocorticids and subsequently disturbs intrauterine environment which may lead to metabolic disorders in the offspring.
Materials and Methods: In this study the effects of gestational 8 and 20 days foot-shock and psychological stress on body weight, plasma corticosterone, insulin, glucose, concentrations and also insulin secretion from isolated pancreatic islets of rats' offsprings were examined. Stress was induced by Communication Box twice a day (1 h/session) for 8 consecutive days beginning on E8 in 8-day stressed group and for 20 consecutive days beginning on E1 in 20-day stressed group.
Results: The results obtained from this investigation indicate that 8 and 20-day chronic foot-shock stress arises maternal plasma corticosterone concentration. Prenatal stress induces lower birth weight and body weight gain in offspring. Insulin secretion from isolated pancreatic islets of offspring in 8 and 20-day foot-shock stress groups in presence of 8.3 and 16.7 mM glucose increased as compared to the control and psychological groups. Furthermore, prenatal stressed offspring had significant elevation in plasma glucose concentration without marked alteration in plasma insulin and corticosterone concentrations.
Conclusion: These data suggest that prenatal stress could result in impaired glucose metabolism in the offspring which is independent of timing of the stress exposure.
Key words: Prenatal stress, Corticosterone, Insulin, Glucose, Langerhans isolated islets.
P-45
In vitro culture of vitrified mouse ovarian tissue derived preantral follicles in two and three dimensional systems
Sadr SZ1, Ebrahimi B1, Shahhoseini M2, Fatehi R1, Favaedi R2, Maroufizadeh S3.
- Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Epidemiology and Reproductive Health at Reproductive Epidemiology Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: Cryopreservation is the best choice to preserve fertility in patients exposed to premature ovarian failure. Designing an appropriate system for in vitro follicle culture that resembles in vivo conditions is really valuable and could improve the usage of cryopreserved tissue.
Materials and Methods: Ovaries of 12-day-old female NMRI mice were exposed to EG and DMSO combination (7.5% and 15%) in two steps for 15 and 30 minutes respectively, then cryopreserved by needle immersed method (NIV). Afterwards, middle sized preantral follicles (110-130 µm) were mechanically isolated and were distributed into two different groups: two dimensional culture system (2D) and three dimensional culture system (3D; alginate encapsulation). Both groups were cultured for 12 days in α-MEM that supplemented with 10 mIU FSH, 1% ITS, 50 ng/ml activin A and 5% FBS. Finally, follicular morphology, survival, growth rate and also quantitative expression of oocyte maturation genes (Gdf9, Bmp15 and Bmp6) were studied on the first and last days of culture.
Results: At the end of culture period, although follicular morphology in 3D culture system was better preserved as compared to the 2D one, survival rate of cultured preantral follicles was significantly (p<0.05) lower than in 2D group. Expression analysis of oocyte maturation genes indicated a reduction process in both groups during 12 days of culture. In the last day of culture period, genes expression level in 3D culture system was significantly lower than 2D group.
Conclusion: Although the better survival rate was seen in 2D culture system, morphologic characteristics including antrum formation and follicle development was extremely better preserved in 3D culture system. Overall, as three dimensional culture system mimic in vivo conditions, so could be used as a more appropriate system for follicle culture.
Key words: Ovarian tissue, Preantral follicle, Two dimensional culture system, Three dimensional culture system, Vitrification.
P-46
Routine use of EmbryoGlue® as embryo transfer medium does not improve the ART outcomes
Safari S1, Razi MH1, Safari S2, Razi Y3.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Qom University of Medical Sciences, Qom, Iran.
- Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: The assisted reproductive technology (ART) success rates have been significantly improved over last decades; however, embryo implantation still remains a major limiting factor. The composition of an embryo transfer (ET) medium is important for interaction between embryo and endometrium at the time of implantation. Some modifications to the embryo culture media are developed to mimic the in vivo conditions. One of the examples for that is supplementation of ET media with hyaluronan (HA, hyaluronic acid), a major glycosaminoglycan in uterine fluid, which assumed to improve the process of implantation.
Materials and Methods: A cohort of total 229 patients was retrospectively enrolled for the present study. They were subjected for ET on day 2 either in EmbryoGlue® (n=117) as study group or in conventional ET medium with low concentration of HA as control group (n=112).
Results: Patients in the both groups, in regards to the mean level of day 3 FSH, the etiology of infertility, the history of implantation failure and the rate of good quality embryos showed similar characteristics. There were no significant differences between two groups in terms of clinical and ongoing pregnancies, implantation, delivery and live birth rates. In spite of a decreased abortion and increased multiple pregnancy rates in the study group compared to the control group (15.8 vs. 19% and 20.6 vs. 15.6 respectively), the differences were not statistically significant.
Conclusion: Routine use of EmbryoGlue® as a HA enriched ET medium for cleavage stage embryos does not have advantage to the conventional one for infertile patients undergoing ART.
Key words: Hyaluronan, EmbryoGlue®, Embryo transfer, Pregnancy outcome.
P-47
The relation between vitamin C in follicular fluid with the morphology of oocyte and the quality of embryo in IVF patient
Saffari S1, Bahadori MH2, Sharami SH3, TorabZadeh P1, Goudarzvand M1.
- Department of Developmental Biology, Karaj Branch, Islamic Azad University, Karaj, Iran.
- Cellular and Moulicular Research Center, Guilan University of Medical Sciences, Rasht, Iran.
- AlZahra Reproductive Health and Medical Research Center, Rasht, Iran.
Introduction: Oxidative stress and the inappropriate effect of reactive oxygen in the body or laboratory circumstances can decrease the quality of embryo and sexual cells. Ascorbic acid is a natural antioxidant which has a protective effect in the body.
Materials and Methods: This is a cross-sectional analytical study which was conducted on 50 women with IVF referred to Al Zahra Hospital, Rasht, Iran. 20-45 years old women with infertility caused by tubal dysfunction, ovarian factors or male factors were included. Patients underwent same protocol for stimulating; the ovulation and injection of 10,000 units of hCG. After 36 hr, follicles were suctioned and matured oocytes were separated for fertilization. Vitamin C was assessed biochemically and the morphology of oocyte and the quality of embryo were evaluated by by inverted optical microscope.
Results: Among patients 583 oocytes and 275 embryos were assessed. There was no significant relation between, age, BMI and duration of infertility with vitamin C (p>0.05). Also there was no significant relation between vitamin C, the maturity of oocyte and the quality of embryo (p>0.05). But MII oocytes were more in patients with vitamin C less than 1 in comparison with ≥1 (p=0.038). Also the mean 2PN embryos in 0-0.05 level of vitamin C was higher than other groups but no significant relation was noted (p=0.719).
Conclusion: According to our results vitamin C at special level can improve the morphology of oocytes and the quality of embryos.
Key words: Infertility, Follicular fluid, IVF, Vitamin C, Oocyte, Embryo, Antioxidants.
P-48
Polscope analysis of meiotic spindle and zona pellucida birefringence of metaphase ΙΙ oocytes in polycystic ovarian syndrome patients
Safian F1, Khalili MA1, 2, Ashourzadeh S2, 3, Omidi M2.
- Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Afzalipoor Clinical Center for Infertility, Afzalipoor Hospital, Kerman University of Medical Sciences, Kerman, Iran.
Introduction: Polycystic ovarian syndrome (PCOS) is one of the most common causes of infertility in women of reproductive age. Currently, one of the best therapeutic options for PCOS patients is ICSI. Moreover, IVM can be a useful technique for women with PCOS who are at the risk of ovarian hyperstimulation syndrome (OHSS). On the other hand, the oocyte quality can be a determining factor for outcome of ICSI cycles.
Materials and Methods: This prospective study included immature oocytes (30 GV and 5 MI) undergoing IVM, and MII oocytes obtained from PCOS patients (29.64±5.31 years) in ICSI program. Using a PolScope, the presence of MS and ZP birefringence was assessed in both in vivo-matured (n=32) and matured oocytes after IVM (n=24). Oocytes were classified as high birefringent (HB) ZP and low birefringent (LB) ZP. Furthermore, the rates of fertilization and embryo development were evaluated.
Results: The maturation rate was 68.5% after IVM. Analysis revealed that the percentage of a HB ZP was significantly higher in the IVM oocytes than in vivo-matured ones (58.3% vs. 31.2%, p=0.04). There was insignificant relationship between spindle detection and either in vivo-maturation or IVM oocytes (p=0.53). Likewise, there were similar outcomes for the rates of fertilization and embryo development after ICSI between two groups (p=0.80 and p=0.13, respectively).
Conclusion: Clinical IVM is a safe technology for the maturation and maintenance of oocytes integrity. Furthermore, the use of non-invasive PolScope is recommended for the detection of healthy oocytes in ICSI.
Key words: PCOS, IVM, PolScope, ZP birefringence, Meiotic spindle.
P-49
History and application of public bank of umbilical cord blood
Seyfipoor Naghne E1, Bolorytabar F1, Khoshravash S2.
- Department of Nursing and Midwifery, Islamic Azad University, Felavarjan Branch, Isfahan, Iran.
- Department of Nursing and Midwifery, Isfahan University of Medical Sciences, Isfahan, Iran.
Introduction: Umbilical cord blood is rich of hematopoietic stem cells. Today, umbilical cord blood constitutes about 20% of stem cells transplant. 75% of umbilical cord blood banks in the world are public and the remaining are private (trade). 27 years after the first successful umbilical cord blood transplant, more than 555,000 umbilical cord blood units have been made available around the world after performing qualitative study in public bank of umbilical cord blood in order to treat patients who need hematopoietic stem cells transplant. The first program of public umbilical cord blood bank was implemented by the New York in 1991 and in Asia in 1999 in Japan. In Iran in late 2008, the Stem Cells Association has started its activity by receiving financial supports. In this type of storing, umbilical cord blood does not only belong only to the family and public organizations are responsible for paying related costs. Cells in this banking are kept safe and number of their alive cells remains unchanged during the freezing process. Samples saved in bank in terms of health indices such as viral and microbial contaminations are evaluated by colony measurement method. With regard to high prevalence of blood malignancies and thalassemia in Iran, it has been very important to establish public bank of umbilical cord blood and support it spiritually and materially.
Materials and Methods: Subjects of this review study are derived from articles and sites related to collecting, maintain and transplanting umbilical cord blood.
Results: Development of umbilical cord blood banks to develop transplantation centers and treat patients is of unavoidable necessities in the society health system. In societies that the government has ability to establish public bank and supports this bank, development of public banks has preference to development of private banks.
Conclusion: In year 2011, research and development department established public bank of umbilical cord blood in order to scientific and practical promoting and proceeded to amniotic membrane, standardization of tissue adhesive, production and standardization of amniotic extract and platelet extract by establishing bank. Collecting this bio garbage and transforming it to biologic bands, eye drop to improve cornea wounds and mixing hydrogen with amniotic membrane extract to treat wounds for therapeutic consumptions is economical and is effective to decrease therapeutic costs. Also fibrin glue is produced only via umbilical cord blood. Fibrin glue as cell carrier and tissue adhesive is widely used in surgery and cell therapy. Transplant of stem cells of umbilical cord has opened bright and promising horizons for medical society to treat patients with different chronic and acute disorders. So, saving umbilical cord blood finds high importance and its necessity feels further.
Key words: Umbilical cord, Blood bank
P-50
Protective effect of alcoholic extract of Garden cress (Lepidium sativum) seeds on the histopathological changes of epididymis and fertility in diabetic rats
Seyed Hoseini ES1, Nikzad H1, Amini Mahabadi J1, Haddad Kashani H2.
- Gametogenesis Research Center, Kashan University of Medical Sciences, Kashan, Iran.
- Anatomical Sciences Research Center, Kashan University of Medical Sciences, Kashan, Iran.
Introduction: Diabetes mellitus (DM) is a frequent and serious metabolic illness all over the world and plants have been a desirable source of medicine recently. Diabetes has unpleasant effect on male reproductive system and it may lead to male infertility. It causes erectile dysfunction and reduces ejaculate volume by affecting the health of small blood vessels and the small nerves that control ejaculation and also decrease libido by decreasing testosterone levels. Current study evaluated the possible protective efficiency of Garden cress (Lepidium sativum) seed extract on fasting blood sugar (FBS) and then assessed epididymal histopathologic changes in streptozotocine (STZ)-induced diabetic rats.
Materials and Methods: 50 male adult Wistar rats were randomly categorized into five groups (each 10 rats). Groups 1 were control placebo group receiving only 0.1 ml normal saline via gastric gavages, Group 2 as control diabetic rats received an intraperitoneally injection of STZ 60 mg/kg body weight. Rats with FBS >250 mg/dl were considered as diabetic. Group 3 were diabetic rats receiving insulin in dose 3U/100 g body weight and Groups 4 and 5 were diabetic rats that received 0.1 cc of 200 and 400 mg/kg‚ ethanol extract of Lepidium sativum seed by gavages daily. One day after the last gavages, rats were anesthetized by chloroform. Epididymis duct was removed from abdomen and weighed with a digital scale. Afterwards, samples were putted in Bouin's solution for histological measurement.
Results: Administration of 200 and 400 mg/ml doses of Lepidium sativum seed extract increased epithelium height and decreased interstitial volume density and fibro muscular thickness significantly. Also, volume density of epithelium, fibro muscular, lumen and interstitial were decreased significantly. Tubular and lumen diameter did not change significantly in different groups.
Conclusion: It seems Lepidium sativum seed extract has benefit as a supplementary protective agent against bad effects of diabetes on reproductive system in diabetic male.
Key words: Diabetes‚ Lepidium sativum seed extract, Epididymis‚ Streptozotocine‚ Insulin.
P-51
New insights into the effect of aflatoxin on infertility of animal models
Sheikhpour R, Jabbari S.
Yazd Medical Science Branch, Islamic Azad University, Yazd, Iran.
Email: R.sheikhpour@yahoo.com
Introduction: Aflatoxins are produced via Aspergillus flavus and Aspergillus parasiticus, species of fungi. Toxic and carcinogenic properties of Aflatoxin are determined. After entering the body, Aflatoxins may be metabolized by liver to a reactive epoxide intermediate or hydroxylated to become the less harmful. Nowadays, infertility properties of Aflotoxine have been known in studies.
Materials and Methods: Literature review was conducted using the following databases: SciELO, Lilacs, Medline, PubMed, Scopus and science direct from 2000-2014. The key-words used were Aflatoxin, infertility, fertility, animal, human.
Results: When Aflatoxin level increased in diet of male goats, glucose and total protein concentration were decreased in the testes and testosterone level was significantly reduced. The study suggests that exposure of male goats to dietary Aflatoxin will reduced testicular biochemical and testosterone with resultant depression in sperm storage capability and daily sperm production in the animals. Chronic Aflatoxin exposure in animals can result in impaired reproductive efficiency. One study showed that Aflatoxin decreased fertility, abortion, lowered birth weights and caused disturbances in hormonal metabolism in sheep. Another study showed that the fertility of treated mice with Aflatoxin at a daily dose of 50 µg/kg body weight was reduced drastically. Sperm concentration in the epididymis and sperm motility decreased whereas sperm abnormalities increased.
Conclusion: The results of studies showed that Aflatoxin can cause infertility in animal models, but these results need to be evaluated by more clinical trials.
Key words: Aflatoxin, Infertility, Animal.
P-52
ART cycles outcome in donor oocyte recipient couples: A case-control study
Tahajjodi S, Khalili MA, Nabi A, Omidi M.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Science, Yazd, Iran.
Email: s.tahajjodi@yahoo.com
Introduction: Oocyte donation is an acceptable alteration in women infertility treatment for different indications. Several factors including the age of the oocyte donor and recipient, oocyte and embryo quality and characteristics of endometrium can influence on ART outcome.
Materials and Methods: Outcome of 202 cycles, consist of 113 DO cycles and 89 AO cycles, were compared. Both DO and AO groups were divided to two subgroups according to fresh and frozen embryo transfer. First DO and AO groups included the fresh embryo transfer (fDO, fAO) and another DO and AO groups included the frozen embryo transfer (fzDO, fzAO). Comparison was performed in regard to chemical pregnancy, clinical pregnancy, implantation rate, live birth and abortion rate.
Results: DO (mean=28.29 years) and AO (mean=29 years) groups were significantly matched by age. No significant differences were observed between fDO and fAO groups and also between fzDO and fzAO groups with regard to chemical pregnancy, clinical pregnancy, implantation rate, live birth and abortion rate. But, there was a significant increase in oocyte number, MII oocyte number and embryo number in DO compared with AO groups (p<0.001).
Conclusion: Despite of increasing in total number of oocyte, MII oocyte and embryo, our findings showed that oocyte donation program has no impact on ART pregnancy outcome.
Key words: Oocyte donation, ART outcome, Women infertility.
P-53
Dose stress always has a negative effect on sperm cell?
Vaseghi-Dodaran H1, Zhandi M2, Daghighkia H1, Sobhani A3.
- Department of Animal Sciences, Faculty of Agriculture, Tabriz University, Tabriz, Iran.
- Department of Animal Sciences, College of Agriculture and Natural Resources, University of Tehran, Tehran, Iran.
- Department of Animal Sciences, Faculty of Agriculture, Mohaghegh Ardabili University, Ardabil, Iran.
Introduction: Cryopreservation of semen requires optimized condition to minimize the harmful effects of various stresses. Oxidative stressors are known to be major mediators of cell damages during cryopreservation. An important perspective is that free radicals are not exclusively beneficial or exclusively detrimental. Rather, they need to be maintained at appropriate levels to ensure physiological function, while preventing pathological damage. Recent studies showed that placing the cells in a controlled stress conditions could affect cell survival in later stages.
Materials and Methods: This article discusses some of the new approaches employed for improve spermatozoa before and after freezing-thawing. A review of recent bibliography was carried out in PubMed, Google scholar and SID by the use of relevant keywords.
Results: Osmotic, oxidative, hydrostatic and mechanical stressors before freezing induce expression a series of heat shock proteins (HSPs), decrease apoptotic sperm amount and increase live sperm during treatment and high mitochondrial activity observed in treatments. Cell's reaction to environmental stresses is depended on domain, concentration and acts time of this reactions.
Conclusion: Although cellular and sub-cellular mechanisms, supposedly contributing to these processes, require further research, the new principle may outline a completely new strategy in mammalian embryology, as well as cryopreservation of other cells and tissues with remarkable theoretical and practical consequences.
Key words: Cryopreservation, Sperm, Stress, Sublethal.
P-54
The effect of KIT ligand (KITL) and leukemia inhibitory factor (LIF) on maturation and apoptosis of follicles after vitrification of mouse ovary and three dimensional culture
Abdi Sh1, Salehnia M1, Hosseinkhani S2.
- Department of Anatomy, Tarbiat Modares University, Tehran, Iran.
- Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Introduction: In vitro ovarian follicle culture techniques not only provide a model for research into the mechanism of folliculogenesis but also, in combination with cryopreservation of ovarian tissue, it may have clinical applications in preserving of fertility.
Materials and Methods: This experimental study is carrying out on 7-days-old female mice (NMRI). In the first step, the ovaries were vitrified with a solution containing ethylene glycol and then their morphology, ultrastructure and apoptosis were compared with non-vitrified ovaries. In the second step, the non-vitrified and vitrified ovaries were cultured in base medium α-MEM supplemented with KL and leukemia inhibitory factor (LIF) for 7 days then their morphology, area, apoptosis, hormone assay were analyzed. In third step, mechanically isolated preantral follicles were cultured in two dimensional and three dimensional systems in α-MEM supplemented with 5% FBS, 100 mlU/ml, 1% ITS, 10 ng/ml rEGF and KL for 12 days then the ovulation were induced. At the end of culture survival and maturation rate, apoptosis and hormones were assayed. In the fourth step, the level of reactive oxygen species (ROS) and ATP, distribution of mitochondria and fertilization rate in MII oocyte were assessed.
Results: The results of first step showed that the morphology, ultrastructure and apoptosis of vitrified ovaries were similar to non-vitrified ovaries. The results of second step showed that the percentage of preantral follicles, ovarian area, production of hormone in the non-vitrified ovaries cultured in medium supplemented with KL were significantly higher and the level of caspase 3/7 was lower in comparision to other groups (p<0.001). The results of next step showed that the survival, maturation rates and production of hormone of follicles in KL supplemented in three dimensional system groups were higher than other groups. The level of caspase-3/7 activity was lower in three dimensional culture systems (p<0.05). The results of fourth step showed that the level of ROS and ATP in MII from in vitro culture of follicle had significant different with control in vivo group and the rate of embryo development were significantly higher in three dimensional culture system.
Conclusion: Our results demonstrated that the culture of ovarian tissue and three dimensional culture of ovarian follicle in combination of KL improved the growth and development of ovarian primordial follicle.
Key words: Vitrification, In vitro maturation, Apoptosis, Mitochondria, KIT Ligand (KITL).
P-55
Evaluating the levels of expression of TLR 9 in women with endometriosis and its comparison with normal endometria
Aflatoonian Ali1, Janan A2, Ramazanali F2, Moieni A1, 2, Aflatoonian R2.
- Tehran University of Medical Sciences, Tehran, Iran.
- Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: Endometriosis is a benign condition which endometrial glands and stroma appear outside the uterine cavity which presents by pelvic pain and infertility. Endometriosis is associated with changes in cellular and humeral immunity and impaired immune response, leading to inefficient removal of debris after a menstrual cycle. Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system. Based on recent studies TLRs are increased in endometriosis and initiate immune responses.
Materials and Methods: This case-control study assessed and compared expression of Toll like Receptor 9 in the endometrium of three type tissues: 1) Eutopic endometria of women with endometriosis 2) Ectopic endometria women with endometriosis 3) Endometrium of women without endometriosis. Eutopic and ectopic endometrial samples were taken from 10 patients with endometriosis (Case Group). Also endometrial samples were taken from 10 patients without endometriosis or infertility history whom operated for other gynecological cause. The level of TLR9 gene expression in ectopic samples, eutopic and controls were evaluated by Real Time-PCR. Patients were chosen randomly from Arash Hospital. Data was analyzed by SPSS.
Results: Real Time-PCR showed that TLR9 expressed in all three groups of eutopic, ectopic and control. According to statistical analysis, level of TLR9 was higher in ectopic group, but not significantly (p=0.13). There was no significant difference between eutopic and control groups.
Conclusion: Considering the role of TLR9 in the innate immune system, such as pathogen detection and set up a cascade of inflammatory response associated with cell proliferation and inhibition of apoptosis, this study approves the association between TLR9 and endometriosis. Because of lack of information about this new issue, we suggest further researches and more studies.
Key words: TLR9, Endometriosis, Inniate immune system.
P-56
Congenital uterine malformations: A widespread phenomenon among infertile women with recurrent miscarriages and IVF failure
Javam M, Ahmadi F.
Department of Reproductive Imaging at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: f_ahmadi@royaninstitute.org
Introduction: Uterine malformations are a various group of congenital uterine disorders found in women with recurrent abortions or IVF failure.
Materials and Methods: A narrative review was performed within articles published at “PubMed”, “Elsevier”, "EBSCO", original text books and etc. to reach the aim. Several unique high-quality images were provided in this article, using the archive of infertile patients referred to imaging department of Royan Institute.
Results: Congenital uterine anomalies are originated from development defects of mullerian ducts during fetal growth. They are also associated with higher incidences of infertility, preterm birth, intra uterine fetal death and etc. However, manifestations and severity of the obstetric/ gynecologic complications and treatment procedures vary depending on the type of anomaly. Thus, accurate diagnosis of uterine malformations and differentiation between various types of them, have a vital role in decision about treatment procedures and management of these patients. Several imaging modalities are used to investigate women suspected to have uterine anomalies. In this article, we described about various types of uterine malformations and imaging evaluation of them prior to IVF treatment cycles.
Conclusion: Congenital anomalies of the uterus are a major cause of recurrent miscarriage and pregnancy failure. Several imaging methods help midwives and obstetricians evaluate this group of infertile women to detect suspected uterine anomalies.
Key words: Congenital uterine malformation, Infertility, Recurrent miscarriages.
P-57
Study on the impacts of anti-histamine dimenhydrinate on testicular tissues and sperm production of male mature rat
Ahmadifar M1, 2, Vahidi-eyrisofla N3, Taghvaei F4, Jafarzade Rastin S4, Yousef Zadeh AR6, Daliri L5, Gharanfoli M1.
- Department of Biology, College of Science, University of Science and Culture, ACECR, Tehran, Iran.
- Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Biology, Damghan Branch, Islamic Azad University, Semnan, Iran.
- International Campus, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Epidemiology and Reproductive Health at Reproductive Epidemiology Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Scientific Promotion, Tehran, Iran.
Introduction: In this study we did a research about the effects of AHDH on testicular tissues and sperm production of rats. Dimenhydrinate (DH) is used as anti-nausea as well as an anti-gastric reflux in cases of nausea caused during travelling. It is easily taken orally and it will be excreted by the kidneys within 24 hr.
Materials and Methods: This survey is conducted in laboratory on experiment basis among 30 mature male rats categorized in 3 different groups; two experimental groups and one control group. The control group was watered normally for 60 days and the first experimental group was watered with 200 mg DH blended in their water equal to one kg of their body weight for 60 days. In the end, after the rats were anesthetized, testicles were removed, sectioned and painted with Hematoxilin and Eozine and were investigated.
Results: The obtained results have revealed that in the experimental groups, testicles weight decreased substantially in comparison to those of the control group (p<0.001). The reduction of the number of sperm cells within the experimental group, in comparison to those of the control group was significant (p<0.001). The reduction of the Leydig cell was very substantial in the second experimental group (p<0.001). The difference of the Sertoli cells was not noticeable in neither of the experimental groups.
Conclusion: DH has impact on testicular tissues and weakens their mechanism.
Key words: Dimenhidranate, Testis.
P-58
Investigating association of G103T polymorphism of Coagulation factor XIII and recurrent pregnancy loss
Ahmadpour Nazm S1, Bonyadi M2, Taghavi S3, Ajamian F1.
- Biology Group, Science faculty, Guilan University, Rasht, Iran.
- Biology group, Science Faculty, Tabriz University, Tabriz, Iran.
- Department of Obstetrics and Gynecology, Medical faculty, Tabriz University of Medical Sciences, Tabriz, Iran.
Introduction: Recurrent miscarriage (RM) occurs in 1-3% of couples that attempting to bear children. Thrombophilia is one of the suspected cause of recurrent miscarriage. At the end of coagulation cascade there is Factor XIII that makes blot stable. The polymorphism G103T of factor XIII gene is the most common polymorphism that affects FXIII’s activity.
Materials and Methods: The study groups consisted 50 patients with two or more consecutive miscarriage. The control group included 50 women with at least two successful delivery and no history of pregnancy loss. By using PCR-RFLP, DNA from both groups analyzed for carrying mutation of FXIII.
Results: 4% in the case group were homozygote (TT) for 34 Leu mutation whereas no homozygote (TT) was found in control group (p<0.05). 28% patients in the case group and 26% women in the control group were found to be heterozygote for G103T polymorphism (p>0.05). No significantly differs was observed between patients with RPL and healthy women for G103T mutation.
Conclusion: No statistically differs between case group and control group was observed.
Key words: Recurrent miscarriage, Thrombophilia, F XIII.
P-59
Effect of pumpkin seed and ginger extracts on adult rat’s sperm characteristics, biochemical parameters and epididymal histology treated with cyclophosphamide
Amini Mahabadi J1, Nikzad H1, Aghaie S2, Taghizadeh M3, Azami-Tameh A2, Taherian SS2.
- Gametogenesis Research Center, Faculty of Sciences, Kashan University of Medical Sciences, Kashan, Iran.
- Anatomical Sciences Research Center, Faculty of Sciences, Kashan University of Medical Science, Kashan, Iran.
- Research Center for Biochemistry and Nutrition in Metabolic Disorders. Faculty of Sciences, Kashan University of Medical Science, Kashan, Iran.
Introduction: Infertility is one of the problems of human society and 10-15% of couples have experienced some forms of infertility problems. Reproductive toxicity is one of cyclophosphamide (CP) side effects in cancer treatment.
Materials and Methods: Male adult Wistar rats were randomly divided into six groups. Group 1 as control received an isotonic saline solution injection intraperitoneally (IP). Group 2 was injected intraperitoneally with a single dose of CP (100 mg/kg) once. Group 3 and 4 received CP plus 300 and 600 mg/kg combined pumpkin seed and Zingiber officinale extract (50:50). Group 5 and 6 received only 300 and 600 mg/kg combined pumpkin seed and Zingiber officinale extract. Six weeks after treatment, sperm characteristics, histopathological changes and biochemical parameters were assessed.
Results: Results showed that in CP treated rat’s sperm characteristics were diminished significantly. Biochemical analysis showed that the administration of combined extracts could increase the Total Antioxidant Capacity (TAC) level significantly in groups 3, 4, 5 and 6. Also, in these groups, the sperm viability, motility, count, normal sperm morphology, epididymal epithelium and fibromascular thickness were improved compared to control and CP groups. Interestingly, the mixed extract could improve histopathological changes such as vacuolization, disorganization and separation of epididymal tissue in CP treated rats as well.
Conclusion: Overall our findings indicated that the combined extracts might be used as a protective agent against CP-induced reproductive toxicity.
Key words: Pumpkin seed extract, Zingiber officinale extract, Cyclophosphamide, Total antioxidant capacity (TAC), Sperm parameters, Rat epididymis.
P-60
Effect of vitamin D insufficiency treatment on fertility outcomes in frozen-thawed embryo transfer cycles: A randomized clinical trial
Aflatoonian A, Arabjahvani F, Eftekhar M, Sayadi M.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: F_arabjahvani@yahoo.com
Introduction: Frozen- thawed embryo transfer is an essential part of ART treatment and outcomes of this procedure are associated with several clinical factors. Several studies have showed an increase level of IVF outcomes in women with sufficient vitamin D. whether treatment of vitamin D insufficiency can improve pregnancy rates in frozen-thawed embryo transfer cycles.
Materials and Methods: This is an interventional, randomized clinical trial. Serum 25-(OH) vitamin D level of 128 women who had undergone IVF/ ICSI with cryopreservation of embryos was checked. One hundred fourteen infertile women with insufficient serum vitamin D (less than 30 ng/ml) were included in the study. Fifty seven women were treated with supplementary vitamin D, 50000 IU weekly, for 6-8 weeks and 57 women were received no supplementation. 106 women completed frozen thawed embryo transfer cycles and included in the final analysis. Primary and secondary outcomes were chemical and clinical pregnancy respectively.
Results: Our study did not show any significant difference between vitamin D insufficient and treated women in term of chemical (29.40% vs. 29.10% respectively, p=1.00) or clinical (25.50% vs. 21.80% respectively, p=0.81) pregnancy rates.
Conclusion: Vitamin D insufficiency treatment is not associated with higher pregnancy rate in frozen-thawed embryo transfer cycles.
Key words: Vitamin D, Embryo transfer, Pregnancy rate.
P-61
Association of rs10954213 polymorphism of IRF-5 gene with idiopathic recurrent miscarriage
Arjmand F1, Ashrafzadeh Mehrjardi HR2, 3, Ghasemi N3.
- Department of Immunology, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Faculty of Medicine, Yazd, Iran.
- Department of Biology, Faculty of Science, Islamic Azad University, Ashkezar Branch, Yazd, Iran.
- Recurrent Abortion Research Center, Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: Interferon regulatory factor (IRF) 5 is a transcription factor that can induce transcription of IFN-α mRNA. Recent studies have shown that IRF-5 promotes the proliferation of T cells and the activation of TH1 and TH17 cells but does not induce TH2 or Treg differentiation. TH17 cells produce inflammatory cytokine IL-17 which plays an important role in the induction of inflammation via neutrophil infiltration and stimulation of inflammatory cytokines. There are many reports that inflammatory processes play a major role in recurrent miscarriage in a manner that an uncontrolled and persistent inflammatory response during pregnancy can harm placental growth. We analyzed the rs10954213 polymorphism of IRF-5 gene in normal pregnancy and recurrent miscarriage patients in order to discover a possible mechanism for the genetic control of immune regulation in patients with recurrent miscarriage.
Materials and Methods: Genomic DNA from 100 recurrent miscarriage (RM) patients and 100 normal fertile control individuals using the routine salting out method were isolated. DNA fragments were then analyzed by real time PCR with SYBR Premix Ex Taq II master mix. Statistical analysis used SPSS 19 software. IRF-5 allele frequencies and genotypes in RM women and the fertile control group were compared using a Chi-square test.
Results: Our results so far indicate that there is a detectable relationship between rs10954213 polymorphism of IRF-5 and susceptibility to recurrent miscarriage.
Conclusion: IRF-5 gene plays a possible role in the induction of inflammation and therefore leads to recurrent miscarriage. However, additional studies are needed in this regard.
Key words: Rs10954213, IRF-5, Recurrent miscarriage.
P-62
Role of 45,X mosaicism in couples with fertility problems
Bahman I1, Bagherizadeh I1, Abdi A1, Shajarehpour L1, Hadipour F1, Hadipour Z1, Saremi A2, Shafaghati Y1, 3, Behjati F1, 3.
- Department of Medical Genetics and Sarem Cell Research Center (SCRC), Sarem Women’s’ Hospital, Tehran, Iran.
- Department of Perinatology and Sarem Cell Research Center (SCRC), Sarem Women’s’ Hospital, Tehran, Iran.
- Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran
Introduction: Infertility is typically defined as the biological inability of a person to contribute to conception after one year of attempting to have a child. Genetic factors such as chromosomal abnormalities are one of the major causes of infertility and spontaneous abortions. The objective of this study was to establish chromosome X abnormality rate among women referred for infertility problems to Sarem Women's Hospital in Tehran between October 2007 and January 2015.
Material and methods: A total number of 1764 women with the age range of 12-60 years were referred for chromosomal investigation. Referral reasons included recurrent abortions, primary infertility, secondary infertility, Turner syndrome, premature ovarian failure, amenorrhea and ART failures. Cytogenetic analysis was performed on cultured peripheral blood lymphocytes stimulated with phytohaemagglutinin M, using standard techniques. Karyotype was done in all patients using high resolution GTG banding technique. For each patient, at least 15 chromosome spreads were examined by light microscopy, and extended to 100 in the case of mosaicism.
Results: Cytogenetic investigation was performed on 1764 patients. The overall chromosome X abnormality rate in infertile and subfertile women was 4% (74 out of 1764). The cytogenetic result for these patients with fertility problems is as follows: 74 patients had a numerical chromosome abnormality and 65 (85%) of them had mosaicism of 45,X with other different cell lines. The age range of women with 45,X mosaicism was 12-53 years old.
Conclusion: Accurate genetic diagnosis is the most important prerequisite for genetic counseling in patients with fertility problems. Finding of chromosome X numerical abnormality are of great value in better management of the patients. However, the attribution of chromosome X instability due to age should be considered in Genetic counseling of these couples.
Key words: Fertility problems, Chromosome X abnormality, 45,X cell line.
P-63
Effects of mitochondrial gene deletions on human sperm motility
Bahrehmand Namaghi I, Vaziri H.
Department of Biology, Faculty of Science, Guilan University of Medical sciences, Rasht, Iran.
Email: i_man_bahrehmand@yahoo.com
Introduction: Asthenozoospermia is an important cause of male infertility. It has been primarily characterized by reduced sperm motility owing to a variety of factors, including ultra-structural abnormalities, abnormal semen liquefaction, anti-sperm antibodies, varicocele and endocrine abnormality, etc. The mitochondrion is the major energy provider for sperm motility. Mitochondrial DNA contains several genes encoding for proteins that play an important role in oxidative phosphorylation and in ATP production. Mutations in sperm mtDNA result in either functionless or malfunctioning proteins, subsequently affecting sperm motility leading to asthenozoospermia.
Materials and Methods: To detect 4977 bp deletion in spermatozoa mtDNA, semen samples of 42 asthenozoospermic infertiles and 50 controls from northern Iran were collected. After extraction of spermatozoa total DNA, Gap-PCR was performed.
Results: 4977 bp deletion was observed in 73.80% of patients with asthenozoospermia, compared with 36% in controls (OR=5.0101, 95% CI: 2.0408-12.2998, p=0.0004).
Conclusion: Large-scale mtDNA deletions in spermatozoa may induce bioenergetic disorders and cause subfertility or infertility in men. It is concluded that there is a strong association between sperm mtDNA 4977 bp deletion and asthenozoospermia-induced infertility in the population examined. Nevertheless, to validate our results broader research may be needed.
Key words: Asthenozoospermia, mtDNA deletion, Male infertility, Motility.
P-64
Effects of Nano TiO2 on chromatin, apoptosis and parameters of sperm in mice
Danafar AH1, 2, Khoradmehr A1, Hosseini M3, Halvaei I1, Golzadeh J1, Anvari M1.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Biology, Ashkezar Branch, Islamic Azad University, Ashkezar, Iran.
- Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran.
Introduction: Titanium dioxide nanoparticles (TiO2 NPs) are manufactured worldwide and used in a broad range of applications. In mammals, there are only limited reports regarding the effect of TiO2 NPs on male reproductive system. It has been demonstrated that TiO2 NPs taken up by mouse Leydig cells reduced the viability and proliferation of these cells. Also it is improved that titanium compounds can disrupt the mouse blood-testis barrier. In this study we decide to evaluate the effects of nano-TiO2 on chromatin, apoptosis and sperm parameters of male mice.
Materials and Methods: 35 NMRI male mice were classed in five groups: control, sham and three treated groups. Low, medium and high treated groups were gavaged 2.5, 5 and 10 mg/kg doses of TiO2 respectively for thirty five days. Sham group only received saline instead. The mice were weighted before and after administration. Sperm count, motility, morphology and viability were analyzed accordingly. Sperm DNA integrity and apoptosis were assessed using acridine orange, aniline blue, toluidine blue, CMA3 and TUNEL assay.
Results: Non-significant decrease in body weight gain was observed. Sperm analysis showed no count, motility and viability changes but morphological changes were significant. Most of the morphological abnormalities were observed in sperm neck and tail. Tail morphological changes in treated groups were significant in comparison to non-treated ones (p<0.001). Compared to non-treated groups, sperm neck changes in medium and high treated groups were significant but not in low treated group (p<0.001). Results of acridine orange, aniline blue, toluidine blue and CMA3 were non-significant in all groups. Also increasing apoptotic cells in treated groups were discovered by TUNEL assay but there were non-significant statistically.
Conclusion: TiO2 significantly may increase sperms with abnormal morphology especially in tail and neck part. Also apoptosis boosting occurred but the exact mechanism is still controversial.
Key words: Nano-TiO2, Sperm parameters, Apoptosis.
P-65
Protective role of vitamin C on hazardous effects of acrylamide in rat offspring
Dortaj H, Anvari M, Yadegari M.
Shahid Sadoughi University of Medical Scienses, Yazd, Iran.
Email: hengameh.dourtaj@gmail.com
Introduction: Acrylamide (ACR) is a substance chemical used in industrial and laboratory procedures. Acrylamide according to the method of cooking foods are increasingly used and its adverse effects on multiple organ systems have been described sporadically in the literature. The purpose of this study was to evaluate the effects of ACR during pregnancy and lactation on the development and changes in cortical layer of the cerebellum and cerebellar Purkinje cells in fetal and neonatal rat by using histological and morphometric and stereological technique.
Materials and Methods: In this study 20 adult female Wistar rats weighing 180 gr and aged two months were used. Animals were randomly divided into four groups. Female pregnant rats were orally administered 10 mg/kg ACR and/or 200 mg/kg vitamin C (vit C). Pregnant rats were sacrificed on the 15th day of gestation and mother’s weight was measured. After that, their fetuses were taken out and were evaluated for fetus number, weight, crown-rump length (CRL) and cerebellar development. To study the neonatal period, 6 infants at day 21 were randomly selected and placed under deep anesthesia and transcardial perfusion. The cerebellum was taken out and fixed and cerebellum changes were evaluated by crysel violet and Hematoxylin-Eosin staining method. The cerebellar cortex layers volume were investigated by Cavalieri's principle method. Data were analyzed by SPSS software and by ANOVA and LSD Test. P<0.05 considered as statistically significant.
Results: The results showed that ACR decreased fetal weight and CRL, but this reduction in weight and volume of the cerebellum p˂0.001 and the number of embryos with p<0.05 was significant. Histological and stereological examinations revealed that the cerebellar volume was decreased in ACR and ACR+vitC group vs control (p˃0.001). While in vitamin C group the cerebellar volume was inversed (p˃0.05). ACR in newborn decreases body weight, brain weight, thickness of cerebellum with p˃0.001. The extent of this reduction in the weight of the cerebellum was significant with p˂0.05.
Conclusion: ACR exhibits a harmful effect on the development of the cerebellar cortical layers, which may be prevented by administration of vit C as an anti oxidant.
Key words: Acrylamide, Vitamin C, Cerebellum, Development, Rat.
P-66
Robust stem cell isolation from human dental pulp
Ebrahimi B, Banaei M, Namiranian G, Tabatabaei SA.
Yazd Cardiovascular Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: bhnmebrahimi@yahoo.com
Introduction: Dental pulp stem cells (DPSCs) are a population of clonogenic and highly proliferative cells derived from enzymatically digested Dental pulp tissue. Providing scaling-up of stem cells at early passages is of importance for regenerative medicine purposes. Typically, two protocols are employed to isolate stem cells from human dental pulp: tissue explants culture and enzyme digestion of pulp tissue and culture of released cells.
Materials and Methods: Dental pulp was extracted from third molars of 60 healthy subjects. In the first method pulp was digested with 1 mg/mL collagenase/ dispase (Roche) for 30 min and released cells obtained using a 70-µm cell strainer for culture, in the second method intact pieces of pulp were cultured and in the third method digested pulp pieces were immobilized and cultured. The cells and tissues maintained in alpha-MEM supplemented with 20% fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, and 25 ng/mL amphotericin B and incubated in humidified incubator with 5% CO2 at 37oC. In each group cells and colonies were counted and compared.
Results: Results showed that treating pulp segments with enzymes and culturing them (combinatory method) increased the efficiency of cell isolation up to 60% significantly in 3-4 days of culture compared with other methods which this value was <20% in 10-15 days.
Conclusion: According to the small size of pulp tissue and its low stem cell contents, acquiring substantial quantities of cells in primary culture will facilitate the in vitro expansion and providing adequate production of the stem cells at early passages with minimum risk of losing their ‘stemness’ and aberrant genetic changes for use in research, tissue engineering and regenerative medicine. Optimized method increase efficiency of cell isolation and provides significant quantities of stem cells in primary culture more than other methods.
Key words: DPSCs, Cell isolation, Cell culture.
P-67
Evaluation of exon 8 of DPY19L2 gene in total globozoospermic patients referred to Royan Institute
Ebrahimi Nasab M1, Totonchi M2, Sabbaghian M3.
- Department of Biology, Faculty of Basic Sciences and Advanced Technologies, Science and Culture University, ACERCR, Tehran, Iran.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Andrology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: Lack of acrosome decreases the capacity of the sperm for penetrating in the oocyte which consequently results in infertility. Globozoospermia is a rare and severe teratozoospermia characterized by round-headed spermatozoa lacking acrosomes. DPY19L2 is one of the genes which are dominantly expressed in the testis and it has been shown that is involved in the cause of this phenotype. Recent studies have shown that in large majority of globozoospermia patients a 200 kb deletion including DPY19L2 gene occurs. Different mutations in exon 8 of this gene have been also observed.
Materials and Methods: In total 24 men with total globozospermia and 24 men with normal spermogram referring to Royan Institute were selected. Then we sequenced exon 8 and intron boundaries in the non-deleted patients using specific primers and PCR technique.
Results: In our results no mutations were detected in exon 8 of the patients without the gene deletion, which were 29.17% of the total globozospermia patients.
Conclusion: According to our data, exon 8 mutations or polymorphisms has no effect on globozospermia but whereas 70.83% of globozospermia patients had whole DPY19L2 deletion and on the critical role of DPY19L2 protein in acroplaxome attachment to the nucleus, it can be concluded that the absence of this protein is one of the major causes of globozoospermia in Iranian infertile men. In addition, exploring other coding exons of this gene is our next aim.
Key words: DPY19L2 gene, Globozoospermia, Male infertility.
P-68
Anti-oxidant effects of herbal supplements on seminal parametmeters
Eghbal Eftekhaari T, Fallahi S, Malekzadeh K.
Molecular Medicine Research Center, Hormozgan University of Medical Sciences, BandarAbbas, Iran.
Email: tasnim.eftekhaari@gmail.com
Introduction: Varicocele is one of the leading causes of male infertility, causing a decrease in sperm parameters such as motility, count, concentration, and abnormal sperms such as azoospermia and asthenospermia.
Materials and Methods: A herbal compound capsule containing 4 herbs was prescribed 5 times perday (3 times during morning and evening and 2 times at night), and because of the warm nature of these herbs, 3 herbal extracts including Cichorium intybus L, Salix Aegyptiaca and Fumaria Parviflora 3 cups/day were added to this medication.
Results: After 4 weeks of consuming these medications, the body warmness decreased , ejaculated sperm volume and other sperm parameters increased including motility, concentration and morphology of sperm changed form astenia and oligospermia to normal morphology.
Conclusion: The herbal compound containing antioxidant supplements and enriched in minerals and fat soluble vitamins showed promising effects on improving sperm parameters and increase the chance of male fertility.
Key words: Varicocele, Increased sperm motility, Increased sperm count, Sperm paramteters, Antioxidants.
P-69
The effect of brown algae Sargassum extract on freezing of sperm
Fardmanesh H1, Fallahi S2, Shakerizadeh S3, Masoudi M3, Fadaei- Arsanjan M4.
- Department of Student Research Committee , Hormozgan University of Medical Sciences, Bandar Abbas, Iran.
- Department of Fertility and Infertility of Hormozgan, Hormozgan University of Medical Sciences, Bandar Abbas, Iran.
- Department of Molecular Medicine, Hormozgan University of Medical Sciences, Bandar Abbas, Iran.
- Department of Biology, Islamic Azad University, Arsanjan Branch, Fars, Iran.
Introduction: Oxidative stress process resulting from freezing and thawing of sperm, influences parameters of sperm and decreases its fertility. Antioxidants play a conservative role against oxidative damages during freezing of sperm. Using an appropriate freezing bank effects on fertility and motility of sperm, so we decided to evaluate effect of antioxidant extract of brown algae Sargassum on reactive oxygen species (ROS) and parameters of frozen sperm.
Materials and Methods: 11 normal semen samples were divided in to 3 groups, including A treated with 250 µg/ml and B with 500 µg/ml of extract of algae Sargassum and C group without any treatment (as a control group) then froze throughout rapid freezing method. Using CASA software and oxisperm kit, the motility/morphology and ROS levels of sperm were measured, respectively. Finally data were analyzed with SPSS software assumed significant level of p<0.05.
Results: The analysis of sperm parameters demonstrated that general motility (p=0.006) and advanced motility (p=0.007) significantly increased in both A and B groups, after treatment compared with C group. Moreover, the level of ROS notably declined in both treated group. However this treatment revealed any changes in sperm morphology.
Conclusion: The brown algae Sargassum extract is rich in antioxidant component that leads to decrease oxidative damage via neutralizing of ROS and improve sperm motility. Our results suggested that the extract can be used as a potential antioxidant factor in sperm freezing bank.
Key words: Sperm motility, Antioxidants, Reactive oxygen Species, Brown algae sargassum.
P-70
Immunohistochemical evaluation of a testis-specific histone demethylase, JMJD1A, in tissue samples of infertile men referred to Royan Institute
Favaedi R1, Shahhoseini M1, Kordestani shargh E2, Sodeifi N3, Sadighi Gilani MA3.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
- Department of Andrology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: Post-meiotic stages of mammalian spermatogenesis require unique and dynamic epigenetic events leading to histone removal followed by chromatin condensation. Through these events, histone demethylases such as JMJD1A play an important role in compaction of sperm chromatin due to regulation of histone methylation dynamics and alteration of chromatin structure. As “histone methylation” is one of the best-characterized modifications in the study of germ cell development, evaluation of presence/ absence of JMJD1A protein as in impaired spermiogenesis were aimed in this study.
Materials and Methods: For this respect, consent was obtained from azoospermic infertile men referred to Royan Institute according to local ethical approval, and then testis tissue samples were collected from three groups including complete maturation arrest, Sertoli cell only syndrome, and hypo spermatogenesis as positive control. Immunohistochemical analysis of paraffin embedded tissue samples was performed qualitatively using anti-JMJD1A antibody to elucidate presence/ absence of this protein in nucleus of germ cells.
Results: Immunohistochemical analysis data showed absence of JMJD1A protein in nucleus of germ cells in groups with spermatogenesis impairment (complete maturation arrest and Sertoli cell only syndrome groups) compared to control group.
Conclusion: It can be concluded that there is an obvious association between absence of histone demethylation as a chromatin condensing state with impairment of spermatogenesis and male infertility.
Key words: Spermatogenesis, Male infertility, Epigenetic, JMJD1A, Demethylation.
P-71
Pregnancy outcome of intracytoplasmic injection with epididymal and testicular sperm retrieval in azoospermic patients
Moein MR, Ghasemi-EsmailabadS, Ghasemzadeh J, Fesahat F, Tabibnejad N.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: qasemisaeed@yahoo.com
Introduction: Intra cytoplasmic sperm injection (ICSI) is an assisted reproductive technique for treatment of infertility in azoospermic men. It seems that pregnancy outcome in ICSI following percutaneous epididymal sperm aspiration (PESA) is better than using testicular sperm retrieving from testis due to greater motility and better morphology of epididymal spermatozoa. To determine clinical pregnancy outcome following ICSI with epididymal sperm comparing to that of testicular sperm in men with azoospermia.
Materials and Methods: 60 men with azoospermia who were candidate for ICSI have been selected. Sperm retrieval was performed using PESA (n=30) or testicular sperm extraction (TESE) (n=30). The number of embryos and live births were analyzed and evaluated between two mentioned groups and also thawed sperms after freezing in TESE group (n=30).
Results: No difference was seen in age and duration of infertility between groups. The number of embryos were not different significantly between TESE and PESA groups (p>0.05), but the different was significant between PESA and TESE freeze groups (3.67±2.89 vs. 2.10±1.53 respectively; p<0.05). The live birth rate was higher in the PESA group compared with the TESE group p<0.05).
Conclusion: Intracytoplasmic sperm injection using sperm from epididymis is more effective than testicular sperm injection and can successfully be performed to treat men with azoospermia.
Key words: ICSI, TESE, PESA, Freeze, Live birth rate.
P-72
Prevalence and risk factor of congenital malformation in Kashan
Haddad Kashani H1, Seyed Hoseini ES2, Dadkhah M3, Nikzad H2.
- Anatomical Sciences Research Center, Kashan University of Medical Science, Kashan, Iran.
- Gametogenesis Research Center, Kashan University of Medical Science, Kashan, Iran.
- Faculty of Medicine, Kashan University of Medical Sciences, Kashan, Iran.
Introduction: Congenital malformation (CM) is a major childhood health difficulty. Treatment and rehabilitation of children with congenital malformations is costly and complete recovery is usually impossible. The prevention and treatment of congenital malformations are basic concerns for child health. The purpose of this study was to define rate of CM in Beheshti Hospital in Kashan, Iran to find out if there has been any variation in the rate and types of CM in this area.
Materials and Methods: This descriptive-observational study carried on 2700 births delivered from Beheshti Hospital in Kashan during 2012 and determines the prevalence of CM and type of it in this city.
Results: Prevalence of CM was 1.851% (2% in male and 1.61 % in female). Out of the 50 cases, 28 (56%) were males and 21 (42%) were females and 1 with uncertain genitalia. 13 members of the family were CM positive in musculoskeletal system (26%). Overall, anomalies of the cardiovascular system were second in frequency which involved 10 out of 50 patients (20%), also Down syndrome and skin malformation were the lowest rate anomaly in this study in Kashan (4% and 2% respectively). There wasn't statistical difference between prevalence of CM and neonatal gender and mother’s age, father’s age, mother’s number of abortion, and mother’s number of live children. But there was statistical difference between prevalence of CM with gestational age and Apgar number.
Conclusion: In this study the overall prevalence of congenital malformation among the newborn was less than those previous reported in Tehran and Yazd and higher than the rate of malformation in Gorgan and Arak. This difference determining the needs of more extensive studies.
Key words: Congenital malformation, Newborn, Beheshti Hospital, Kashan.
P-73
Relevance between testicular tissue vitrification and short term culture with degeneration and apoptosis genes expression
Hajiaghalou S1, 2, Ebrahimi B2, Shahverdi AH2, Sharbatoghli M2.
- Department of Developmental Biology, Faculty of Basic Sciences and Advanced Technology in Biology, University of Science and Culture, ACECR, Tehran, Iran.
- Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: Cryopreservation of testicular tissue has been recommended as a promising technique for fertility preservation in pre-pubertal boys who scheduled to undergo gonadotoxic treatment. Our aim was finding the quota of apoptosis genes involved in extrinsic pathway in testicular cell death after vitrification and during short term culture.
Materials and Methods: Testes were obtained from 7 days old NMRI male mice, divided and randomly distributed into control and vitrification groups. Vitrification was performed in 3 step by increasing concentration of vitrification solution (DMSO, EG). Both fresh and vitrified-warmed testes was cultured in RPMI and 10% KOSR for 20 hr. Real-time PCR, flow cytometery and light microscopy were used respectively for evaluation of gene expression, cell death and tissue integrity at 0, 3 and 20 hr of culture.
Results: Decreasing of tissue integrity was obvious in vitrification group as compared to the control one at all times of culture. Mean percentage of cell death was significantly (p<0.05) higher in vitrification group in comparison with control during culture period. Although expression of Fas was significantly (p<0.05) higher in vitrification group at 0 and 3 hr of culture, it was significantly lower at 20 hr of culture as compared to the control group. Significant (p<0.05) increase of Fas ligand was found in vitrification group at 3 and 20 hr of culture. Mean percentage of Caspase 3 was significantly (p<0.05) higher in vitrification group than control group.
Conclusion: Concurrent increment of cell death and apoptosis genes expression in vitrification group during culture period, could be a reason for extrinsic pathway involvement in degeneration of testis tissue after vitrification and also during culture period.
Key words: Extrinsic pathway of apoptosis, Tissue degeneration, Vitrification, Short term culture.
P-74
Magnetic activated cell sorting and its application for selection of human non apoptotic spermatozoa in ART
Hassani Bafrani H1, Catt S2, Pangestu M2, Smith Peter T2.
- Kashan University of Medical Sciences, Kashan, Iran.
- Monash University, Melbourn, Austuralia.
Introduction: Hydrogen peroxide has been implemented in literature to induce a significant increase in caspase. Activation of caspase 9 triggers a cascade of caspase activation, including caspase 3, which promotes cellular apoptosis. Magnetic cell sorting (MACS) using annexin V-conjugated microbeads eliminates apoptotic spermatozoa with annexin V-positive.
Materials and Methods: A total of 20 semen samples were obtained from male partners of couples for analysis. One aliquot (0.5ml) of the sperm suspension was subjected to MACS. Motility and concentration was checked, and a sample taken for tunnel before and after MACS. The remaining sperm suspension was divided into 6 tubes (2x control, 2x peroxide, 2x peroxide/ melatonin). DNA fragmentation was evaluated using the TUNEL assay (Roche, Indianapolis, IN, USA) (14), with some modifications. Data were analyzed using Graph Pad Instat Ver. 3.10.
Results: Results of the TUNEL assay in pretreatments of human spermatozoa with 100uM peroxidase for 24 hrs revealed that the percentage of sperm with fragmented DNA was significantly lower on the sored sperm after sorting MACS (p˂0.001 vs. control). Following the pretreated human spermatozoa with peroxidase for 24 hr, percentage of sperm motility and progressive motility were significantly reduced (p˂0.001 vs. control). Pretreatments of human spermatozoa for 24 hr revealed that the percentage of sperm motility and progressive motility were significantly reduced by 100 µM peroxidase and peroxidase with MACS, versus zero hour in control group (p<0.001).
Conclusion: The sperm was treated with 100 µM peroxidase sorting using MACS retain appropriate spermatozoa and select sperm good quality. The use of MACS will select only sperm with intact this result in high percentage motility and progressive motility.
Key words: Apoptosis, Human spermatozoa, MACS.
P-75
Bibliometric mapping and clustering analysis of Iranian-based research on reproductive medicine
Bazm S1, Ramezani A2.
- School of Public health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Faculty of Management and Information Sciences , Iran University of Medical Sciences, Tehran, Iran.
Introduction: Nowadays infertility is a major problem in the world. Increasing the rate of infertility in the world and Iran led our study to assess the trends in Iranian research output related to reproductive medicine through the year of 2010-2014.
Materials and Methods: We used bibliometric mapping and clustering analysis method to visualize representing bibliographic data of Iranian production. All publication data from Scopus was retrieved for 2010-2014.
Results: Analysis of data showed that a total number of 3035 papers had been indexed in Scopus through the period of study.
Conclusion: The study indicated that scientific production in the field of reproductive medicine had increased in the past 5 years.
Key words: Reproductive medicine, Bibliometric analysis.
P-76
Menstrual pattern following tubal ligation: A historical cohorts
Jahanian Sadatmahalleh Sh1, Ziaei S1, Kazemnejad A2, Mohamadi E3.
- Department of Reproductive Health and Midwifery, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
- Department of Biostatistics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
- Department of Nursing, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Introduction: Tubal ligation (TL) is recommendable for women completed their family. The existence of the menstrual disorders following this procedure has been the subject of debate for decades.
Materials and Methods: A historical cohort study was carried out on 140 women undergoing tubal ligation and on 140 women who used condom as the main contraceptive method. They aged between 20 and 40 years and were selected from a health care center, during 2013-2014. The two groups were comparable in demographic and personal characteristics. Data collection tool was a questionnaire including questions regarding demographic, menstrual and obstetrical characteristics. A validated Pictorial Blood Loss Assessment Chart (PBLAC) was also used to measure the menstrual blood loss. All statistical analyses were carried out using software package used for statistical analysis (SPSS) version 20 (SPCC Inc., Chicago, IL, USA). Student’s t-test and chi-square test were carried out to reveal the statistical differences between the groups. Logistic regression was done to build a prediction model in menorrhagia.
Results: Women with TL had more menstrual irregularity than those without TL (24.3% vs. 10% respectively, p=0.002). Women with TL had more polymenorrhea (9.3% vs. 1.4%; p=0.006), hypermenorrhea (12.1% vs. 2.1%; p=0.002), menorrhagia (62.9% vs. 22.1%; p<0.0001) and menometrorrhagia (15.7% vs. 3.6%; p=0.001) than those without TL. There is a significant difference in the PBLAC score between women with and without TL (p<0.0001). According to logistic regression, age (OR=1.08, CI:1.07-1.17; p=0.03), TL (OR=5.95, CI:3.45-10.26; p<0.0001), and cesarean section (OR=2.72, CI:1.49-4.97; p=0.001) were significantly associated with menorrhagia.
Conclusion: We found significant differences in menstrual disorders between women with and without TL. Therefore, women should be informed by the health providers regarding the advantages and disadvantages of TL before the procedures.
Key words: Historical cohort study, Tubal ligation, Menstrual disorders, PBLAC.
P-77
Effect of nano silver on morphological and chromosomal abnormality of NMRI mouse fetus
Mozafari M1, Kalantar SM2, Khoradmehr A2, Danafar AH2, 3, Miresmaeili M4.
- Department of Biology, Islamic Azad University, Ashkezar Branch, Yazd, Iran.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Young Researchers Club, Islamic Azad University, Yazd, Iran.
- Yazd Academic Center of Education, Culture and Research Higher Education Institute, Yazd, Iran.
Introduction: Development of nanotechnology caused the use of the materials in nano sizes. Nowadays, special biological properties of nano silver are playing an important role in our life. Some studies suggest a potential for adverse effects of nano silver on fetal development of mammals, but additional research is needed. In this study we decide to evaluate the effects of nano silver on fetus chromosomal structure and its development.
Materials and Methods: 24 pregnant mice were divided into four groups. Nano silver (1 mg/kg, 70 nanometer) were gavaged to the first, second and third group, from the 1th-7th, 8th-14th and 1th-14th gestational days respectively. Nothing was gavaged to the control group. On 14th day the pregnant mice were dislocated and the liver of fetus was used for karyotyping analysis. The fetuses were weighed and their crown-rump length and head circumference were measured by caliper.
Results: According to the effects of nano silver, the results indicated that body weight of embryos were significant decreased in third group in comparison with the others (p<0.001). Also there were significant reduction of crown-rump length of fetus in third and second group compared to other groups (p<0.05). There was lower fetus head circumference among nano silver treated and controls (p<0.05). Karyotyping analysis of fetal liver was normal in all groups.
Conclusion: It seems that abuse of nano silver during pregnancy can reduce weight, crown-rump length and head circumference in mice fetus, but its mechanism is not completely clear.
Key words: Pregnant mice, Nano silver, Karyotype.
P-78
Neuronal markers expression of induced human adipose-derived stem cells in alginate hydrogel
Khosravizadeh Z1, Khosravizadeh Z1, Razavi Sh2.
- Tehran University of Medical Sciences, Tehran, Iran.
- Isfahan University of Medical Sciences, Isfahan, Iran.
Introduction: Hydrogels provide appropriate three-dimensional environment for cell cultures and cell encapsulation in hydrogels is a promise plan for tissue engineering applications. Alginate is a biocompatible hydrogel that provides a supportive system for the encapsulated cells. Moreover human adipose derived stem cells (hADSCs) might be a suitable source of cells for use in autologous cell therapy; also these cells can differentiate into neuron-like cells. Therefore, in this study we evaluated effect of alginate hydrogel on the neural potential of induced hADSCs.
Materials and Methods: Isolated hADSCs were induced in neural medium and encapsulated in alginate hydrogel. Using Immunocytochemical and real-time RT-PCR analysis, some neural markers were evaluated in differentiated hADSCs.
Results: The expression of Nestin, GFAP and MAP2 markers significantly increased in alginate cell cultures relative to monolayer induced cells (p<0.001).
Conclusion: These findings showed that alginate hydrogel can provide a suitable environment for neural differentiation of hADSCS.
Key words: Human adipose-derived stem cells, Alginate hydrogel, Neural differentiation, Tissue engineering.
P-79
Protective effect of N-acetyl-L-cysteine on testicular tissue in mice treated with Para-Nonylphenol
Malmir M, Soleimani Mehranjani M, Naderi Noreini S.
Faculty of Science, Arak Univercity of Medical Scinecs, Arak, Iran.
Email: mehhdi66@yahoo.com
Introduction: Para-Nonylphenol (p-NP) is an alkylphenol considered as an environmental pollutant with estrogenic and toxic effects. It can also cause morphological and functional alterations in the male reproductive system inducing infertility. The purpose of this study was to investigate the protective effects of N-acetyl-L-cysteine (NAC), as a powerful antioxidant, on p-NP-induced testicular toxicity in mice.
Materials and Methods: Adult male NMRI mice (32±4 gr) were divided randomly into 4 groups (n=6), control, NAC (150 mg/kg/day), p-NP (250 mg/kg/day) and p-NP+NAC, and they were treated orally for 35 days. Finally, mice were killed, their right testis were removed and fixed followed by sectioning, tissue processing and Heidenhain azan staining. Testicular tissue sections were then evaluated using stereological method. The level of Malondialdehyde (MDA), a lipid peroxidation index, was also measured in the testis of mice. Data were statistically analyzed using one way ANOVA and means were considered significantly different at p<0.05.
Results: A significant decrease in the mean total volume of testis, diameter, length and the volume of the seminiferous tubules, height of the germinal epithelium and the thickness of the basement membrane along with a significant increase in the MDA level was observed in the p-NP group compared to the control (p<0.01) while the above parameters were compensated to the control level in the p-NP+NAC group (p<0.01).
Conclusion: Our findings indicate that N-acetyl-L-cysteine can protect the testicular tissue against the tissue damage and oxidative stress induced by para-Nonylphenol in mice.
Key words: Para-nonylphenol, N-acetyl-L-cysteine, Stereology, Testis.
P-80
Effects of dietary canola oil on sperm quality parameters at Afshari Ram Breed
Mansouri S, Pirestani A.
Islamic Azad University, Khorasgan Branch, Isfahan, Iran.
Email: nmansorrin@yahoo.com
Introduction: In mammalian and non-mammalian spermatozoa, there are natural fatty acids, cholesterol, phospholipids (mainly lecithin, cephalin and sphingomielin) and glycolipids. Phospholipids of mammalian sperm cell membranes characteristically contain very high proportions of long-chain polyunsaturated fatty acids, particularly n-3 series
Materials and Methods: 18 Kurdish rams were selected with weight average 54.47±2.58 kg and with the age of 3-4 years approximately. They were divided to two experimental groups randomly. Experimental groups were control and canola oil (2.5% of DMI). Before of study, 10 day was considered as adaptation period and then Sperm was collected by electro ejaculation at 6 week and 11 week after begging of adaptation period and sperm motility was analyzed by using CASA software at zero time and 24 hours post ejaculation. Also, percentage of live/dead sperm and morphology was evaluated by staining of Eosin-Nigrosin and Papanicolaou, respectively.
Results: The results showed that motility parameter wasn’t significantly different between whole experimental groups at first time (week 6) but PM% and TM% was significantly different in canola oil group compare to control group at second time (week 11), separately. Percentage of live/dead sperm and morphology was higher significantly in canola oil group compare to control group.
Conclusion: It was concluded that canola oil can improve sperm motility, morphology and viability in Afshari ram.
Key words: Canola oil, Motility, Afshari Ram, Sperm.
P-81
Mutation analysis of bone morphogenetic protein-15 gene in Iranian patients with polycystic ovarian syndrome
Mehdizadeh A, Kalantar SM, Sheikhha MH.
Department of Genetics, International Campus, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: anahita.mz@gmail.com
Introduction: With the prevalence on the order of 6-10%, polycystic ovarian syndrome (PCOS) is considered the most common endocrinologic disorder affecting women in their reproductive age. Major criteria that have been proposed for the diagnostic characteristics of PCOS are clinical and/ or biochemical hyperandrogenism, ovulatory dysfunction and polycystic ovary in ultrasound. It has been suggested that genetic factors participate in the development of PCOS. Follicular development has been considered as one of the impaired processes in PCOS. BMP-15 gene is a candidate gene in follicular development and its variants may play role in pathogenesis of PCOS. Previous investigations have revealed controversial results on BMP-15 mutations in PCOS women among various racial groups.
Materials and Methods: A cross-sectional study was carried out on 70 PCOS patients. Following taking the informed consent, 5ml venous blood was taken from each participant. Genomic DNA was extracted from the blood sample by salting out Method. Then a set of PCR reactions for BMP15 gene was performed using specific primers followed by genotyping with direct sequencing.
Results: As for exon1 in BMP-15, 20 heterozygote (G/C) and 2 homozygote (G/G) cases were found in 70 PCOS patients. Also, one -9 C>G polymorphism in 5’UTR and 3 cases of A308G mutation (A/G) were discovered in our patients.
Conclusion: Result of this study indicates that variants of BMP-15 gene could be related to susceptibility of development of PCOS and may be used as genetic markers for detecting PCOS in the future.
Key words: Polycystic ovarian syndrome, Bone morphogenetic protein 15.
P-82
Effect of adding human chorionic gonadotropin to frozen thawed embryo transfer cycles with history of thin endometrium
Davar R1, Farid Mojtahedi M1, Miraj S1, 2.
1. Department of Obstetrics and Gynecology, Research and Clinical Center for Infertility, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Department of Obstetrics and Gynecology, School of Medicine, Shahrekord University of Medical Sciences, Shahrekord, Iran.
Email: miraj.sepideh@gmail.com; dr.s.miraj@gmail.com
Introduction: Embryo implantation process is a complex phenomenon and depends on fetal and maternal factors interaction. Endometrial thickness is needed for successful implantation. Increasing endometrial thickness, raise the chance of clinical pregnancy. The triple line pattern, with thickening more than 7mm, is indicating the greater chance of successful implantation. We designed this study to assess adding HCG to the conventional protocol in endometrial preparation in women with thin endometrium and history of IVF-ET failure.
Materials and Methods: This non-randomized clinical trial study (quasi experimental design) was performed in Yazd Research and Clinical Center for Infertility on 28 patients. Participants were women who candidate for frozen thawed embryo transfer and had 2 previous failed ET cycle because of thin endometrium. All patients received 8 mg estradiol valterate on second day of menstrual cycle and continued during the study. HCG was administrated (150 IU, IM) from 8th days of cycle. In 12th-13th day Trans-vaginal sonography was done, when endometrial thickness reached at least 7mm, HCG was discontinued and frozen thawed embryo transfer was done.
Results: Totally 28 patients were included. The mean age of participants was 30.39±4.7 years. The mean of endometrial thickness before and after HCG were 5.07±0.43 and 7.85±0.52 mm which were significantly different (p=0.00). After HCG administration 100% patient's endometrial thickness reached more than 7mm. The frequency of 20% improvement after HCG was 89.3% (25 patients). Also there were 5 (17.8%) clinical and chemical pregnant women after HCG.
Conclusion: Our study findings suggest that adding HCG to the conventional preparation method is an effective protocol and significantly improved endometrial thickness and pregnancy outcomes in women with previous embryo transfer failure because of thin endometrium.
Key words: Human chorionic gonadotropin, Frozen thawed embryo transfer, Thin endometrium.
P-83
Glycyrrhiza glabra and vitamin C can reduce toxic effects of acrylamide on sperm parameters in rat
Mirjalili A1, Talebi AR2, Mangoli E2, Ghasemzadeh J2.
- Shahid Sadoughi University of Medical Science, Yazd, Iran.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: Acrylamide (AA) is a chemically reactive substance used in various industries. Recently, acrylamide was discovered in a variety of human foods including heat-processed starchy foods such as potato chips and bread. AA is able to induce sperm damage in male mice.
Materials and Methods: In this experimental study, 30 male Wistar rats of 28 days of age were divided in to five groups: Acrylamide, Acrylamide+ Vitamin C, Acrylamide+ Glycyrrhiza glabra, Acrylamide+ Vitamin C+ Glycyrrhiza glabra and Control. All treatments were administered (oral Acrylamide 10 mg/kg, Vitamin C 200 mg/kg and Glycyrrhiza glabra 150 mg/kg) daily for two months. Thereafter, the cauda epididymis of each rat was dissected and placed in 1 mL of pre-warm Ham’s F10 culture medium for 30 min. The swim-out spermatozoa were analyzed for count, motility, morphology and viability.
Results: The results showed that almost all of the sperm parameters except non progressive motility were significantly different between groups (p=0.001). Also, the mean of sperm parameters in Acrylamide+ Vitamin C+ Glycyrrhiza glabra group was higher than other groups.
Conclusion: This study showed that the co-administration of vitamin C and Glycyrrhiza glabra as antioxidant can reduce the detrimental effects of acrylamide on sperm parameters in rats.
Key words: Glycyrrhiza glabra, Vitamin C, Acrylamide, Sperm, Rat.
P-84
Infertility in male by neurological disorders
Mollaei AR, Seihei E.
Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
Email: ptalireza@yahoo.com
Introduction: Normal sexual and reproductive functions depend largely on neurological mechanisms. Neurological defects in men can cause infertility through erectile dysfunction, ejaculatory dysfunction and semen abnormalities.
Materials and Methods: Science Direct, PubMed, Cochrane, CINAHL, Embase, ProQuest Dissertations, Scopus, (2000-2015) were searched for English-language studies using a list of keywords. The books about physical therapy and medical and neurological were studied too.
Results: Among the major conditions contributing to these symptoms are pelvic and retroperitoneal surgery, diabetes, congenital spinal abnormalities, multiple sclerosis and spinal cord injury. Erectile dysfunction can be managed by an increasingly invasive range of treatments including medications, injection therapy and the surgical insertion of a penile implant. Retrograde ejaculation is managed by medications to reverse the condition in mild cases and in bladder harvest of semen after ejaculation in more severe cases.
Conclusion: An ejaculation might also be managed by medication in mild cases while assisted ejaculatory techniques including penile vibratory stimulation and electro ejaculation are used in more severe cases. If these measures fail, surgical sperm retrieval can be attempted. Ejaculation with penile vibratory stimulation can be done by some spinal cord injured men and their partners at home, followed by in-home insemination if circumstances and sperm quality are adequate. The other options always require assisted reproductive techniques including intrauterine insemination or in vitro fertilization with or without intracytoplasmic sperm injection. The method of choice depends largely on the number of motile sperm in the ejaculate.
Key words: Ejaculation, Electro ejaculation, Infertility, Nervous system diseases.
P-85
Adenosine deaminase G22A polymorphism and recurrent spontaneous abortions
Mohammadi A1, Bonyadi M1, Khalaj kondori M1, Taghavi S2.
- Department of Animal Biology, Faculty of Natural Sciences, Tabriz University, Tabriz, Iran.
- Maternal Fetal Medicine Unit, Department of Obstetrics and Gynecology, Alzahra Hospital, Tabriz University of Medical Sciences, Tabriz, Iran.
Introduction: Adenosine deaminase (ADA) is an enzyme of purine salvage pathway and has two important isoenzymes ADA1 and ADA2. The adenosine deaminase G22A polymorphism (ADA*2) increases the level of adenosine. Adenosine may play a protective role against recurrent spontaneous abortions (RSA), since it regulates blood flow into the uterus and placenta.
Materials and Methods: A total of 100 women were classified in two groups: First group, with a history of recurrent spontaneous abortions (n=50), and second one, without a history of abortions (n=50). Genomic DNA was extracted from peripheral blood with a commercial kit and PCR-RFLP analysis was used to identify the G22A genetic polymorphism.
Results: The frequency of homozygotes (AA) was 2% in control group, whereas no homozygote (AA) was found in the case group (p>0.05). The frequency of heterozygotes (AG) was 20% in the control group and 8% in the case group (p<0.05). The frequency of homozygotes (GG) was 78% in the control group and 92% in the case group (p<0.05). A significant increase in the frequency of AG genotype in controls (p<0.05, OR=0.348) relative to women with the history of RSA demonstrates the protective effect of AG genotype in controls.
Conclusion: The data suggest that women carrying the ADA*2 allele which is associated with the lower enzymatic activity are better protected against recurrent spontaneous abortions. Because decreased enzymatic activity increases adenosine levels. High adenosine levels may play a protective role against recurrent spontaneous abortions, since it regulates blood flow into the uterus and placenta.
Key words: PCR-RFLP, Recurrent spontaneous abortion, Adenosine deaminase.
P-86
Seminal bacterial contaminations: Probable factor in unexplained recurrent pregnancy loss
Nabi A, Khalili MA, Halvaei I, Ghasemzadeh J, Zare E.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: ali.nabi67@yahoo.com
Introduction: It is estimated that about 50% of causes of recurrent pregnancy loss (RPL) cases remain unknown. Sperm factors are suggested to have probable role in cases with RPL. The goal was to determine the possible relationship between semen bacterial contaminations with unexplained RPL. Also, the correlation between number of bacterial colony and sperm chromatin condensation was examined.
Materials and Methods: This study consisted of 30 fertile men (group A) and 30 infertile men (group B) with unknown RPL in their wives. Semen collection and analysis were done according to WHO manuals. Sperm count and motility were evaluated by Makler chamber. Eosin-Nigrosin and Papanicolaou staining methods were applied for viability and morphology assessment, respectively. The semen samples from both groups were cultured for aerobic bacteria. Aniline blue (AB) and toluidine blue (TB) staining methods were applied for evaluating sperm chromatin condensation.
Results: The numbers of colonies were significantly higher in group B when compared to group A. Also, S. aureus and E. coli contaminations showed significant differences between two groups. Both AB+ and TB+ sperm cells showed significant increase in group B compared to group A. There was a significant negative correlation between colony number and progressive motility (p=0.01), and sperm viability (p=0.007). In addition, positive correlations were found between colony number and AB+ (p=0.001) and TB+ (p=0.004) as well.
Conclusion: Bacterial contaminations in semen of men from RPL couples had significantly higher levels when compared to fertile controls. Presence of microorganisms in semen may be correlated with irregular sperm parameters and quality.
Key words: Recurrent pregnancy loss, Bacteria, Semen.
P-87
Precedent infertility and attitudes towards preferred Cesarean
Naeimi N.
Department of Biology, Faculty of Science, University of Sistan and Baluchestan, Zahedan, Iran.
Email: nnaeimi@rocketmail.com
Introduction: Prevalence of natural delivery and caesarean section in pregnant women of a country is one of the indicators of health system performance. Currently, we are exposure to increasing elective Cesarean with different reasons and attitudes.
Materials and Methods: In this descriptive-analytical study, 271 pregnant women completed a researcher-made questionnaire including, demographic characteristics (age, precedent of infertility) and 10 attitude questions about the influencing factors on choosing Cesarean, based on five Likert scale which was a score of 10-30 negative attitude (opposite preference for Cesarean) and 31-50 positive attitude (positive preference for Cesarean). Therefore, to analyze the obtained data, Chi-square test was used through applying SPSS19 software.
Results: Overall 74 subjects (28%) had negative attitudes, while 192 subjects (72%) had positive attitudes. In total 7 subject of pregnant women group (2.6%) had a precedent of infertility and 259 pregnant women had not (95.6%). During the study, 5 subjects did not answer questions. In addition, 5 subjects with precedent infertility (73.4%) vs. 69 subjects (26.6%) with no precedent, showed negative attitudes. 2 subjects (26.6%) vs. 190 pregnant women (73.4%) had positive attitude toward preference of Caesarean section. There was a significant relationship between precedent infertility and attitude.
Conclusion: According to the results, women with precedent infertility who are using assisted reproductive techniques, are more aware of advantages and disadvantages of different methods of termination of pregnancy and most of them opposed natural delivery. Therefor giving them information through related groups of women's health, can change their attitudes.
Key words: Precedent infertility, Attitude, Preference cesarean.
P-88
Protective effects of BDNF against oxidative damage and apoptosis in human spermatozoa
Najafi A1, Sobhani AGH1, Amidi F1, Sadighi Gilani MA2, Moawad AR3, Mikaeili S1, Fallah P2, Rezaiian Z2.
- Department of Anatomy, Faculty of Medicine, Tehran University, Tehran, Iran.
- Infertility Center, Shariati Hospital, Tehran University, Tehran, Iran.
- Department of Surgery, Royal Victoria Hospital, McGill University, Quebec, Canada.
- Department of Urology, Shariati Hospital, Tehran University, Tehran, Iran.
Introduction: Oxidative Stress (OS) is a condition when there is an improper balance between the amount of pro-oxidant substances and the amount of seminal plasma antioxidant factors that can be induced by repeated cycles of centrifugation and seminal plasma antioxidants, removal during the sperm preparation in Assisted Reproductive Technology (ART) Reactive Oxygen Species (ROS) can cause decrease of motility, increase of apoptosis and impaired sperm function, and ultimately, influence on fertilization and pregnancy rate. Therefore, currently there is a great interest to the use of antioxidants to prevent ROS generation and ROS-induced apoptosis during the sperm preparation processes. Brain-derived neurotrophic factor (BDNF) is member of neurotrophin family that has anti- oxidant and anti-apoptotic effects on nervous system. Recent researches show that it also plays key role in male and female reproductive system such as spermatogenesis, nuclear and cytoplasmic maturation and embryo development.
Materials and Methods: Liquefied semen samples obtained from normozoospermic men (n=25) after preparation by double wash (400× gr, 5 min) swim-up technique were divided in to two groups of control and treated group (with BDNF). Then motility was evaluated by means of CASA. Reactive oxygen species (ROS) and apoptosis were assessed by flowcytometry.
Results: Addition of BDNF to the sperm media significantly increased mean total motility and progressive motility (75.54±2.63% vs. 66.81±2.48%, p<0.001, and 46.18±2.2 vs. 39.631±1.97%, p<0.002, respectively). Also BDNF treatment caused decrease of H2O2 (50.01±6.08 vs. 38.31±5.250, p<0.02), necrosis (23.26±2.71 vs. 27.52±2.27, p<0.001) and apoptosis (3.26±0.4 vs. 3.76±0.55, p<0.49).
Conclusion: Considering this result, BDNF treatment can be a potential tool against oxidative damage and apoptosis in human spermatozoa.
Key words: Brain-derived neurotrophic factor (BDNF), ROS, Apoptosis, Flowcytometry.
P-89
Iranian couple’s sexual compatibility during the time: A qualitative study
Nekoolaltak M1, Keshavarz Z1, Simbar M2, Nazari AM3, Baghestani AR4.
- Department of Midwifery and Reproductive Health, Faculty of Nursing and Midwifery, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
- Safe Motherhood Research Center, Department of Midwifery and Reproductive Health, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
- Department of Counseling, Faculty of Psychology and Education, Kharazmi University, Tehran, Iran.
- Department of Biostatistics, School of Allied Medical Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Introduction: Since sexual compatibility is facilitators for marital compatibility and the root of many divorces is the sexual incompatibility, and due to the limited data available in this topic, this qualitative study was designed to exploring the changes of Iranian couple’s sexual compatibility during marriage years.
Materials and Methods: In-depth interview and written narrative methods were used. Purposive sampling with maximum variation (age, education, years of marriage, number of children, etc.) was conducted. Ale Yasin Clinic in Tehran and Fatemieh Clinic in Varamin were the setting of research. Times of interviews were almost 40 min. Data reached to saturation after 14 semi-structured depth interviews and 54 written narratives with the 68 married men and women. Conventional content analysis was done in MAXQDA10 software. Lincoln and Guba criteria were used to validate qualitative data.
Results: Emerged categories included: “duration to reach sexual compatibility" and “mainly increasing sexual compatibility during the time" and "rarely decreasing sexual compatibility during the time". Couple’s sexual compatibility increases over time due to “Getting to know each other more", “increasing sexual awareness and experience”, “talking about sex with spouse”, “skill acquisition for mutual pleasure and satisfaction”. Couple’s sexual compatibility may decrease over the time because "children growing and conflict parental role with spousal role" and “financial difficulties”.
Conclusion: With knowing the impact of passing the time on couple’s sexual compatibility many divorces can be prevented by suitable planning and counseling.
Key words: Sexual compatibility, Iranian couples, Qualitative study.
P-90
Effect of non-polar extract of Phaleria macrocarpa (Mahkota Dewa) on spermatogenesis in rats
Parhizkar S1, Aziz Dollah M2, Ferdaus Binti N2.
- Medicinal Plants Research Centre, Yasuj University of Medical Sciences, Yasuj, Iran.
- Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra, Malaysia.
Introduction: Fertility is the natural capability of giving live. In male, fertility requires the production of large numbers of normal and mature spermatozoa by testes through a complex process call spermatogenesis. Evidence shows increase male age is associated with the decline of mature sperm in seminiferous tubule. Aqueous extract of Phaleria macrocarpa (PM) has been shown to improve fertility in rats by increasing the spermatogenesis.
Materials and Methods: Hexane extract of Phaleria Macrocarpa was prepared by extracting grinded dried slices of fruit followed with drying of the extract using rotary evaporator. A total of 30 male rats were divided into 5 different groups and subjected to daily treatments with PM extract at one of the following concentration: 0, 6, 12, 60 mg/kg of hexane extract, or commercial testosterone hormone for seven consecutive weeks. They will be fed with pellet once a day in the morning and drinking water will be given ad libitum. Body weight was measured once a week. On the last day of supplementation period, the rats were sacrificed and the testis was isolated for histological evaluation.
Results: The result showed that no significant effect of non-polar extract of PM on testes size and volume, thickness of seminiferous tubules and spermatogonia cell number. The mean thickness of seminiferous tubules were 62, 68, 62, 60, 60 µm for 60, 12, 6, 0 mg/kg of PM extract and commercial testosterone respectively. The mean for spermatogonia cell count in rats treated with 60, 12, 6 and 0 mg/kg and commercial testosterone were 52.8, 43.8, 47.0, 52.6, 52.0 cells respectively.
Conclusion: The study concludes that the non-polar extract of PM has no effect on the fertility of male.
Key words: Phaleria macrocarpa (PM), Spermatogenesis, Rat.
P-91
The functional SNP analysis of CYP2D6 gene in patients with Polycystic ovary syndrome (PCOS)
Peirosabet M1, 2,Khosravifar M2, Chekini Z2, Rahimi A1, 2, Shiva M3, Afsharian P2.
- Department of Basic Science and Advanced Technologies in Biology, University of Science and Culture, Tehran, Iran.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Endocrinology and Female Infertility at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: PCOS is a common ovulatory disturbance, which influents about 5-10% women of reproductive age. Clomiphene citrate is used for ovulatory induction in PCOS, that is metabolized by Cytochrome P450 2D6 (CYP2D6). More than 120 variable alleles have been reported for CYP2D6 that some of them are poor metabolizer and cause resistance to drugs that are substrates of CYP2D6.
Materials and Methods: Whole blood was collected from drug control, PCOS and fertile women (60 in each group). RFLP method was used to determine CYD2D6*34 (2850C˃T). Patients should have the Rotterdam criteria. Inclusion criteria’s were age under 35 years old, candidates for IUI, not using other drugs and not have ovary cautery.
Results: Genotype distribution of CC, CT and TT in PCOS group was 47.4%, 40.4% and 12.3% respectively (p=0.479), and frequency of these genotypes in drug control group was 36.7%, 50% and 13.3% respectively (p=0.479), which was very close to results in fertile group: 33.3%, 56.7% and 10% respectively. The number of antral follicles (follicle diameter≥15mm) was measured as drug response which was 1.21 (p=0.118) in clomiphene citrate administered PCOS patients (64%) while this was 1.64 (p=0.09) in 36% of patients treated with letrozole (follicle diameter≥15mm). Same results were observed in drug control group: antral follicles were 1.66 in 80% of clomiphene citrate treated whereas it was 1.77 when they received letrozole (20% of patients).
Conclusion: Although genotype frequencies of 2850C˃T polymorphisms were not significant between three groups, we observed cases bearing this polymorphism, showed resistance to clomiphene citrate, in both groups, which is the first time to report.
Key words: CYP2D6, Polycystic ovary syndrome, Clomiphene citrate.
P-92
Does L-carnitine therapy add any extra benefit to standard inguinal varicocelectomy in terms of deoxyribonucleic acid damage or sperm quality factor indices: A randomized study
Pourmand GH1, Movahedin M2, Dehghan S1, Mehrsai A1, Ahmadi A1, Pourhosein M1.
- Tehran University of Medical Sciences, Tehran, Iran.
- Tarbiat Modares University, Tehran, Iran.
Introduction: Varicocelectomy and anti-oxidant therapy are both documented to have positive effect on spermatogenesis and improving semen quality.
Materials and Methods: 100 patients enrolled in this study and were randomly divided into 2 groups (50 patients in each group). In group 1, standard inguinal varicocelectomy and, in group 2, standard inguinal varicocelectomy plus oral antioxidant therapy (oral L-carnitine, 250 mg 3 times a day) were performed for 6 months. For all patients, routine semen analysis and DNA damage test of spermatozoa (by 2 methods of terminal deoxynucleotidyl transferase dUTP nick end labeling and protamine damage assay) were performed at baseline and at 3 and 6 months postoperatively.
Results: In both groups, the improvement in semen analysis parameters and DNA damage was observed, but there was not any statistically significant difference between the 2 groups in these parameters, although the slope of improvement in DNA damage was slightly better in group 2 (that was not statistically significant).
Conclusion: We observed that addition of 750 mg of L-carnitine orally daily to standard inguinal varicocelectomy does not add any extra benefit in terms of improvement in semen analysis parameters or DNA damage.
Key words: DNA damage, Varicocelectomy, L-carnitine therapy.
P-93
Different haplotype frequency in AKAP3 gene in Iranian patients with short tail sperm
Poursafari E1, Mohseni Meybody A2, Sabbaghian M2, Modarresi T2, Hosseini H1, Gourabi H2.
- University of Science and Culture, Tehran, Iran.
- Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: One kind of sperm abnormalities that leads to men infertility is short flagella of sperms. In this defect, fibrous sheath and axoneme are disorganized, the sperms tail is short, the numbers of sperms in the semen fluid reduce and the sperms are immotile. A Kinas anchoring protein 3 (AKAP3) gene encodes a protein that is involved in the fibrous sheath structure, regulation of sperm motility and head-associated functions such as capacitation and the acrosome reaction. AKAP3 interacts with the regulatory subunit of Protein Kinas A via its dimerization/ docking domain. In the present study, 30 patients with short tail sperm defect and 40 males with normal spermogram referred to Royan Institute were enrolled as case and control groups respectively. The genetic variation in exon 5 of AKAP3 gene which encodes the functional domain of this protein was studied.
Materials and Methods: PCR- sequencing was done on extracted DNA from blood samples of control and patient groups.
Results: According to the results, four haplotype polymorphisms 1378 T>C, 1391 C>G, 1437 T>C and 1573 G>A were observed in all samples studied. These polymorphisms were all observed as the mutant alleles. 92% C allele and 8% G allele of 1391 C>G polymorphism has been reported in East Asia. In the present study, 100% G allele was observed in this polymorphism.
Conclusion: This difference in frequency of mutant allele can be due to different ethnic of Asian population and ours. 1391 C>G alternation is located on the outside of the AKAP3 binding domain to AKAP4 and seems that it cannot have a role in the interaction between these two proteins.
Key words: Short tail sperm defect, Fibrous sheath, AKAP3 gene, PKA, Polymorphism.
P-94
Study of genetic alterations of STK11 in patients with polycystic ovary syndrome (PCOS) and their response to ovarian stimulation
Rahimi A1, 2, Khosravifar M2, Peirosabet M1, 2, Chekini Z2, Shiva M3, Afsharian P2.
- Department of Basic Science and Advanced Technologies in Biology, University of Science and Culture, Tehran, Iran.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Endocrinology and Female Infertility at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: PCOS is the main reason of anovulatory caused infertility. Metformin use for PCOS patients to reduces hyperinsulinemia and androgens production through activation of STK11. We aimed to study the associations between c.842C>T and c.996G>A polymorphisms and risk of PCOS susceptibility in women plus the relations of two polymorphisms and response to ovarian stimulation in PCOS patients.
Materials and Methods: Blood samples were collected from patients and drug control group (male infertility) and fertile group (60 in each group). For genotype analysis, we used RFLP method. We used Rotterdam criteria for patient selection. Inclusion criteria's were age below 35 years old, candidates for IUI, not using other drugs and not have ovary cautery. Our data shown that frequency distribution of CT, TT,CC genotypes in exon6, in PCO Patients was 1.7%, 0%, 98.3% respectively (p=0.236), these frequencies in fertile group was 5%, 0%, 95% respectively and in drug control group was 0%, 1.7%, 98.3% respectively (p=0.236). We have not detected c.996 G>A polymorphism in any groups.
Results: The mean of age and BMI were 26.31±3.68 years and 27 kg/m2 in PCOS and control group. Most of PCOS patients (64%) were received clomiphene citrate and 36% used letrozole whereas 80% of drug control was treated by clomiphene citrate and 20% used letrozole. In both groups their responses to drugs was measured by the number of antral follicles (follicle diameter ≥15mm) by ultrasonography that was 1.64 in PCOS, p=0.09 and 1.77 in drug control treated with letrozole that were better than clomiphene citrate (1.21 in PCOS, p=0.118 and1.66 in control).
Conclusion: Our data did not find any significant differences between these polymorphisms and response to ART medicines in PCOS women. According to our study, PCOS patients had higher response to letrozole rather than clomiphene citrate.
Key words: STK11, Polycystic ovary syndrome, Polymorphism.
P-95
Effects of electromagnetic fields of magnetic resonance imaging on male mice fertilization
Rostamzadeh A1, 2, Anjamrooz SH1, Fathi F1, Rezaie MJ1,3.
- Department of Anatomical Sciences and Genetics, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran.
- Brain and Spinal Repair Research Center, Tehran University of Medical Sciences, Tehran, Iran.
- Clinical Infertility Research Center, Kurdistan University of Medical Sciences, Sanandaj, Iran.
Introduction: Currently, the use of electromagnetic waves in medicine, especially in diagnostic devices such as magnetic resonance imaging (MRI) has increased and many of its biological effects have been reported. The aim of this study was to investigate the adverse effects of 1.5T MRI on fertility and reproductive parameters of male mice.
Materials and Methods: 40 NMRI adult male mice were randomly divided into two groups of control and experimental. The mice in the experimental group were exposed to MRI at 1.5T for 36 min once a week for a period of 3 weeks. Then, in the 1st day and 35th day after the final exposure, 10 mice were used for IVF and 10 mice for In vivo studies. MRI effects on testis weight, the duration of pregnancy, the number of newborns, sperm count, and fertility were evaluated. The obtained data were analyzed using ANOVA and Tukey’s tests.
Results: According to the present study, one day after MRI exposure, testis weight, sperm count, and the number of born children were significantly decreased in the experimental group (p<0.05). Moreover, a significant number of the embryos failed to develop to the blastocyst stage. In contrast, 35 days after exposure, no statistically significant difference was found (p>0.05).
Conclusion: Based on the results of the present study, it seems that although the MRI at 1.5T has adverse effects on fertility and reproductive parameters of the adult male mice, these side-effects are reversible.
Key words: Electromagnetic fields, MRI, Sperm, Fertility.
P-96
Interference of histone modification with aberrant expression of HOXA10 gene in eutopic and ectopic endometrial tissues of women with endometriosis during menstrual cycle
Samadieh Y1, Shahhoseini M1, Favaedi R1, Mahdian S3, Ramezanali F2, Ashrafi M2, Afsharian P1, Aflatoonian R2.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Endocrinology and Female Infertility at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: Disruption of the balance of epigenetic networks, which involves DNA and histone modifications, can cause several pathologies, including reproductive disorders such as endometriosis. HOXA10 gene expressed in endometrium plays an important role in uterine receptivity at the time of implantation, uterine organogenesis and functional endometrial differentiation.
Materials and Methods: Epigenetic analysis were assayed by Chromatin Immunoprecipitation (ChIP), using anti- H3K9ac, H3K9Me2, H3K27Me3, H3K4Me3 antibodies and quantitative expression analysis was performed by real-time PCR technique. For this respect, eutopic and ectopic endometrial samples were collected using laparoscopy from 36 women with documented endometriosis, and also endometrial biopsies were obtained from 22 healthy women with male factor problem as a control group. Ethical approval and informed patient consent was gained for the use of tissue samples.
Results: Data showed a harmonious pattern between mRNA expression of HOXA10 and epigenetic state of its promoter region, in the way that, in secretory phase the activating epigenetic marks, H3k9ac and H3k4me3 were higher in ectopic endometrial tissues and H3K9ac itself were lower in eutopic endometrium. In contrast, H3K9me2 and H3k27me3, the epigenetic marks, known to be associated with gene repression, showed a different pattern in which that, H3K9me2 were higher in eutopic endometrium and H3K27me3 were lower in ectopic endometrial tissues.
Conclusion: Our findings suggest epigenetic might be greatly responsible for aberrant expression of HOXA10 gene in eutopic and ectopic endometrial lesions of patients with endometriosis.
Key words: HOXA10 gene, Endometriosis, Menstrual cycle, Histone modifications, Epigenetics.
P-97
Evaluation of action and mechanism of L-Carnitine in improved sperm quality in male infertility
Sheikhpour R.
Yazd Medical Science Branch, Islamic Azad University, Yazd, Iran.
Email: R.sheikhpour@yahoo.com
Introduction: About 15% of married couples are infertile and that approximately 50% of this is due to male factor infertility. A number of drugs have been proposed as being possible causes of male factor infertility. Estudies showed that a diet supplemented with L-carnitine can improve sperm quality in some mammalian species. In humans, 75% of carnitine derives from diet, while 25% is synthesized from lysine and methionine, although the enzyme that catalyses the hydroxylation of the 4-butirrobetain in L-carnitine is present in few tissues.
Materials and Methods: This review was conducted using the following databases: Medline, Pubmed, Scopus and Science direct from 1990-2014 for better understanding of the mechanism and action of L-Carnitine in male infertility.
Results: L-Carnitine can increase the concentration of this component in the epididymal tubules and spermatozoa. This molecule increases their motility and fertilizing abilities. Both free L-carnitine and acetylated L-carnitine can be accumulated in spermatozoa. In sperm cells, L-carnitine transports fatty acid to mitochondria for oxidation and production of energy for epididymal spermatozoa. Also L-Carnitine has antioxidant property; it via deleting excess acyl-CoA (formyl-CoA), due to toxic effect have important role in cellular detoxification. L-carnitine as antiaging protects cellular membranes against oxidative damage. It prevents protein oxidation, pyruvate and lactate oxidative damage. During sperm maturation, a reduction in total lipid content has been seen due to the changing composition of fatty acids in sperm cells. This leads to an increase in the fluidity of spermatozoa’s plasmalemma. Moreover, L-carnitine reduces lipid availability for peroxidation which guards against potential peroxidative damage.
Conclusion: The result of studies showed that addition of L-carnitine to the diet of male infertility is effective in increasing semen quality, but these results need to be confirmed by more clinical trials.
Key words: L-Carnitine, Sperm quality, Male infertility.
P-98
Effects of cell phone radiofrequency electromagnetic waves (RF-EMW) on human spermatozoa
Soltani S1, Khalili MA1, Omidi M1, Nabi A1, Shamsi F2.
- Department of Biology, Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Biostatistics and Epidemiology, College of Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: Cellular phones emit radiofrequency electromagnetic waves (RF-EMW) in the low frequency microwave. The detrimental effects of RF-EMW on the reproductive system and human fertility have been debated in recent years. The goal was to assess the influence of cell phones RF-EMW on different parameters and DNA integrity of human spermatozoa.
Materials and Methods: 50 semen samples were categorized in two groups: a) normospermia (n=24); and b) asthenozoospermia (n=24). After liquefaction, each sample was divided into two aliquots. One aliquot (experimental) was exposed to mobile radiation for 1 hr, and the second aliquot (unexposed) served as control. The sperm parameters and chromatin/ DNA integrity were examined and compared between groups.
Results: Normal samples exposed to RF-EMW showed insignificant differences in sperm motility, viability, and chromatin/DNA integrity. Regarding sperm morphology, normal morphology was significantly decreased in experimental group (p=0.04). Moreover, sperm parameters as well as DNA structure showed no significant differences in astenozoospermic samples.
Conclusion: Data did not show any noticeable impact of RF-EMW on human sperm quality. Further studies should be considered for confirmation of the results.
Key words: Cell phone, Electromagnetic radiation, Sperm parameters, DNA integrity.
P-99
Assessment of genetic variations in intron4 and exon5 of RABL2B gene in Iranian infertile men with immotile short tail sperm defect
Hosseini H1, Sabbaghian M2, Mohseni Meybodi A2, Hosseinifar H2, Poursafari E1, Sadighi Gilani MA2.
- Department of Basic Science and Advanced Technologies in Biology, University of Science and culture, Tehran, Iran.
- Department of Andrology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: The immotile short tail sperm (ISTS) defect, is a syndrome which causes male infertility. Patients with ISTS disorder have immotile short-tailed sperm with disorganized axonem, and a significant decrease in sperm counts too. Numerous proteins are involved in sperm tail formation. One of these proteins is RAB Like 2B (RABL2B), which recently its essential role in sperm intra-flagellar transport and fertility in male mouse has been demonstrated. So its gene, which called RAB Like 2B (RABL2B), is an appropriate candidate gene in human studies. RABL2B protein has 4 GTP binding domains which have important roles in protein function. Exon 5 of RABL2B gene, codes one of these main domains and intron 4 is the location for binding to some important transcription factors. The purpose of this study was to evaluate the genetic variations of exon 5 and intron 4 of RABL2B gene in infertile men with ISTS defect and controls
Materials and Methods: In this study, 30 infertile men with ISTS defect and 30 normozospermic men as controls were recruited. Remarkably it took 2 years to collect patients samples. To study the genetic variations, DNA was extracted from peripheral blood, then PCR sequencing was done.
Results: Sequence analysis results did not identify any mutations or single-nucleotide polymorphisms (SNPs) in exon 5, but an intronic variant (rs:144944885), was found in heterozygote form in one patient. No mutations or SNPs was identified in controls.
Conclusion: Although our data just revealed an intronic variations and no mutations or SNPs was detected in exon 5, due to the high expression of RABL2B gene in testis, and considering the fact that RABL2B is evolutionarily conserved and not many studies have been conducted about the exact role of this gene in human male fertility, evaluation of other exons and regulatory areas of this gene is strongly recommended.
Key words: RABL2B gene, ISTS, Male infertility.
P-100
Localization of septin 14 protein in sperm
Vahabi Barzi N, Hosseinifar H, Nickhah Kalashami Z, Sadighi Gilani MA, Sabbaghian M.
Department of Andrology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: nastaranbarzi@yahoo.com
Introduction: In mammals, 14 septin genes have been identified so far. Disruption of septin functions has been implicated in the pathology of many diseases, including male infertility. Here, we study about one of the new members of the septin family called septin14 that is specially expressed in testis. This gene has two transcripts but only one of them transcript into proteins.
Materials and Methods: In this study, semen obtained from subjects attending Royan Institute that had normal spermogram. After that, immunocytochemistry, which is a common laboratory technique, was used to anatomically localize presence of a specific protein in cells by a specific primary antibody. The primary antibody allowed visualization of the protein under a fluorescence microscope when it was bound by a secondary antibody with a conjugated fluorescence.
Results: The protein expression was detected in the sperm from head to tail and highly localized in the front of the acrosome and the neck.
Conclusion: This is the first report on the localization of septin 14 in sperm. Septin 14 plays an important role in sperm morphology. Regarding the presence of this protein in sperm acrosome and neck, it can be concluded that the probable decrease of septin 14 protein expression in sperm may be associated with the pathogenesis of male infertility.
Key words: Septin 14, Immunocytochemistry, Sperm, Male infertility.
P-101
Molecular studying of sperm surface proteins and their cellular functions
Vaseghi-Dodaran H1, Daghighkia H1, Atshan M2, Jafari S3, Sobhani AGH4.
- Department of Animal Sciences, Faculty of Agriculture, Tabriz University, Tabriz, Iran.
- Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Midwifery and Fertility Health, School of Nursing and Midwifery, Ardabil University of Medical Sciences, Ardabil, Iran.
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Introduction: Fertilization in mammalian is a complicated process with some connections among wide range of glycolipid, glycoprotein and antigenic indexes on sperm and egg surface of every species. We can improve our understanding in fertilization mechanisms and also get some information on molecular or immunological defects responsible for infertility by recognizing the antigens on sperm surface and investigating their role on cell physiological performance. The key challenge is to move from lists of identified proteins to informed understanding of biological function. Studying the sperm surface molecules and investigating their biochemical, biophysical and physiological properties to understand fertilization process.
Materials and Methods: A review of recent bibliography collected from internet database as PubMed, Google scholar, SID by the use of relevant keywords on different markers of cell surface and Sperm cell proteomic published in 2000-2014.
Results: Sperm membrane proteins contain an important part of membrane and are considered as special antigenic index to each species. Every change, increase or decrease in these proteins alters sperm performances and abilities. The important events which occurs during the interaction of sperm and egg and the reactions between them for fertilization relates to identification of these two cells by connecting receptors to ligands which exist on their cell membrane. Discovering the proteins on sperm surface which act as fertility biomarkers and participate in fertilization leads to more apprehension of this cell physiology and performance, so understanding molecular events like sperm acrosome reaction, capacitation and motility.
Conclusion: Sperm surface proteins can use as fertility biomarkers, therefore identification and supplementation these proteins to cryopreservation medium can improve post-thaw parameters of sperm and prevent sperm quality during cryopreservation.
Key words: Proteomics, Sperm, Marker, Fertilization.
P-102
Frequency of chromosome inversion (pericentric and paracentric) in recurrent abortions in patients referred to cytogenetics laboratory of Sarem Hospital
Abdi A1, Bagherizadeh I2, Bahman I2, Shajarehpour L1, Hadipour F3, Hadipour Z3, Shafagh Y4, Behjati F5.
- Azad Islamic University, Tehran, Iran.
- University of Social Welfare and Rehabilitation Seiences, Tehran, Iran.
- University of Ghom, Ghom, Iran.
- School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
- Department of Medical Genetics, Tehran University of Medical Sciences, Tehran, Iran.
Introduction: Recurrent abortion affects almost 15% of diagnosed pregnansis. More than 50% of miscarriages in the 1st trimester are due to chromosome abnormality. The aim of this study is to present the role of chromosomal inversions in recurrent miscarriages in patients referred to the Cytogenetics Laboratory of Sarem Hospital in Tehran. One group of inversions is pericentric inversion of chromosome 9, which is usually regarded as a normal population variant.
Materials and Methods: The samples were studied using high resolution GTG banding technique. For each patient, a minimum of 15 metaphases was examined by light microscopy.
Results: Pericentric inversion around centromere of chromosome 9 was observed in 29 patients (1.37%) and pericentric inversion in hetrochromatin region was found in chromosome 1 in one patient and chromosome Y in another patient, and one patient had pericentric inversion of chromosome 2. Chromosomal inversion involving other autosomal chromosomes included pericentric inversion of chromosomes 1, 5, 11 and 12 (1.9%), and paracentric inversion of chromosomes 3, 6, 7, 8, and 12 (0.23%).
Conclusion: The chromosomal imbalance of gametes may produce spontaneous abortions and malformed offsprings. This suggests that such inversions should not be ignored and they can play an important role in reproduction failure. However, we have shown that the rate of pericentric inversion of chromosome 9 is similar in different referral groups (1-2%) and similar to normal population and thus of no clinical significance.
Key words: Pericentric and paracentric chromosome inversions, Recurrent abortions, inv (9).
P-103
Kit Ligand (KL) promotes the primordial follicle growth in mouse vitrified ovaries
Abdi Sh1, Salehnia M1, Hosseinkhani S2.
- Department of Anatomy, Tarbiat Modares University, Tehran, Iran.
- Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Introduction: Following cryopreservation of ovarian tissue, the primordial follicles are well preserved with minimal damage. An alternative technique to improve the development of follicles within ovarian tissue is ovarian organ culture following cryopreservation. Kit ligand (KL) known as stem cell factor, steel factor or mast cell growth factor is involved in the activation of primordial follicles, oocyte growth and proliferation of granulosa and theca cells.
Materials and Methods: One week old mouse ovaries were collected and divided into vitrified and non-vitrified groups. Then they were cultured in the presence or absence of KL for 7 days. The development of ovarian follicles was evaluated by histology and also the mean area and hormonal level was analyzed during culture period. Apoptosis assessment was done using DNA laddering, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) method and caspase -3/7 activity assay.
Results: The proportion of preantral follicles and the level of hormones were increased in all cultured groups and it was significantly higher in KL treated groups than their control (p<0.001). DNA fragmentation and TUNEL positive signals were seen in vitrified cultured ovaries. The level of caspase -3/7 activity was higher in vitrified cultured ovaries.
Conclusion: KL could improve the development of follicles in vitrified cultured ovaries also it could act as anti-apoptotic factor during culture of vitrified samples. The development potential of follicles in vitrified groups was lower than fresh ovaries.
Key words: In vitro culture, Kit Ligand, Vitrification, Caspase -3/7.
P-104
Association between rs1264457 A/G polymorphism of HLA-E gene and recurrent pregnancy loss
Zarezade Z1, 4, Ashrafzadeh Mehrjardi HR2, 4, Zaimy MA3, Ghasemi N4.
- Department of Medical Genetics, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Biology, Faculty of Science, Azad University, Ashkezar Branch, Yazd, Iran.
- Department of Medical Genetics, Tehran University of Medical Sciences, Yazd, Iran.
- Recurrent Abortion Research Center, Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: Recurrent pregnancy loss (RPL) is defined as two or more consecutive miscarriages before twentieth week of pregnancy. The cause of 50% recurrent miscarriages is unexplained. The goal of this research was to investigate the association between RPL and rs1264457 A/G polymorphism of HLA-E gene.
Materials and Methods: In this case-control study we used Polymerase Chain Reaction Restriction Fragment Length Polymorphism (RFLP-PCR) to determine frequency of this polymorphism in 105 women with RPL in comparison with 109 healthy controls. The collected data were analyzed by the statistical package for the social sciences software (SPSS) and the chi-square test.
Results: The finding showed clear correlation between this polymorphism and RPL. The results will be presented in the congress.
Conclusion: If distinctive data increase frequency of this polymorphism in RPL, it can exponent influence of this polymorphism in generating RPL. We hope result of this investigation to be useful for finding some causes of unexplained recurrent pregnancy loss.
Key words: Recurrent pregnancy loss, HLA-E, Polymorphism.
P-105
Evaluation of the meiotic spindle and zona pellucida after vitrification of mouse MII oocytes
Akyash F, Khalili MA, Safari S, Ashourzadeh S, Agha-Rahimi A, Khoradmehr A.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: fakyash@gmail.com
Introduction: Oocyte vitrification is used as fertility preservation method for young aged females with cancer. The oocyte is one of the biggest mammalian cells with large volume of cytoplasm, compares with other cells. Also, oocyte has less permeability to cryoprotectants (CPAs), since high concentration of CPAs used in vitrification procedure with possible toxic effects. Evaluation of the effect of vitrificaiton on meiotic spindle (MS) and zona pellucida (ZP) of mouse MII oocytes using Polyscope technology was the subject of this study.
Materials and Methods: Ovulation induction performed for 6-8 weeks old NMRI mice by injection of 10 IU PMSG (IP) and 48 hr later with 10 IU HCG. After oviduct removal, COCs were denuded and MII oocytes were retrieved aseptically. The equilibration (7.5% EG, 7.5% DMSO) and vitrification (15% EG, 15% DMSO) solutions were prepared in Hams F10 supplemented with 20% HSA as base medium. For 3 steps of thawing, base medium plus 1, 0.5 and 0.25 M sucrose were used respectively. Assessment of ZP and MS performed at different time intervals after thawing.
Results: After warming, vitrified oocytes showed a significantly fast ZP digestion timing compared to the control group (p<0.05). Presence of MS was detected in 69.23% of vitrified oocytes 1 hr after warming, and presence of MS was showed in 11.11% of vitrified oocytes 2 hr after warming and 26.92% of vitrified- warmed oocytes showed sign of degeneration.
Conclusion: ZP digestion was faster in vitrified group rather than control and recovery of MS structure after vitrification/ warming at 37oC. Vitrification technique still needs more modification before its application in fertility preservation program.
Key words: Oocyte, Meiotic spindle, Zona pellucida, Vitrification.
P-106
Protective effect of vitamin E on testicular germ cells and serum Malondialdehyde concentration in rats fallowing exposure to bisphenol A
Amjadi M, Soleimani Mehranjani M, Naderi Noreini S.
Department of Biology, Faculty of Sciences, Arak University, Arak, Iran.
Email: m.amjadi1369@gmail.com
Introduction: Bisphenol-A (BPA), used in plastic industries, has toxic effects on the reproductive system. It can cause testis injury through the production of Reactive oxygen species (ROS).
Materials and Methods: 24 adult male rats (220±15 gr) were divided into 4 groups (n=6): control, BPA (250 mg/kg/day), Vit E (150 mg/kg/day) and BPA+ Vit E. All groups were orally treated for 56 days. By the end of the treatment, animals were killed, their right testis were taken out, fixed, sectioned, processed and stained with heidenhain azan method. The total number of germ and Sertoli cells was estimated using the optical dissector technique. Serum MDA levels were also measured with the spectrophotometric method. Data were statistically analyzed using one way ANOVA and Tukey's test and means were considered significantly different at p<0.05.
Results: A significant decrease in the total volume of testis, number of long and round spermatids, spermatocytes and Sertoli cells (p<0.05) and a significant increase (p<0.03) in the serum MDA level were found in rats treated by BPA compared to the control group. Histopathology observations revealed morphological changes in BPA-exposed rats including atrophy and vacuolation in the germinal epithelium. The above parameters were compensated to the control level in the BPA+ Vit E group.
Conclusion: Due to the antioxidant activity of vitamin E, it may ameliorate the damaging effects of bisphenol A on spermatogenesis.
Key words: Bisphenol A, Vitamin E, Sertoli and germ cells number, Optical disector.
P-107
Evaluation of follicular genes pattern and growth of preantral follicles after culture in alginate hydrogel following vitrification of the mouse ovarian tissue
Asgari F1, Valojerdi MR1, 2, Ebrahimi B 2.
- Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
- Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: This study was set up to evaluate the effect of ovarian tissue vitrification on the in vitro growth and pattern of follicular genes expression in mouse preantral follicles encapsulated within alginate hydrogel.
Materials and Methods: Ovaries of 12-14 days old female NMRI mice allocated into fresh control and vitrification groups. For cryopreservation, ovaries equilibrated with a solution (ES) that composed of 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 15 min and vitrification was performed by a solution (VS) that composed of 15% EG and 15% DMSO for 30 min then ovaries loaded to nitrogen with needle. Descending concentrations of sucrose (1, 0.5, 0.25 M) used for warming step. After histologic assessment of ovaries, in the next stage, pre-antral follicles was mechanically isolated from control and vitrified ovaries and was cultured for 12 days in 0.7% sodium alginate. Preantral follicles survival rate, growth, antrum formation and relative expression of oocyte- specific genes (Bmp15, Gdf9،Fgf8, Igf1, Kit, Kit ligand) was assessed after 1, 8 and 12 days of culture and finally maturation rate of oocytes was studied.
Results: Preantral follicles in vitrified group showed a lower survival rate on 8 and 12 days of culture (p<0.05) but could retain a comparable morphological appearance, growth and antral formation with the control group. Reduction of Bmp15, Gdf9, Fgf8, Kit, Kit-l showed during 12 days of culture (p<0.05). Although the expression of Gdf9, Kit, Kit-l in vitrification group was more than control group at 1st day of culture but all genes in both groups showed same expression after 12 days of culture (p<0.05).
Conclusion: Although vitrification of ovarian tissue reduces the survival rate, it is a safe method for preservation of preantral follicles and could not modify the relative expression of follicullar genes and oocytes maturation capacity.
Key words: Follicular genes, Preantral follicle, Vitrification of ovarian tissue, Alginate.
P-108
Effects of morphine on sperm parameters, protamine deficiency and DNA integrity in mice
Bahari H1, Talebi AR1, 2, Nahangi H1, Anvari M1, 2, Abbasi Sarcheshmeh A1, Khoradmehr A2, Danafar AH2.
- Department of Biology and Anatomy, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: Morphine as a natural alkaloid (opiate) is the most effective pain-relieving drugs and can be abused because of its high addictive potential. Opiate abuse is considered as one of the problems associated with poor semen production and sperm quality. Therefore, for the first time, this experimental study was carried out to evaluate the effect of intraperitoneal injection of morphine on sperm parameters, protamine deficiency and DNA integrity of spermatozoa aspirated from cauda epididymis of mice.
Materials and Methods: Totally 24 adult male Balb/c mice (8 weeks old. 30g) were equally divided into 3 groups each containing 8 mice. Mice of group 1 served as control fed on basal diet, group 2 received basal diet and normal saline and group 3 received basal diet and morphine (15 mg/kg/daily, intraperitoneal) for 35 days. Finally right tail of epididymis of each mouse was cut and placed in Ham’s F10 for 30 min. Released sperm were used to analyze count, motility, viability (eosin-nigrosin staining), morphology (Papanicolaou), protamine deficiency with chromomycin A3 (CMA3) and apoptosis via TUNEL assay.
Results: In morphine-treated mice a significant decrease was found in sperm viability, normal morphology, count and motility compared to other groups (p<0.05). In relation with protamine deficiency the rates of CMA3-reacted spermatozoa were similar in groups (p>0.05). In addition, in morphine-treated mice there was a significant increase in apoptosis compared to other groups (p<0.05).
Conclusion: The results showed that morphine abuse disturbs sperm parameters and DNA integrity but not protamine content of the sperm nucleus.
Key words: Mice, Morphine, Sperm parameters, Apoptosis, Protamine deficiency.
P-109
Study the protective role of jujube extract on growth index of mice fetuses exposed to Carbamazepine
Doostabadi MR1, Afshar M1, Nahangi H2, Hasanzadeh Taheri MM1, Hassanpour M1.
- Birjand University of Medical Sciences, Birjand, Iran.
- Shahid Sadoghi University of Medical Sciences, Yazd, Iran.
Introduction: Carbamazepine (CBZ) is an anticonvulsant drug that is widely used, primarily to treat seizures. One of the side effects of this medication during pregnancy is the reduced fetal growth. The jujube has been widely used in traditional medicine because of its special compound. The purpose of this study was to evaluate the preventive effects of aqueous extract of jujube on growth index of mice fetuses exposed to Carbamazepine.
Materials and Methods: In this study, 100 Balb/C mice (28-30 gr; 8-9 weeks-old) were divided into eight experimental and two control groups. Experimental groups (I, III, V) and (II, IV, VI) received daily intraperitoneal injections of 50 and 100 mg/kg of CBZ, respectively. Experimental groups (III, IV) and groups (V, VI) were gavaged doses of 200 and 400 mg/kg of aqueous extract of jujube respectively. Groups VII and VIII received only doses of 200 and 400 mg/kg of aqueous extract of jujube respectively. Control groups I and II were gavaged to normal saline and tween respectively. Injections were done on 0-15 gestational days (GD), and aqueous extract was gavaged along with CBZ starting 10 days prior to gestation. On GD 18, fetuses were removed for weighing and crown-rump measuring. Data were analyzed by ANOVA and Tukey tests using SPSS (version 18) at 0.05%.
Results: Average weight and body length of fetuses in experimental groups I and II, that received CBZ, significantly reduced compared with control groups. In the fetuses of experimental groups V (1.20±0.38) and VI (1.21±0.21 gr) that recieved jujube extract (400 mg/day) with CBZ, the mean weight of these fetuses increased meaningfully in comparison with the experimental groups I (0.93±0.19 gr) and II (0.92±0.26 gr) respectively.
Conclusion: This study showed that aqueous extracts of jujube can have preventive effects on reducing effect of CBZ on birth fetal weight.
Key words: Growth index, Jujube, Carbamazepine, Mice.
P-110
Effectiveness of mindfulness-based cognitive therapy on improvement of perceived stress and infertility in infertile women undergoing IVF irrational cognitions
Dortaj A1, Mehdizadeh A1, Dortaj H2.
- Payame Nour University, Dubai, UAE.
- Shahid Sadoughi University of Medical Scienses, Yazd, Iran.
Introduction: Effectiveness of mindfulness-based cognitive therapy on improvement of perceived stress and infertility in infertile women undergoing IVF irrational cognitions.
Materials and Methods: This clinical trial with pre-test post-test was done in 1331 infertile women referred to the Reproductive Health Research Center, Crescent Hospital, UAE who were treated with IVF. In total, 24 infertile women filled an irrational cognitions questionnaire related to childbearing and infertility. They were divided into experimental and control groups. The experimental group received mindfulness-based cognitive therapy for 8 sessions of 2 hr each. The control group did not receive any mental health services. Those two questionnaires were completed before and after the intervention. Data were analyzed by SPSS and p<0.05 was considered significant.
Results: There were no significant differences between the experimental and control groups in terms of reduction of perceived stress of infertility. But, the improvements in the recognition of irrational for childbearing between two groups was significantly different.
Conclusion: Teaching mindfulness-based cognitive therapy on improvement of perceived stress and irrational cognitions related to fertility in infertile women undergoing IVF treatment is effective.
Key words: Perceived stress, Infertility, IVF treatment, Mindfulness-based cognitive therapy, Irrational cognitions of parents.
P-111
Genotype and phenotype frequencies of paraoxonase 1 in fertile and infertile men
Fattahi A1, Tavilani H1, Latifi Z1, Esfahani M2, Khodadadi I2, Karimi J2,Bahrayni E2, Vatannejad A3, Vaisi-Raygani A4, Ghorbani M2.
- Department of Clinical Biochemistry, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
- Department of Clinical Biochemistry, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran.
- Student’s Scientific Research Center, Tehran University of Medical Sciences, Tehran, Iran.
- Fertility and Infertility Research Center, Kermanshah University of Medical Science, Kermanshah, Iran.
Introduction: Paraoxonase1 (PON1) is a glycoprotein associated with high density lipoprotein and has antioxidant activity. The impact of PON1 in various stages of spermatogenesis has also been suggested.
Materials and Methods: Q192R variants of PON1 were determined in 150 fertile and 150 infertile men using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Plasma arylesterase and paraoxonase activities were detected by spectrophotometry and malondialdehyde (MDA) level was measured using thiobarbituric acid.
Results: Our results showed no significant difference in the distribution of PON1 genotypes and alleles between fertile and infertile groups. However morphology and motility of sperm were associated with various genotypes of PON1. The number of fertile males with the BB phenotype (high activity) was significantly higher than that of infertile males, whereas the number of individuals with the AB phenotype (moderate activity) was statistically higher in infertile men compared with the fertile group. Additionally, MDA and arylesterase activity levels were significantly higher in infertile subjects compared with fertile men.
Conclusion: We speculate that the low activity of PON1 can be a risk factor for male infertility probably due to a decrease in antioxidant activity of PON1 and increase in lipid peroxidation.
Key words: Genetic polymorphism, Male infertility, Paraoxonase 1, Reproduction.
P-112
Saccharin consumption increases sperm DNA fragmentation and apoptosis in mice
Rahimipour M, Talebi AR.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: marziehrahimipour@yahoo.com
Introduction: Saccharin is an artificial non-caloric sweetener that used to sweeten products such as drinks, candies, medicines, and toothpaste, but our bodies cannot metabolize it. Sodium saccharin is considered as an important factor in tumor promotion in male rats but not in humans.
Materials and Methods: Totally 14 adult male mice were divided into 2 groups. Group 1 served as control fed on basal diet and group 2 or experimental animals received distilled water containing saccharin (0.2% w/v) for 35 days. After that, the left cauda epididymis of each mouse was cut and placed in Ham’s F10. Swimmed-out spermatozoa were used to analyze count, motility, morphology (Pap-staining) and viability (eosin-Y staining). Sperm DNA integrity, as an indicator of apoptosis, was assessed by sperm chromatin dispersion (SCD) and terminal deoxynucleotidyl transferase (TUNEL) assay.
Results: Following saccharin consumption, we had a reduction in sperm motility with respect to control animals (p=0.000). In addition, the sperm count diminished (17.70±1.11 in controls vs. 12.80±2.79 in case group, p=0.003) and the rate of sperm normal morphology decreased from 77.00±6.40 in control animals into 63.85±6.81 in saccharin-treated mice (p=0.001). Also, we saw a statistically significant increase in rates of sperm DNA damage and apoptosis in experimental group when compared to control one (p=0.001, p=0.002 respectively).
Conclusion: Saccharin consumption may have negative effects on sperm parameters, and increases the rate of sperm DNA fragmentation and apoptosis in mice.
Key words: Sperm, Saccharin, Apoptosis, Mice.
P-113
Increased telomeric repeat containing RNA (TERRA) levels in cumulus cells of infertile polycystic ovary syndrome patients
Ghobadi N1, 2, Shahhoseini M2, Favaedi R2, Hassani F3, Movaghar M3, Karimian L3, Eftekhari Yazdi P3.
- Faculty of Basic Sciences and Advanced Technologies in Biology at University of Science and Culture, Tehran, Iran.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: One of the important reasons of anovulation based infertility is polycystic ovary syndrome (PCOS) which occurs in 5-10% of women in reproductive age. It seems that the length of telomere, TTAGGG tandem repeats, is related to proliferation and differentiation events during follicular development including the mechanisms which regulate successful reproduction. It is widely recognized that bi-directional communications exist between oocytes and the surrounding cumulus cells which are essential for the production of competent oocytes. During the past few years, study on cumulus cells showed that the length of telomere is longer in cumulus cells of mature oocytes than immature ones. Until now, telomeres have been considered to be transcriptionally silent but recent studies have clearly shown that transcription of subtelomere regions produces telomeric repeat containing RNA, named TERRA. In this study, we tried to identify the correlation of TERRA levels with oocytes maturation by evaluating of TERRA transcripts in cumulus cells of PCOS patients.
Materials and Methods: For this respect, cumulus cells were collected from 6 PCOS patients and 9 healthy women with male factor infertility through ICSI/ IVF procedure. TERRA transcripts were measured by using quantitative real- time PCR.
Results: Our data showed that the level of TERRA transcripts was increased significantly in PCOS group vs. control group (p˂0.05).
Conclusion: This finding implies a considerable association between TERRA transcript levels and PCOS, so it can be concluded that any changes in levels of TERRA transcripts can be judged as a potential marker for the quality of oocytes in ART procedure.
Key words: TERRA, Telomere, PCOS, Cumulus cells.
P-114
Epigenetic modification profile of endometrium in endometriosis patients
Hosseini E1, Afsharian P2, Ashrafi M3, 4, Ramazanali F2, Mehraein F1, Shahhoseini M2, Aflatoonian R3.
- Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Endocrinology and Female Infertility at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Obstetrics and Gynecology, Iran University of Medical Sciences, Tehran, Iran.
Introduction: Endometriosis defined as the presence of endometrium-like tissue outside of the uterine cavity, is a common gynecologic disorder. Although endometriosis is a multifactorial disease and the exact etiology is not clearly understood, recently, some evidence suggests that epigenetic is associated with the molecular features of endometriosis. Two main epigenetic regulatory mechanisms, DNA methylation and histone modifications regulate expression of genes and recognize the states of diseases. The aim of this study was to investigate alterations of DNA methylation and histone acetylation and methylation levels in eutopic and ectopic endometrium of endometriosis patients.
Materials and Methods: Informed consents were gained from all patients then eutopic and ectopic endometrium samples (n=5) were collected from endometriosis patients undergoing surgery and biopsy, as well as endometrial tissues from healthy fertile women (n=5) during the proliferative phase of menstrual cycle. Chromatin extracts from samples were prepared following fixation and then shearing into fragments by sonication. Nucleosome ELISA was performed on chromatin extracts, in order to identify global histone H3K9 acetylation/ methylation and DNA methylation, using antibodies against H3K9ac, H3K9me and MeCP2, respectively.
Results: We have identified global histone H3K9 hypermethylation in ectopic and eutopic endometrium, compared with controls. A significant hyperacetylation at histone H3K9 was observed in eutopic samples compared to ectopic and control groups. Furthermore, eutopic endometrial samples were globally DNA hypermethylated in comparison with controls.
Conclusion: These results clearly show an epigenetic switch in endometrial and endometriotic tissue of patients with endometriosis, in the way that aberrant DNA methylation and histone acetylation/ methylation status may play a dynamic role in occurrence of endometriosis and support the opinion that epigenetic abnormalities have causative functions in endometriosis.
Key words: Endometriosis, Endometrium, Epigenetic, Methylation, Acetylation.
P-115
The effects of chronic and acute ethanol administration on sperm chromatin parameters in mice
Hosseini A, Borzouei Z, Faramarzi A, Ghasemi-Esmailabad S, Talebi AR.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: hosseinia30@yahoo.com
Introduction: Sperm chromatin can be damaged by effects of some toxic materials. Among this, ethanol is one of the reproductive toxins that consumes abuse in men and may be associated with poor sperm quality. The purpose of this investigation was to examine the acute and chronic effects of ethanol consumption on sperm parameters and chromatin assay through testing the effects in mice.
Materials and Methods: Animals were randomly divided into 3 groups consisting of 10 mice each. Group 1 and 2 served as alcohol group and were received a daily dose of (3 g/kg body weight as 25%, v/v) ethanol by I.P. for four and eight weeks respectively. Group 3 (control group) was given normal access of food and water. The subdivisions and dosages were based on past works. At the end of treatments, laparotomy was conducted to expose the reproductive system, cauda of epididymis were immediately dissected out for evaluations of sperm parameters and various tests were used to analysis of the chromatin integrity of sperm.
Results: Various tests such as aniline blue (AB), chromomycin A3 (CMA3), toluidine blue (TB), and acridine orange (AO) were used to analysis the sperm quality. We did not find any statistically significant differences in acute and control groups. Although sperm count and motility were increased but viability, normal morphology, DNA and chromatin integrity were unaffected by ethanol in acute group and were identical with control group. Our results demonstrated that count, motility and viability of sperms were dramatically decreased in chronic treated animals. Besides of that, DNA and chromatin integrity were significantly decreased.
Conclusion: Therefore, ethanol abuse can induce abnormality in sperm structure and function, and this may be one possible cause of male infertility.
Key words: Sperm, Ethanol, Chromatin integrity.
P-116
Comparison of oral dydrogesterone with vaginal progesteronefor luteal support in IUI cycles: A randomized clinical trial (RCT)
Khosravi D1, Taheripanah R1, Khosravi R1, Taheripanah A2, Tarighat Monfared V1, Defaee S1, Mehdighalb S1, Hosseini Zijoud SM3.
- Reproductive Health Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
- Department of Molecular and Cellular Sciences, Faculty of Advanced Sciences & Technology, Pharmaceutical Sciences Branch, Islamic Azad University, Tehran, Iran.
- Hamadan University of Medical Sciences, Hamadan, Iran.
Introduction: Progesterone supplementation is the first line of treatment in ovarian stimulation. This study was conduct to compare the effect of oral dydrogestrone with vaginal cyclogest on luteal phase support in intrauterine insemination (IUI) cycles.
Material and methods: This prospective, randomized, double blind study was performed in a local infertility center from May 2013-2014. It consisted of 150 infertile women younger than 35 years old undergoing ovarian stimulation for IUI cycles. They underwent ovarian stimulation with oral dydrogesterone (20 mg) as group A and vaginal cyclogest (400 mg) as group B in preparation for the IUI cycle. Clinical pregnancy and abortion rates, mid luteal progesterone (7 days after IUI) and patients' satisfaction were compared between the two groups.
Results: The mean serum progesterone levels was significantly higher in group A compared to group B (p=0.001). Pregnancy rate in group A was not statistically different from group B (p=0.58). Abortion rate in group B was higher than this rate in group A, but the difference was not statistically significant (p=0.056). Satisfaction rates were significantly higher in group A compare to group B (p<0.001).
Conclusion: We concluded that oral dydrogestrone is as effective as vaginal progesterone for luteal-phase support in woman undergoing IUI cycles. Moreover, the mean serum progesterone levels and satisfaction rates in dydrogestrone group were higher than cyclogest group.
Key words: Dydrogesterone, Cyclogest, Luteal-phase, Infertility.
P-117
The detrimental effects of alcohol on sperm parameters and DNA integrity in diabetic mice
Mangoli E1, Pourentezari M2, Talebi AR1, Rahimipour M3, Taheri F1.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Biology and Anatomical Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Biology and Anatomical Sciences, Tarbiat Modares University, Tehran, Iran.
Introduction: Diabetes mellitus (DM) is a chronic disease that can affect male reproductive function at multiple levels. Ethanol can suppress reproductive function and sexual behavior in laboratory animals and humans. Alcohol also is considered as one of the problems associated with poor semen production and sperm quality. The main goal of this study is to examine the effect of alcohol on sperm parameters and DNA integrity in diabetic mice.
Materials and Methods: Totally 32 adult male Syrian mice (10 weeks old, 35 gr) were divided into 4 groups, mice of group 1 served as control fed on basal diet, group 2 received streptozotocin (STZ) (200 mg/kg, single dose, intra peritoneal) and basal diet, group 3 received alcohol (10 mg/kg, water-soluble) and basal diet and group 4 received streptozotocin and alcohol. After 35 days, the cauda epididymis of each mouse was dissected and placed in 1 mL of pre-warm Ham’s F10 culture medium for 30 min. The swim-out spermatozoa were analyzed for count, motility, morphology and viability. Also the sperm chromatin quality and DNA integrity, was evaluated with Aniline blue (AB), Toluidine blue (TB), Acridine orange (AO) and Chromomycin A3 (CMA3) staining.
Results: In this study all of the sperm parameters were significantly diffrent between groups (p=0.001). Also, regarding the sperm DNA integrity tests, the results from four tests showed significant differences between groups. Also, in diabetes+ alcohol mice, a significant increase was found in mean tests compare to other groups.
Conclusion: Although the DM may have bad effects on sperm fertility potential and DNA integrity, but, on the other hands, consumption of alcohol in diabetic mice, may have detrimental effects on sperm parameters, sperm function and also sperm chromatin condensation in experimentally-induced diabetic mice.
Key words: Mice, Sperm parameters, DNA integrity, Diabetes, Alcohol.
P-118
Protective effect of Silymarin on plasma membrane integrity, acrosome integrity and mitochondrial membrane potential in cadmium-treated mice
Ali Asgarnejad P, Momeni HR.
Department of Biology, Faculty of Sciences, Arak University, Arak, Iran.
Email: asgarnejad3@gmail.com
Introduction: Cadmium as a heavy metal and environmental pollutant is able to exert numerous undesirable effects on human reproduction by inducing oxidative stress. Silymarin, a polyphenolic flavonoid extracted from the seeds of Silybum marianum, has effective antioxidant properties.
Materials and Methods: Adult NMRI mice were divided into four groups: 1) control 2) cadmium chloride (5 mg/kg, sc) 3) Silymarin (100 mg/kg, ip) and 4) Silymarin + cadmium. Treatment period was 24 hours. After treatment mice were dissected and their epididymis was cut into small pieces in HTF medium in order to swim out spermatozoa. The spermatozoa from different groups were used to evaluate sperm parameters. Sperm plasma membrane integrity, acrosome integrity and mitochondrial membrane potential assessed by Hoechst and propidium iodide, coomassie blue and rhodamin staining respectively. Data were analyzed with one way ANOVA and p<0.05 was considered significant.
Results: In cadmium-treated mice, the percentage of intact membrane and acrosome were significantly decreased compared to the control. In addition, mitochondrial membrane potential was significantly decreased in cadmium-treated mice in comparison with the control. In Silymarin + cadmium group, Silymarin could significantly compensate the adverse effects of cadmium compared to cadmium group.
Conclusion: The present study showed that cadmium induces toxic effects on mice sperm plasma membrane integrity, acrosome integrity and mitochondrial membrane potential and Silymarin can compensate the toxic effects of cadmium on these sperm parameters.
Key words: Cadmium, Silymarin, Sperm parameters.
P-119
Evaluating quality of well-being, marital adjustment and sexual dysfunction between infertile women
Mousavi Nik M1, Saberi Zafarghandi MB2, Birashk B3, Assari A4, Eshaghi Farahman SR1.
- Department of Clinical Psychology, Mental Health Research Center, School of Behavioral Science and Mental Health, Iran University of Medical Sciences, Tehran, Iran.
- Department of Psychiatry, Mental Health Research Center, School of Behavioral Science and Mental Health, Iran University of Medical Sciences, Tehran, Iran.
- Department of Psychiatry, Faculty of Clinical Psychology, Mental Health Research Center, School of Behavioral Science and Mental Health, Iran University of Medical Sciences, Tehran, Iran.
- Department of Architecture and Urbanism, Tehran Shargh Branch, University of Payame Noor, Tehran, Iran
Introduction: Infertility is a stressful experience and has a high impact on the infertile women’s psychological status. Infertility and it is treatment create a major and prolonged crisis for the women and create a heavy psychological trauma for the women. The effectiveness of infertility treatment depends on success rate of the treatment facility, the emotional well-being and sexual health of the women seeking treatment.
Materials and Methods: For the propose of the study, 50 fertile and 50 infertile women were selected randomly (n=100) from Midwife IVF and Fertility Research Hospital in Mysore-India. All subjects were assessed using Quality of Well-Being Scale-Self Administered (QWB-SA), Marital Adjustment Test (MAT) and Massachusetts General Hospital Sexual Functioning Questionnaire (MGH) questionnaire were administered.
Results: The results showed significant difference between fertile and infertile women assessment on Quality of Well-Being and Marital Adjustment score. Compare with the fertile women, the patients with infertility had significantly lower scores in the sexual interest and sexual arousal domains and lower frequency of intercourse. The patients with infertility retrospectively reported an orgasm, lubrication and sexual satisfaction score that was similar to that of the fertile women.
Conclusion: Women with a diagnosis of infertility were found to be at higher risk for sexual dysfunction when compare with fertile women. Further infertile women reported poor marital adjustment and quality of well being compare with controls. The results suggest to apply counseling and psychotherapy services in the infertility centers to reduce the psychological problems, increase Quality of Well-Being, Marital Adjustment and identify the factors that can contribute to the development of sexual dysfunction on women facing infertility to help them to enhance fertility chances.
Key words: Quality of well-being, Marital adjustment, Sexual dysfunction, Infertile women.
P-120
Assessment of aerobic bacterial and fungal contaminations in liquid nitrogen tanks
Nabi A, Khalili MA, Torki AR, Fesahat F, Anbari F.
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: ali.nabi67@yahoo.com
Introduction: Cryopreservation technologies play an important role in assisted reproduction for both medical and research purposes. But, there are some concerns about the technical and biological safety of this technology because of pathogen transmission during cryopreservation and germplasm banking. The aim of this study was to evaluate the presence of microbial contaminations in liquid nitrogen (LN2) and carrier tanks used in different laboratories of our reproductive science institute.
Materials and Methods: In this descriptive study, different parts of 20 carrier and storage LN2 tanks, such as LN2, vapors, cryocans, and inner surface were qualitatively evaluated for microorganism contaminations through the assessment of bacterial and fungal growth in minimal and selective Petri dishes.
Results: Two fungal (Candida albicans and aspergillus flavus) and two aerobic bacterial microorganisms (Ecoli, Pseudomonas aeruginosa) were the most common findings in bottoms, vapors, and inner surfaces of 2 carriers and 11 storage LN2 tanks. There were Pseudomonas aeruginosa growths in 8 cryocans from contaminated tanks. Also, there were staphylococcus aureus and acinetobacter baumannii contaminations in one storage tank.
Conclusion: Theoretically, LN2 tanks can be considered as a possible source of pathogen transmission. Therefore, performing quality control assays, like microorganism detection tests for LN2 and supplementary equipments should be considered as a routine practice in IVF clinics. Sterilization of tanks should be performed on regular basis.
Key words: Bacterial growth, Contamination, Liquid nitrogen.
P-121
Effect of non-polar extract of Phaleria macrocarpa on sperm quality in adult rats
Aziz Dollah M1, Parhizkar S2, Baharudin N1.
- Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University Putra, Malaysia.
- Medicinal Plants Research Centre, Yasuj University of Medical Sciences, Yasuj, Iran.
Introduction: Diminish production of testosterone hormone could affect sperm quality; hence contribute to the infertility in man.
Materials and Methods: 25 Sprague Dawley adult male rats were randomly divided into five groups. Rats were supplemented orally once a day with 0.25 ml Tween 20 solution containing non-polar hexane extract of PM at one of the following concentrations: 0, 6, 12, and 60 mg/kg for seven weeks. Commercial testosterone hormone was used as positive control. On the last day of supplementation period, all rats were sacrificed and orchidectomy technique was performed to collect semen sample from cauda epididymis of the testis. The sperm count, motility, viability and morphology were determined.
Results: The results showed that the sperm motility was significantly improved by non-polar PM extract (p<0.05) with the value of 66.98% (6 mg/kg), 63.96% (12 mg/kg) and 65.21% (60 mg/kg) compared to 44.08% for 0 mg/kg group. The supplementation of PM extract however did not improved the sperm count, sperm viability and sperm morphology. The sperm count was 852 (0 mg/kg), 918 (6 mg/kg), 906 (12 mg/kg), 1030 (60 mg/kg) and 1027 million cells/ml (commercial testosterone). The sperm viability was 85.90 (0 mg/kg), 85.26 (6 mg/kg), 85.45 (12 mg/kg), 87.88 (60 mg/kg), and 89.32% (commercial testosterone). The sperm morphology was 62.5% (0 mg/kg), 63.7% (6 mg/kg), 72.2% (12 mg/kg), 67.0% (60 mg/kg) and 64.5% (commercial testosterone). The results showed that non-polar PM extract only improved the sperm motility and did not have any effect on the sperm count, morphology and viability.
Conclusion: The study concludes that non-polar extract of PM has no compound that can improve sperm quality except on the motility of the sperm.
Key words: Phaleria macrocarpa, Sperm count, Sperm motility, Sperm viability, Sperm morphology.
P-122
Protective effect of ascorbic acid on testicular tissue in mice exposed with sodium arsenite
Pirozmanesh H, Soleimani Mehranjani M, Naderi Noreini S.
Department of Biology, Faculty of Sciences, Arak University, Arak, Iran.
Email: hp457@yahoo.com
Introduction: Arsenic is one of the major environmental contaminants with carcinogenic and toxic effects. Ascorbic acid as an antioxidant is able to restrict oxidative stress. The aim of this study was to investigate the adverse effect of sodium arsenite on the mice testicular tissue as well as to examine whether vitamin c is able to ameliorate this effect.
Materials and Methods: 24 adult male NMRI mice were randomly allocated into four groups (n=6) including control, sodium arsenite (7 mg/kg/day, orally), ascorbic acid (150 mg/kg/day, orally) and finally sodium arsenite+ ascorbic acid. Mice were treated for 35 days. At the end, mice were sacrificed and their right testis were taken out, fixed, processed and stained with Heidenhain azan method. The total volume of testis, volume of interstitial tissue, volume of seminiferous tubules, diameter and of length of seminiferous tubules, germinal epithelium height and basement membrane thickness were estimated using stereological methods. Data were analyzed using one way ANOVA and Tukey's test and means difference were considered significant at p<0.05.
Results: A significant reduction in total volume of testis, volume of seminiferous tubules and its diameter and germinal epithelium height, was found in sodium arsenite group compared to control group (p<0.04). In ascorbic acid + sodium arsenite group, the above parameters were significantly increased compared with sodium arsenite group (p<0.02). In addition, germinal epithelium height and diameter of seminiferous tubules were increased in mice treated with ascorbic acid alone compared to the control group (p<0.002).
Conclusion: The results indicate that ascorbic acid may be useful in reducing the sodium arsenite- induced toxic effects on testicular tissue.
Key words: Sodium arsenite, Ascorbic acid, Stereology, Testis.
P-123
The comparison of anxiety and depression rate between medical staff of Infertility Centers and Obstetrics and Gynecology Centers of Yazd, Shiraz, Isfahan and Kerman Hospitals
Pourmovahed Z1, Yassini SM1, Khalili MA2, Halvaei I2, Nabi A2, Ghasemi M3, Fesahat F2.
- Research Center of Addiction and Behavioral Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Fertility and Infertility Research Center, Isfahan University of Medical sciences, Isfahan, Iran.
Introduction: Mental health is an important issue in personal, social and occupational function of anyone in the life; moreover, mental problems have many negative consequences on the quality of professional work. Regarding the close and continuous interaction of infertility staff with hopeless infertile couples and in the contrary the atmosphere of happiness especially in Obstetric Wards it make sense that anxiety and depression rates would be different between them.
Materials and Methods: This study was a descriptive-correlation study based on cross-sectional method. 199 individuals who were the staff of Infertility Centers and Obstetrics and Gynecology Wards in four provinces enrolled in this study through stratified sampling. Data collection was done by demographic questionnaire, Spiel Berger and Beck depression inventory tests. Data were analyzed by SPSS software and ANOVA test.
Results: The results showed that there is statistically significant higher rate of anxiety in staff of Obstetrics and Gynecology Wards in Isfahan (54.69±13.58) while there was a higher rate of depression in staff of Infertility Center in Shiraz (14.94±10.87). Overall, correlation between anxiety, depression and work place was statistically significant (p=0.047 and 0.008 respectively). According to ANOVA test, the mean value of anxiety level was higher in the staff of Obstetrics and Gynecology Centers of Isfahan, Shiraz, Shahid Sadoughi and Madar Hospitals in Yazd (p=0.03, 0.006, 0.008, 0.012 respectively) and Infertility Center of Yazd (p=0.00).
Conclusion: Comparison of the mean value of anxiety in staff of Obstetrics and Gynecology Wards and Infertility Centers was not significant in these four centers except for Isfahan Center. As long as we know that infertile couples have little chance for success rate and Obstetrics and Gynecology patients have little risk of failure in treatment it could be mentioned that the anxiety and depression in the staff are not correlated with the client illness.
Key words: Anxiety, Depression, Infertility centers, Obstetrics and gynecology centers.
P-124
Developmental competence of immature oocytes aspirated from ovarian antral follicles as a method for fertility preservation
Safian F1, Khalili MA2, Karimi Zarchi M3, Mohsenzadeh M1, Ashourzadeh S1, 4.
- Department of Biology and Anatomical Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Obstetrics and Gynecology, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Afzalipoor Clinical Center for Infertility, Afzalipoor Hospital, Kerman University of Medical Sciences, Kerman, Iran.
Introduction: Advances in the treatment of cancerous women in reproductive age have markedly been increased recently. Also, in vitro maturation (IVM) of immature oocytes collected from ovary has been proposed for fertility preservation. In addition, the morphology of oocytes post IVM is one of the factors determining its developmental competence. By using the non-invasive PolScope system, the meiotic spindle and zona pellucida (ZP) can be assessed in living oocytes.
Materials and Methods: The ovarian cortex from 26 cancer patients (21-45 years old), were obtained directly from collaborating hospitals, and transported to the IVF center on ice. 61 immature oocytes were aspirated, of which 18 (29.5%) were degenerated and discarded. The remaining 43 (70.5%) healthy oocytes were cultured for 48 hr IVM culture media. The rate of maturity was assessed and the ZP birefringence and meiosis spindle (MS) were imaged with Polscope technology.
Results: 43 immature oocytes underwent IVM technology, of which 30.2% reached viable metaphase II (MII) oocytes. The ovarian tissues of 9 (34.6%) women were lacking oocytes at any stage. There was a positive correlation between the recovered number of oocytes and the ovarian volume. During polarized light microscopy examination, MS could be visualized only in one of the MII oocytes, but high ZP birefringences were observed in most of the oocytes post IVM (61.5%).
Conclusion: Oocytes maturation post IVM from unstimulated ovaries showed a good developmental competence in cancerous patients. Further studies should be performed to advance the oocyte maturation program, such as co-culture system, for fertility preservation.
Key words: Fertility preservation, Ovarian tissue, IVM, Polscope.
P-125
Effect of Heracleum persicumm oil and alcoholic extracts on sperm parameters and chromatin quality in mice
Taghizabet N1, Mangoli E2, Anbari F2, Talebi AR2, Masoodi A1.
- Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: Seminal plasma from infertile men has lower antioxidant levels than that of fertile men, particularly of patients whose semen have poor sperm motility. Evaluation of the importance and the effects of plant derived drugs on fertility of laboratory animals have long been recognized. Antioxidant activity has been reported from Heracleum persicum (Golpar).
Materials and Methods: 80 adult male mice (10 weeks old, 35 gr) were divided to 3 groups: group1 received hydroalcoholic extract (1000 mg/kg, ip), group 2 received oil extract (200 ml/kg, ip) and group 3 serving as the sham control group that received water. After 35 days, the cauda epididymis of each mouse was dissected and placed in 1 mL of pre-warm Ham’s F10 culture medium for 30 min. The swim-out spermatozoa were analyzed for count, motility, morphology and viability. The sperm chromatin quality and DNA integrity, was evaluated with Aniline blue (AB), Toluidine blue (TB), Acridine orange (AO) and Chromomycin A3 (CMA3) staining.
Results: In sperm analysis, progressive and non-progressive motility were significantly differences between groups. Regard to sperm chromatin quality, the results of TB and AO tests showed statically significant differences between groups, but in AB and CMA3 staining, we didn’t see any differences between them. So we can say that the Heracleum persicum extracts doesn’t have any detrimental effects on histone-protamines replacement during the testicular phase of sperm chromatin packaging.
Conclusion: According to our results, Heracleum persicum extracts as an antioxidant although improved sperm motility but may influence the male fertility potential via affecting DNA/ chromatin quality in mice.
Key words: Heracleum persicum extracts, Sperm, Mice, Chromatin.
P-126
Effect of vitrification on high magnification morphology, chromatin condensation, and fertility potential of human spermatozoa
Taherzadeh S2, Khalili MA1, 2, Anvari M1, 2, Ghazali Sh1, Agha-Rahimi A1, Anbari F1, Ghasemi-Esmailabad S1.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Anatomy, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: Sperm vitrification is a technique of ice- and CPA- free cryopreservation by direct plunging of a sperm suspension into liquid nitrogen (LN). Sperm characteristics of motility and morphology are important for normal spermatozoa-oocyte interaction. However, defects on chromatin condensation can cause vacuolization on sperm morphology. Motile sperm organelle morphology examination (MSOME) is an unstained real time high magnification that analysis viable sperm morphology. This study investigated the influence of vitrification on human sperm structure by MSOME technique, and fertility potential by zona binding assay (ZBA) and chromatin condensation by toluidine blue (TB) and aniline blue (AB) assessment.
Materials and Methods: 30 normozoospermic ejaculates were prepared by swim up technique, and supernatants were divided into two parts: fresh and vitrified groups. For vitrification, sperm suspension was mixed with equal volume (1:1) of Hams F10 +5% HSA +0.5 M sucrose. Then, 30 µl sperm suspension was dropped into LN. Warming was performed by quick submerging spheres into pre-warmed 5ml Hams F10 with 5% HSA at 37˚C. Sperm motility, stained morphology, MSOME and ZBA were evaluated for each sample. Three classes were considered for MSOME analysis: high quality sperm with a score of 4-6 (Class 1); medium-quality sperm with a score of 1-3 (Class 2); low-quality sperm with a score of 0 (Class 3). 2×10⁶ spermatozoa in each 25µl droplet containing 4 Oocytes were performed for ZBA. In addition, samples were fixed for TB and AB assessments.
Results: Cryopreservation significantly reduced both progressive motility and normal morphology of spermatozoa. There was no significant differences between the rates of MSOME in class 1 (14.93±14.66 vs. 13.56±11.34), class 2 (53.53±13.99 vs. 55.63±12.16), class 3 (31±20.81 vs. 30.80±16.03) pre and post vitrification, respectively. However, vitrification reduced the fertility potential of spermatozoa from normozoospermic samples (13.40±22.73 vs. 9.00±13.87) and chromatin condensation (TB: 60.32±16.60 vs. 57.28±17.19) (AB: 38.32±8.14 vs. 34.35±6.87).
Conclusion: Vitrification had adverse effects on sperm parameters of motility and morphology. However, this technique did not increase the rate of vacuolization of sperm head or severe alteration in fertility potential. Since, the majority of human spermatozoa contained vacuolization in head region, it is highly recommended to use MSOME technology for assessment of sperm fine morphology during clinical microinjection procedure.
Key words: Sperm vitrification, Motile sperm organelle morphology examination (MSOME), Zona binding assay (ZBA).
P-127
Vitamin C attenuates detrimental effects of diabetes mellitus on sperm parameters, chromatin quality and rate of apoptosis in mice
Talebi AR1, Mangoli E1, Nahangi H2, Anvari M2, Pourentezari M2, Halvaei I1.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Biology and Anatomical Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: Diabetes Mellitus (DM) may affect male reproductive functions at multiple levels. It is shown that intake of antioxidants such as vitamin C and E can reinforce the stability of testicular blood barrier and protect sperm DNA from oxidative stress.The main goal was to examine the protective effects of vitamin C on sperm parameters, sperm chromatin condensation and apoptosis in experimentally-induced diabetic mice.
Materials and Methods: 28 adult Syrian mice were divided into 4 groups. In group 1, the mice were diabetic that received a single dose of Streptozocin (STZ) (200 mg/kg) intra-peritoneally (ip). Group 2 included diabetic mice that received vitamin C (10 mg/kg/daily, ip). Mice in group 3 received vitamin C and group 4 was considered as control. After 35 days, sperm analysis was done accordingly. To asses sperm chromatin and DNA quality, we used aniline blue (AB), toluidine blue (TB), chromomycin A3 (CMA3), acridine orange (AO) and terminal transferase mediated deoxyuridine triphosphate biotin end labeling (TUNEL) tests.
Results: All of the sperm parameters (count, motility, morphology and viability) had significant reduction in diabetic mice but, the data showed a significant increase in all of the sperm parameters in diabetic + vitamin C when compared with diabetic and control animals (p<0.05). There were significant differences (p<0.001) between groups regarding TB staining (48.8±5.92 vs. 34.3±4.13), AO test (35.9±6.11 vs. 20.8±2.89) and TUNEL test (39.42±7.18 vs. 22.00±3.65) in diabetic and diabetic + vitamin C groups, respectively. Nevertheless, in CMA3 and AB staining assays, there were not any significant differences between different groups.
Conclusion: Vitamin C, as a potent antioxidant, can attenuate detrimental effects of diabetes mellitus on the sperm parameters, chromatin quality and apoptosis in an experimental model.
Key words: Sperm, Diabetes mellitus, Mice, Vitamin C, Chromatin, Apoptosis.
P-128
DNA fragmentation in mammalian spermatozoa and its relationship with male infertility
Vaseghi-Dodaran H1, Daghighkia H1, Atshan M2, Sobhani A3, Sobhani AGH4.
- Department of Animal Sciences, Faculty of Agriculture, Tabriz University, Tabriz, Iran.
- Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Animal Sciences, Faculty of Agriculture, Mohaghegh Ardabili University, Ardabil, Iran.
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Introduction: It has been suggested that altered nuclear chromatin structure or damaged DNA in spermatozoa is implicated as a possible cause of increased infertility in males. Since the first reports on sperm DNA integrity, this subject has become the focus of numerous studies. Up to 8% of infertile men have been shown to have high levels of sperm DNA fragmentation despite a normal semen analysis. Currently, there are eight major tests of sperm DNA fragmentation, including the comet assay, sperm chromatin structure assay (SCSA), acridine orange test (AOT), tritium-labeled 3H-actinomycin D (3H-AMD) incorporation assay, terminal TdT-mediated dUTP-nick-end labeling (TUNEL) assay, in-situ nick translation (ISNT) assay, DNA breakage detection-fluorescence in-situ hybridizations (DBD-FISH) assay and sperm chromatin dispersion (SCD) test. New studies suggest that sperm with certain levels of DNA fragmentation serve as a strong predictor of reduced male fertility. Diagnosis of the fertilizing ability of a semen sample is important for consistently high reproductive efficiency. This paper aimed at investigating the potential use of sperm DNA fragmentation (SDF) to improve the routine screening of male infertility.
Materials and Methods: This article discusses some of the current techniques employed for evaluating chromatin structure or DNA damage in spermatozoa. A review of recent bibliography was carried out in PubMed, Google scholar and SID by the use of relevant keywords, in order to evaluate the possible correlation between the conventional seminal parameters and sperm DNA fragmentation assessment as diagnostic tools in male infertility evaluation.
Results: A negative correlation was found between sperm characteristics and the proportion of sperm showing DNA fragmentation. For fragmentation >30%, a significant decrease of the fertilization rate was observed.
Conclusion: The proportion of sperm with DNA fragmentation appears to be potentially useful as a predictor of infertility. Sperm DNA fragmentation is a parameter worth integrating in routine clinical practice. However, additional large scale studies based on the optimization of sperm DNA integrity are needed.
Keywords: Spermatozoa, Male, DNA damage, Infertility.
P-129
Establishment of spermatogonial stem cell line in mouse testicular culture
Azizi H1, Amirian M2, Skutella Th2.
- Department of Biotechnology, Amol University of Special Modern Technologies, Amol, Iran.
- Institute for Anatomy and Cell Biology, Medical Faculty, University of Heidelberg, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany.
Introduction: In the seminiferous tubules of the testis spermatogonial stem cells (SSCs), Sertoli cells and differentiating cells during spermatogonesis are present. Materials and Methods: Here we report the establishment of a new VASA/ DAZL/ PLZF/-, Sox9/ Nanog/ GFRa1 low and KLF4, SOX2, VIMENTIN, N-MYC, OCT4+ cell line both from neonate and old mice, which could be expanded on SNL feeders for more than a year.
Results: Unlike undifferentiated SSCs they have a smaller nuclear/cytoplasm ratio in EM. FACS analysis showed the expression of CD49, CD29, CD9, GFRa1 and E-CAD. In Fluidigm analysis the cells expressed pluripotency markers OCT4, NANOG, SOX2 and KLF4 but only partially expressed the typical germ cell profile of SSCs. One month after transplantation in busulphan treated NOD SCID mice we observed localization of GFP labelled cells in the basal compartment of the seminiferous tubule.
Conclusion: These more differentiated SSCs could provide an ideal cell system for studying both pluripotency and in vitro differentiation of SSCs to sperm and also provide a new strategy for isolation of SSCs from neonate and old mice by morphology based selection.
Key words: Germ cells, Spermatogonial stem cells, Testicular culture.
P-130
The association between TNF-alpha -308 polymorphism and susceptibility to spermatogenic failure
Khademi Bami M1, Dehghan Tezerjani M2, Vahidi Mehrjardi MY2, Ashrafzadeh Mehrjardi HR1, Ghasemi-Esmailabad S2, Montazeri F2, SabbaghNejad Yazd S2, Shadram Y2, Golzade M2, Karimi R2, Sheikhha MH2, Kalantar SM2.
- Department of Biology, Islamic Azad University, Ashkezar Branch, Yazd, Iran.
- Research and Clinical Center for Infertility, Shahid Sadoughi Medical Sciences University, Yazd, Iran.
Introduction: Approximately half of infertility reasons are related to male factors among which genetic etiology is the main cause. Signaling molecules can regulate spermatogenesis during regulation of germ cells maturation. Tumor necrosis factor-α (TNF-α) is one of the prominent cytokines that supports signaling molecules and it can affect spermatogenesis.
Materials and Methods: This case-control study includes 50 azoospermic men who referred to Yazd Research and Clinical Center for Infertility and 50 healthy controls. After sperm analysis, DNA was extracted and Restriction Fragment Length Polymorphic-Polymerase Chain Reaction (RFLP-PCR) was carried out for TNF alpha -308 polymorphism.
Results: The frequencies of A allele and G allele were 58% and 42% in azoospermic group, and 34% and 67% among controls, respectively. Among azoospermic patients, 42% presented AA homozygous genotype, 32% AG heterozygous genotype, and 26% GG homozygous genotype. In the control group, 20% presented AA homozygous genotype, 28% AG heterozygous genotype, and 52% GG homozygous genotype. According to our findings, A allele (p=0.001, OR=2.681) and AA genotype (p=0.030, OR=2.89) have a positive association with spermatogenic failure in the cases.
Conclusion: Our data suggest that the polymorphism might be associated with the risk of spernatogenic failure in Iranian azoospermic and it can be used as an infertility marker for screening as well as further treatment.
Key words: Azoospermic, Cytokines, TNF-α, Polymorphism, RFLP-PCR.
P-131
The role of FSH receptor gene alterations in poor ovarian response to gonadotropins
Masoudi N1, Zari Moradi S1, Mohseni Meybodi A1, Mansouri Z1, Totonchi M1, Mirghavam-Aldin N2, Ashrafi M2, Gourabi H1.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Endocrinology and Female Infertility at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: Poor ovarian response is an infertility disorder in which women’s ovaries don’t have proper response to gonadotropins. Follicle stimulating hormone (FSH) has a critical role in the maturation of the ovarian follicles from the antral to the graffian stage. FSH will start a signaling cascade in the granulosa cells after sitting on its receptor (FSHR). Alteration of this receptor may change follicle maturation and therefore result in improper response to gonadotropins. We investigated the association of FSH receptor gene alteration in ovarian response of poor responder patients.
Materials and Methods: The presence of P.Ala307Thr, P.Ser680Ala, P.Ala665Thr and Mut.Val341Ala were analyzed in a case control study. 70 Iranian poor responder patients were selected as the case group. 60 Iranian fertile women were enrolled as the control group. The patients DNA were extracted from their peripheral blood and amplified by relevant primers. For determining allelic variant status all PCR products were analyzed by Sequencing.
Results: The results showed that the homozygous Ser680 and Ala307 variants seem not to be significantly associated with poor response to gonadotropins. The FSHR P. Ala665Thr genotype frequency was similar in all patients and controls. The number of oocytes retrieved was comparable between patients with different FSHR genotype.
Conclusion: Although data are accumulating with evidence suggesting that the ovarian response to gonadotropins may be mediated by different genetic alterations, the optimal biomarkers and the efficacy of the tests still remain to be evaluated.
Key words: Female infertility, Poor ovarian response, Follicle stimulating hormone receptor (FSHR).
P-132
A review of studies about vascular endothelial growth factor gene polymorphisms as genetic cause of infertility
Nabat E1, Farahzadi R2.
- Center of Excellence for Biodiversity, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran.
- Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Introduction: Infertility is defined as an inability of couples to achieve pregnancy after one year of regular, non-contraception intercourse. Diverse factors such as environmental, immunological, endocrine and genetic predispositions are thought to be involved in the development of the infertility. Infertility may be due to male or female factors or both. Causes of female infertility may include ovulation disorders, uterine abnormalities and fallopian tube blockage. Recurrent miscarriage (RM) and endometriosis are the common cause of infertility in female. Genetic factors can play an important role in susceptibility to RM and endometriosis, such as polymorphisms in vascular endothelial growth factor gene (VEGF). Recently researchers have found that endothelial dysfunction and placental ischemia/hypoxia are important factors in miscarriage. VEGF plays a pivotal role in the formation of blood supply of the fetus through the placenta and development of endometriosis. Many recent studies have indicated that a reduced VEGF serum concentration was associated with increased risk of RM and endometriosis. Therefore, the aim of this study was to investigate the association of VEGF polymorphisms such as +405 G/C, -460 C/T in 5’-UTR, +936 C/T in 3’-UTR and -1154 G/A in promoter region, with RM and endometriosis.
Materials and Methods: This review is focused on the most recent laboratory and clinical findings to investigate the association of VEGF polymorphisms with RM and endometriosis.
Results: -1154A and +93 6T alleles are increased in RM, while only in the study of Hsieh et al +405 C/C and -460 T/T genotypes are highest in patients suffering from endometriosis.
Conclusion: According to many functional studies, A allele of -1154G/A, T allele of +936C/T, C/C genotype of +405 G/C and T/T genotype of -460 C/T are associated with reduced VEGF transcriptional levels. These could lead to inadequate angiogenesis thus, lower endometrial vascularization, impaired placentation and subsequent predispose to RM and risk of endometriosis.
Key words: Infertility, Endometriosis, Recurrent miscarriage, Polymorphism, VEGF.
P-133
Association study of GSTO1 gene polymorphism (E155del) among patients with recurrent miscarriage from NorthWest of Iran
Yasari S1, Bonyadi M1, Taghavi S2.
- Center of Excellence for Biodiversity, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran.
- Maternal Fetal Medicine Unit, Department of Obstetrics and Gynecology, Alzahra Hospital, Tabriz University of Medical Sciences, Tabriz, Iran.
Introduction: Recurrent miscarriage (RM) is the occurrence of three or more consecutive pregnancies that end in miscarriage of the fetus before viability. About 1-5% of couples trying to conceive are affected by recurrent miscarriage. RM is considered as a multi-factorial disease and nearly half of RM cases cannot currently be explained clinically. Several studies have indicated that genetic polymorphisms could be factors for causing susceptibility of RM with unknown etiology. GSTO1 protein has a crucial role in detoxification metabolism and it is believed that GSTO1 gene could be involved in RM. In this study the possible association of GSTO1 gene polymorphism [E155del (rs11509437)] with recurrent miscarriage in NorthWest of Iran was evaluated.
Materials and Methods: In this case-control study, forty-eight patients with a history of RM and forty-eight referent women, who had at least two live births without other pregnancy complications, were included. Glutathione S-transferase Omega 1 (GSTO1) gene polymorphism (E155del) was screened using polymerase chain reaction with confronting two-pair primers (PCR-CTPP). Subsequently, PCR products were electrophoresed in 2% agarose gel and visualized by ethidium bromide staining.
Results: This study revealed that there was a significant association with the allelic frequency of del155 between the patient and control groups (p=0.04). However, analysis of genotype’s frequencies between patient and control showed no significant association (p=0.14).
Conclusion: Although some studies have indicated that the genetic polymorphisms of detoxification genes, encoding related enzymes, could be involved in RM diseases, current study showed the weak association of GSTO1 gene polymorphism (E155del) with susceptibility to develop RM in this population.
Key words: NorthWest of Iran, Recurrent miscarriage, GSTO1 gene, E155del polymorphism, Cellular detoxification.
P-134
Effects of in vitro maturation (IVM) on epigenetic changes in oocytes and early embryos
Allahveisi A1, Rezaei MJ2, Nikkhoo B3, Yousefian E4.
- Department of Reproductive Biology, Infertility Center of Beassat Hospital, Kurdistan University of Medical Sciences, Sanandaj, Iran.
- Department of Histology and Embryology, Infertility Center of Beassat Hospital, Kurdistan University of Medical Sciences, Sanandaj, Iran.
- Department of Pathology, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran.
- Department of Anatomical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
Introduction: In vitro maturation (IVM) of oocyte is an effective technique for avoiding ovarian hyperstimulation syndrome in patients with polycystic ovaries (PCOS) during in vitro fertilization (IVF). Pervious study indicated that more than 1000 children have been born from IVM technique, especially in patient with PCOS. However, IVM remains questions even today. Epigenetic reprogramming occurs in the periods of gametogenesis and embryogenesis, which regulates the gene activity without alteration of DNA sequences. This review discuss about effects of in vitro maturation (IVM) on epigenetic changes in oocytes and early embryos.
Material and methods: This article presents result of a systematic review about effects of in vitro maturation (IVM) on epigenetic changes in oocytes and early embryos.
Results: Different studies showed that IVM down regulated the protein expression of enzymes controlling histone acetylation such as histone acetyltransferase GCN5 (GCN5) and histone deacetylase 1 (HDAC1), as well as their common target, acetyl-histone H3 (Ac-H3), in metaphase II (MII) oocytes and two-cell embryos. The significantly decreased HDAC1 mRNA levels in oocytes and early embryos from IVM strongly suggested that IVM technique down regulated the transcription of HDAC1 gene in oocytes before and after fertilization.
Conclusion: Our studies indicated that IVM could affect the protein and gene expression level related to histone acetylation in oocytes and early embryos. Therefore, the impacts of IVM on epigenetic changes may be reasons for the lower rates of fertilization and early embryos in IVM.
Key words: IVM, Oocyte, Epigenetic.
P-135
Association of 14-bp insertion/deletion polymorphism of HLA-G gene with idiopathic recurrent miscarriage
Arjmand F.
Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: fateme.arjmand@yahoo.com
Introduction: HLA-G is supposed to play a pivotal role in tolerance of the semi-allogeneic graft in pregnancy by inhibiting the cytotoxic functions of T- and NK cells. A 14-bp insertion/deletion polymorphism in exon-8 has a possible role in HLA-G expression.
Materials and Methods: Genomic DNA from 200 recurrent miscarriage (RM) patients and 200 normal fertile control individuals were isolated using the routine salting out method. Exon-8 of HLA-G gene of the two groups were amplified using polymerase chain reaction and analyzed by electrophoresis on 10% non-denaturing polyacrylamide gel electrophoresis containing ethidium bromide and visualized under ultraviolet light. Statistical analysis used SPSS 19 software. HLA-G allele frequencies and genotypes in RM women and the fertile control group were compared using a Chi-square test.
Results: There was a difference in allelic frequencies of 14-bp insertion polymorphism between fertile controls and RM patients; frequency of +14/-14 bp heterozygotes was significantly increased in RM patients as compared with fertile controls.
Conclusion: A 14-bp insertion/deletion polymorphism in exon 8 plays a possible role in HLA-G expression in certain cases of recurrent miscarriage. However, additional studies are needed in this regard.
Key words: 14-bp insertion, Deletion polymorphism, HLA-G, Recurrent miscarriage.
P-136
Mutation analysis of exone 2 of Tnp2 gene in varicocele patients
Danafar AH1, Heidari MM2, Khatami M2.
- Department of Biology, Ashkezar Branch, Islamic Azad University, Ashkezar, Iran.
- Department of Biology, Faculty of Science, Yazd University, Yazd, Iran.
Introduction: Varicocele is one of the main causes of male infertility that caused by inflammation of the veins of the pampiniform plexus. Several studies have given great evidences that revealed the relationship between sperm DNA damage and varicocele. Because of the crucial role of Transition Nuclear Proteins (TNPs) and Protamines in sperm DNA condensation and integrity, the mutations in these genes can increase the risk of sperm DNA damage and infertility in varicocele condition.
Materials and Methods: DNA was extracted from total blood of 78 infertile patients with varicocele and 75 fertile control men for PCR amplification and SSCP analysis. DNA from samples with altered band pattern in the SSCP was then sequenced to search for mutations.
Results: The results of sequencing showed one variant at position IVS1-26G>C (rs8043625) in the intronic region of this gene. Comparison of the genotypes between cases and controls showed significant differences in frequencies of GG and CC (p=0.002, p=0.01), but not in GC genotype of this polymorphism (p=0.41). Also it was found that varicocele risk in men who have the CC and GC genotypes is respectively 3.07 and 1.37 fold higher than those who don't have these genotypes (OR=3.07, OR=1.37).
Conclusion: High conservation of this SNP position during evolution can represent the effects of this nucleotide in some important processes associated with the expression of this gene like mRNA splicing; but the exact mechanism is not clear.
Key words: Varicocele, Infertility, Tnp2, SSCP.
P-137
Assessing the polymorphism frequency rate of insulin receptor gene in PCOS
Khazamipour A1, Nejatizade A1, Kalantar SM2.
- Department of Genetic, Faculty of Science, Hormozgan University, Bandar Abbas, Iran.
- Research and Clinical Center for Infertility, Shahaid Sadoughi University of Medical Scinces, Yazd, Iran.
Introduction: Polycystic ovarian syndrome (PCOS) is a common endocrine disorder with prevalence of 5-10% globally among women of reproductive age. PCOS is being characterized by chronic anovulation, polycystic ovaries, hyperandrogenism, hirsutism, overweight, insulin resistance and infertility. It is well-known that PCOS is a complex trait like type-2 diabetes where both genetic and environmental factors play a crucial role in pathogenesis of the disease. Some type-2 diabetes susceptibility genes including those for insulin secretion and action such as insulin receptor showed considerable contribution to genetic predisposition of PCOS. Since insulin receptor gene seems to be a strong candidate gene to PCOS, we aimed to investigate the role of insulin receptor gene polymorphism (T/C) located in a transcription enhancer element of the gene to the disease susceptibility.
Materials and Methods: We carried out a cross-sectional case-control study. Using simple random sampling 90 healthy women were selected. All 90 patients fulfilled the 2003 Rotterdam criteria of PCOS. The subjects were genotyped for insulin receptor gene polymorphism (T/C) using PCR-Sequencing. Differences in genotype distributions between case and control subjects were examined via a chi-square test.
Results: In the present study 180 women between 18-40 years old age were assessed. Clinical, biochemical and metabolic characters of women were compared. Among them, obesity (Waist round more than 85 centimeters), hirsutism and irregular menarche (monthly cycles more than 35 days), LH/FSH >2, BMI>25, (mean: 28.19) in the PCOS group were significantly higher than control group (p<0.05). There was no significant associated between two groups for diabetes mellitus type II, hypertension (>140/90 mmHg) and hormonal level (TSH >5 and prolactin >29.2). Similarly in PCOS and control group, 95% of women had TT genotype and 2.2% had CC genotype. Frequency of C allele in PCOS and control group were 3.3% and 2.2% respectively. Frequency of TC genotype in PCOS and control group were 2.2% and 4.4% respectively. Noted polymorphism had no significant difference between PCOS and control women (p=0.99).
Conclusion: Although PCOS showed high prevalence among women in Bandar Abbas, Iran, insulin receptor gene polymorphism (T/C) didnot show significant influence on susceptibility to PCOS.
Key words: Polymorphism, Insulin receptor gene, Polycystic ovarian syndrome.
P-138
Association of SNP rs.2414096 of CYP19 gene with polycystic ovarian syndrome in Iranian women
Mehdizadeh A, Kalantar SM, Sheikhha MH.
Department of Genetics, International Campus, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: anahita.mz@gmail.com
Introduction: Polycystic ovarian syndrome (PCOS) is the most common endocrinologic disorder of women in their reproductive age. Major characteristics of PCOS are clinical and/ or biochemical hyperandrogenism, ovulatory dysfunction and polycystic ovaries. The etiology of PCOS is unknown, however, it has been suggested that genetic factors play major role in development of PCOS. Of those the ones implicating in metabolism of androgens are important as they are impaired in PCOS. CYP19 gene encodes aromatase which has a crucial role in androgen synthesis and variants of this gene might implicate in the pathogenesis of PCOS. Investigations have indicated the role of single nucleotide polymorphism of CYP19 in hyperandrogenism and PCOS in some racial groups. There is no data on the variants of this gene in PCOS population in Iran.
Materials and Methods: In a case-control study 70 PCOS women and 70 normal controls were selected. Following informed consent, 5 ml blood was taken from each woman of which genomic DNA was extracted by salting out method. Then a set of PCR reactions for CYP19 gene was carried out using specific primers for SNP rs.2414096 followed by a subsequent enzyme digest (RFLP) with HSP92II.
Results: Genotype frequencies of SNP rs.2414096 in PCOS women were as follows: AA (14.4%), AG (44.3%) and GG (41.4%) while in normal group, these genotypes were 24.3%, 52.9% and 22.9%, respectively. While allele frequencies in PCOS group were 49.3% for A and 50.7% for G, normal group had a different percentage of A (36.4%) and G (63.6%). The calculations for both genotype and allele frequencies showed statistical significance.
Conclusion: Variants of SNP rs.2414096 in CYP19 could play a role in development of PCOS in Iranian women.
Key words: Polycystic ovarian syndrome, CYP19, Single nucleotide polymorphism.
P-139
Exposure to tertiary-butyl hydroperoxide (TBHP)- down regulates the expression of Ddx3y in mature mice testis
Mokhtari P, Fatemi N, Golkar Narenji A, Mohseni Meybodi A, Gourabi H.
Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email:gourabi@royaninstitute.org
Introduction: Approximately 40% of infertility is associated with male factors. Two major causative factors of male infertility are oxidative stress (OS) and genetic factors. OS damages the sperm plasma membrane, the genome integrity and alter the expression profile of genes involved in spermatogenesis. Ddx3y gene is one of the important azoospermia factor (AZF) genes in Y chromosome. AZF deletion causes a severe block in spermatogenesis which affects the proliferation of spermatogonia, and consequently male infertility. The expression profile of Ddx3y gene was evaluated in testis tissues of Balb/c mice after OS induction.
Materials and Methods: A model of oxidative stress in adult male Balb/c mice testis by injection of the 1:10 concentration of tertiary-butyl hydroperoxide (TBHP) was created. Case group included treated mice by TBHP for 2 weeks and control group treated only by injection of dH2O. Induced ROS levels in testes tissue samples of all mice were measured by flow-cytometry. Consequently the expression of Ddx3y gene was quantitatively measured in samples of both groups by real-time PCR.
Results: According to flow-cytometry results, an increase of oxidative stress in TBHP treated mice in comparison to control group was observed. The gene expression of Ddx3y in testis was significantly down regulated in OS-exposed and ROS induced mice.
Conclusion: Our results indicated that Ddx3y may be a major target gene of OS and the down regulated expression of Ddx3y can be closely related to male reproductive toxicity induced by TBHP.
Key words: Male infertility, Oxidative stress, TBHP, Ddx3y.
P-140
Association of +331G/A polymorphism and differential expression of PR-B isoform in endometriosis
Mousazadeh S1, Afsharian P1, Shahhoseini B1, Aflatoonian R2.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Endocrinology and Female Infertility at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: It is well accepted that endometriosis is a progesterone resistance disease. The effects of progesterone are mediated by its two progesterone receptor (PR) isoforms, namely PR-A and PR-B. These isoforms are functionally different. The most widely studied polymorphism in the promoter region of PGR is the G to A substitution at position +331 (rs10895068). Recently, many studies have investigated the role of +331G/A polymorphism in the etiology of various types of cancers.
Materials and Methods: Blood samples were recruited from 98 women undergoing laparoscopy for endometriosis and 102 healthy fertile women at Royan Institute, Tehran, Iran in 2013-2014. After DNA extraction, allele and genotype frequencies were determined by PCR-RFLP. Then, RNA was extracted from selected eutopic tissue samples of endometriosis patients. Analysis of PR-B mRNA expression was performed using Real-time PCR.
Results:Our data showed the frequency distribution of GG, G/A and AA genotypes in +331G/A polymorphism was 98.04%, 1.96% and 0.0% in patients and 97.96%, 2.04% and 0.0% in control groups respectively (p=0.968). Although our data didn’t show any significant association with +331G/A in our groups, however, we were able to demonstrate higher expression level of PR-B in patients with G/A compared to patients with GG genotypes.
Conclusion: Many studies have been shown that the expression level of PR-B reduced severely during endometriosis, which can affect the function of progesterone. Our findings support this observation, patients with G/A genotypes have high expression level of PR-B compared to patients with GG genotypes. Therefore, +331G/A have been found to lead increased transcriptional activity of PR-B in patients by favoring G/A or AA genotypes that may be able to influence the function of progesterone and reduce the susceptibility and symptoms of endometriosis.
Key words: Endometriosis, PGR, Polymorphism.
P-141
Association between GSTT1 null mutation and endometriosis in an Iranian population
Rastegar M1, Seifati SM1, Sheikhha MH2.
- Department of Biology, Islamic Azad University, Ashkezar Branch, Yazd, Iran.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: Endometriosis is a common gynecological disease that involves growth of endometrial tissue outside its normal location. It seems that genetic factors have an important role in the pathophysiology of endometriosis. In addition, there are reports suggesting a role for environmental pollutants in developing endometriosis. GSTTl is one of the phase II enzymes involved in detoxification of some environmental carcinogens.
Materials and Methods: Genomic DNA from 95 patients with endometriosis and 141 healthy controls was collected. GSTT1 null genotyping was performed using polymerase chain reaction (PCR) method. Results: Frequency of GSTT1 null deletion in both patients and controls was 22%, which shows no significant difference between two groups (p=0.983).
Conclusion: These results suggest that GSTT1 null mutation is not associated with endometriosis in our population.
Key words: Endometriosis, Glutathione s-transfers, GSTT1.
P-142
The low frequency of breast and ovary cancer protective allele (D302H) of CASP8 gene among Iranian patients
Rasti A1, Farashahi Yazd E2, Shiryazdi SM3, Nematzade H4, Rastegar M1, Sheikhha MH1.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Genetic, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
- Department of Surgery, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
- Hematology, Oncology and Genetic Research Center, Shahid Sadoughi Hospital, Yazd, Iran.
Introduction: Suppression of apoptosis is one of the major mechanisms underlying the origin and progression of cancer. The CASP8 plays a key role in the initiation of apoptosis. Previous case-control studies have indicated that the D302H genotype of CASP8 is associated with a reduced risk of breast and ovarian cancer. However, the frequency of this polymorphism in the Iranian people has not been reported yet. Therefore, this polymorphism was genotyped among patients with breast cancer and healthy women.
Materials and Methods: PCR-RFLP technique was used to genotyping of CASP8 D302H polymorphism among 100 female patients with breast cancer and 100 healthy women in this case-control study. Fisher's exact test and SPSS software were used to analyze the data.
Results: The mean age of patients and healthy groups was 51.37±1.27 and 52.57±1.21 years respectively. The frequencies of the GG and GC genotypes in case group were 91% and 9%, respectively, and among the control group, the frequencies were 97% and 3%, respectively )p=0.2). CC genotype was not found in any of the groups. Furthermore, the C allele frequency was 4.5% among the cases and 1.5% among the control (p=0.14). There was no significant difference between cases and controls. In total, the frequency of the C allele was observed in about 3% of people in our study (breast cancer patients and healthy women).
Conclusion: Previous case-control studies have indicated that the D302H genotype of CASP8 was associated with a reduced risk of breast and ovary cancer. In the present study the frequency of allele C was detected 3%. Indeed, the frequency of C allele in Iranian study population is less than one-fourth the frequency of this allele in Caucasian populations (13.29%). According to the low frequency of this allele in Iranian population and the protective effect of allele C, one of the conclusions that can be raised is that the Iranian population is more prone to breast and ovarian cancer than Caucasian populations in relation to this polymorphism. Therefore further studies would be needed with appropriate sample size to investigate this matter.
Key words: CASP8 D302H, Polymorphism, Apoptosis, Breast cancer.
P-143
“Cell-free fetal DNA” a novel promising biomarker for prenatal diagnosis and complicated pregnancies
Sadeghi F1, Khalaj kondori M1, Sadeghi M2.
- Department of Biology, Faculty of Natural Science, University of Tabriz, Tabriz, Iran
- Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
Introduction: Cell free fetal DNA (cff DNA) is a novel promising molecular biomarker that has been applied in various aspects of obstetrical research, notably in prenatal diagnosis and complicated pregnancies.
Materials and Methods: Data base was browsed using key words “cell-free fetal DNA” and “noninvasive prenatal diagnosis” for obtaining related reports. Articles were screened for relatedness and the recent progresses in the field.
Results: Cell-free fetal nucleic acids can be detected in the maternal circulation during pregnancy, potentially offering an excellent method for early non-invasive prenatal diagnosis (NIPD) of the genetic status of a fetus. Using molecular techniques, fetal DNA and RNA can be detected from 5 weeks gestation and are rapidly cleared from the circulation following birth. Cell-free fetal DNA comprises only 3-6% of the total circulating cell-free DNA, therefore diagnoses are primarily limited to those caused by paternally inherited sequences as well as conditions that can be inferred by the unique gene expression patterns in the fetus and placenta. Broadly, the potential applications of this technology fall into two categories: first, high genetic risk families with inheritable monogenic diseases, including sex determination in cases at risk of X-linked diseases and detection of specific paternally inherited single gene disorders; and second, routine antenatal care offered to all pregnant women, including prenatal screening/diagnosis for aneuploidy, particularly Down syndrome (DS), and diagnosis of Rhesus factor status in RhD negative women. Already sex determination and Rhesus factor diagnosis are nearing translation into clinical practice for high-risk individuals.
Conclusion: In conclusion, the discovery of Cell free fetal DNA (cff DNA) has revolutionized the field of non-invasive prenatal diagnosis (NIPD) and has opened a new avenue in the field of obstetrical research and may in future form part of national antenatal screening programmes for DS and other common genetic disorders.
Key words: Cell free fetal DNA, Noninvasive prenatal testing (NIPT), Prenatal diagnosis, Pregnancy complications.
P-144
Identification of a time window for spontaneous establishment of pluripotency in mouse spermatogonial stem cells in vitro
Azizi H1, Conrad S2, Hajizadeh Moghaddam A1, Baharvand H3, Skutella T4.
- Department of Biotechnology, Amol University of Special Modern Technologies, Amol, Iran.
- Institute of Anatomy, University of Tübingen, Tübingen, Germany.
- Department of Stem Cells and Developmental Biology at the Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
- Institute for Anatomy and Cell Biology, Medical Faculty, University of Heidelberg, Im Neuenheimer Feld, Heidelberg, Germany.
Introduction: Although testis-derived embryonic stem cell-like (ES-like) cells have been obtained in several studies, the time window for the shift to pluripotency is not clear yet.
Materials and Methods: Here we describe, that only during a special time window (41 until 125 days) after initiation of germ line stem cell (GSCs) cultures from neonate and adult promoter-reporter Oct4-GFP transgenic mouse the spontaneous appearance of germline-derived pluripotent stem (gPS) cells from both neonate and adult GSCs occurred.
Results: The isolated and long-term cultured (more than one year) GSCs which were isolated by a morphology based selection procedure expressed germ cells markers and exhibited a similar morphology with a high nucleus/cytoplasm ratio in comparison to undifferentiated SSCs (spermatogonial stem cells) in vivo. The generated gPS cells expressed pluripotency marker, in-vitro differentiated into all three germ lineages, formed complex teratoma after transplantation in SCID mice and produced chimeric mice.
Conclusion: Although the exact mechanism of the development of gPS cells from GSCs is still unclear, this new information could provide an ideal strategy for scheduling natural conversion mechanisms of ES-like cells from mouse testis.
Key words: Germ cells, Spermatogonial stem cells, Pluripotency.
P-145
Association between MspI polymorphism of CYP1A1 gene and endometriosis in an Iranian population
Babaki A, Seifati SM.
Department of Biology, Ashkezar Branch, Islamic Azad University, Yazd, Iran.
Email: babakiaz@yahoo.com
Introduction: Endometriosis is defined as the presence of endometrial tissue outside the uterine cavity. It is associated with dyspareunia, dysmenorrhea, pelvic pain and infertility and about 3-10% of women suffer from it in reproductive age. It seems that genetic and environmental factors play a role in the development of endometriosis. The relationship between genetic polymorphisms and endometriosis has been investigated in several genetic studies. CYP1A1 gene is a member of the cytochrome P450 gene family. It is involved in estrogen metabolism and in detoxification.
Materials and Methods: In a case-control study, DNA extraction was performed from the blood specimens of 93 cases of endometriosis and 139 healthy controls. The polymorphism was determined by PCR-based restriction fragment length polymorphism (RFLP) method.
Results: There was no significant difference in frequency of TT, TC, and CC genotypes of MspI polymorphism of CYP1A1 gene between case and control groups (p=0.961).
Conclusion: Results of this study revealed no association between the MspI polymorphism of the CYP1A1 gene and susceptibility to endometriosis in the studied population.
Key words: Endometriosis, MspI polymorphism, CYP1A1 gene.
P-146
Preimplantation genetic screening: Which stage? Which technique?
Bazrgar M.
Department of Genetics at Reproductive Biomedicine Research Centre, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: bazrgar.masood76@gmail.com
Introduction: Preimplantation genetic screening (PGS) techniques have a wide range of resolution, complexity and costs. Additionally each of biopsy stages has advantages and disadvantages.
Materials and Methods: Recent publications certainly systematic reviews and randomized clinical trials were considered in details.
Results: Polar body biopsy (PB) is the least invasive however provides the least capture of abnormalities. PB focuses on female meiotic errors, fails to identify approximately 40% of aneuploidies from postzygotic and paternal origins. PB is suitable in countries with legal restrictions for embryo biopsy. Cleavage stage biopsy (CB) of few cells (1 or 2) does not affect differentiation potential, however removal of 2 cells can result in an impaired implantation potential while mosaicism is not detectable in one cell biopsy. In trophectoderm biopsy (TB) several cells are available for analyses. In day 5 TB when the genetic results cannot be obtained within 24 hours, cryopreservation should be considered. The least and the most aneuploidy rates has been reported for TBs and CBs, respectively (1 versus more than 3). TB increases absolute implantation rate about 20% in comparison with CB. Blastocoel fluid aspiration is a new source of DNA for PGS with few publications. FISH-based PGS is low resolution due to limitation in number of both chromosomes and probes per chromosome which could be tested. Therefore it has not been effective in improvement of clinical outcomes while PGS methods with ability for detection of all 24 chromosomes (PGS-24) result in increased implantation and pregnancy rates and diminished effect of advanced maternal age. Concerning PGS-24 methods, digital PCR is mostly applied for PBs; real time qPCR is only applicable to TBs and limited by number of samples, two on each 384-well plate. Array CGH is robust and scalable, turnaround time as short as 12 hours, decreased cost per sample as increased case numbers. SNP arrays and NGS-based methods for copy number analysis are likely to be the most accurate and informative but complex and high-cost equipment. Low pass NGS-based PGS is moving to be cost effective in addition to performance same as array CGH.
Conclusion: Clearly, the largest reported increases in implantation and live birth rates to date have been with TB. TB is a good choice for good-prognosis patients particularly for those wishing to have elective single-embryo transfer. However, the cleavage-stage embryos of some poor-prognosis patients may implant and develop in utero but not develop to the blastocyst stage in vitro. At the moment, array CGH seems to be the most applicable method for PGS however future would be with higher resolution methods following decrease of related costs, hence ethical concerns on designer babies will need more serious considerations.
Key words: PGS, Biopsy, Stage, Technique.
P-147
Expression of CYP19A1 in patients with endometriosis and normal endometrium during the menstrual cycle
Khateri E1, 2, Shahhoseini M2, Mahdian S3, Aflatoonian R3, Afsharian P2.
- Department of Molecular and Cellular Science, Faculty of Advanced Sciences and Technology, Islamic Azad University, Tehran, Iran.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, Tehran, Iran.
- Department of Endocrinology and Female Infertility at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, Tehran, Iran
Introduction: Endometriosis is an estrogen-dependent inflammatory disease defined by the growth of endometrial tissues outside of the uterus. Epidemiological and clinical studies show that estrogen is essential for the growth of endometriosis. Molecular studies have revealed the presence of aromatase P450, the key enzyme in the biosynthesis of ovarian estradiol, inside the endometriotic tissues, indicating local synthesis of estradiol. So, it is proposed that the enzyme aromatase P450, which is coded by CYP19A1 gene, will be expressed aberrantly in endometriosis.
Materials and Methods: To evaluate the expression profile of CYP19A1 gene in women with endometriosis during menstrual cycle, ectopic endometriotic lesions and eutopic endometrium samples were collected using laparoscopy from 20 women with endometriosis and endometrial biopsies were obtained from 10 healthy fertile women as a control group. For this respect, ethical approval and informed patient consent was gained for the use of tissue samples. Quantitative expression analysis was performed using real-time PCR technique.
Results: Data showed that mRNA expression of CYP19A1 was significantly higher in ectopic endometrium of patients with endometriosis, in both proliferative and secretory phase, in comparison with eutopic and control group. CYP19A1 expression in eutopic endometrium was significantly higher in comparison to control group in proliferative phase.
Conclusion: Higher expression of CYP19A1 mRNA in the endometriotic tissues, may contribute to the etiology and progression of endometriosis and it is involved in disease pathogenesis.
Key words: Endometriosis, CYP19A1, Menstrual cycle.
P-148
A headache in prenatal diagnosis: A case of vanished twin or hermaphroditism?
Mousavi F1, Mohammad Khani N2, Bagherizadeh E1, Hadipour F3, Hadipour Z3, Shafeghati Y4, Behjati F5.
- Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran.
- Islamic Azad University, Karaj Branch, Karaj, Iran.
- Qom University, Qom, Iran.
- School of Medicine, Tehran University of Medical Sciences,Tehran, Iran.
- Department of Medical Genetics, Tehran University of Medical Sciences,Tehran, Iran.
Introduction: The finding of a mixture of both 46,XX and 46,XY cells in amniotic fluid culture has been frequently described. In the great majority of cases, the finding is followed by the birth of a normal male infant, leading to a consensus that the finding is the result of contamination with maternal cells in a normal male fetus. There are, however, several other possible explanations including the presence of cells from an undiagnosed twin pregnancy, cross contamination in the laboratory, the presence of cells from a “vanished” male twin, and true fetal chimerism. An accurate obstetric history and thorough sonography can be of great value in correct assessment of such situations.
Materials and Methods: A 29 Years-old woman was referred for genetic counseling. Her gestational age was 16 weeks and maternal biochemical serum screening test indicated high risk for Down Syndrome. She underwent amniocentesis for chromosome study using standard high resolution GTG banding technique.
Results: Karyotype result was 46,XY and sonography revealed a female fetus with normal internal genital organs. A normal baby girl was borned with 46,XX karyotype. Upon reviewing of amnio GTG slides both 46,XY and 46,XX cell lines were detected. The patient admitted that she had experienced a miscarriage and heavy bleeding early in pregnancy. The sonography at the time of amniocentesis had shown a shrinking cyst next to the fetus. These findings strengthened the vanished or resorbed twin as the reason for such a finding.
Conclusion: The fetus was most likely a non-identical twin. Most of grown cells were from the vanished male fetus which led to misdiagnosis. Thorough genetic counseling with the view of obstetric history is of great value in such situations.
Key words: Amniocentesis, Resorbed twin, Hermaphroditism, Genetic counseling.
P-149
+49A/G CTLA-4 gene polymorphism in NorthWest Iranian population with recurrent pregnancy loss
Parsa S, Jabbarpour bonyadi M, Zeinal zadeh N, Taghavi S.
University of Tabriz, Tabriz, Iran.
Email: sara.parsa6928@gmail.com
Introduction: Recurrent pregnancy loss is defined as three or more pregnancy loss before 20th gestational weeks. There are several factors which involved in pregnancy loss including anatomical, hormonal, immunological and infectious factors. Immunological factors are important due to the interaction between mother and fetus. In spite of allogeneic proteins encoded by paternal genes, it is clear that a series of regulatory mechanisms should exist in maternal immune system not to reject fetus during pregnancy. T regulatory cells which are one of the important component of humeral immune response plays role in the fetu-maternal interface. One of the regulatory ways for these cells is mediated by antigen independent co-stimulatory signals. Interaction of B7/ CTLA-4 is one of these signals. Cytotoxic T Lymphocyte Antigen 4(CTLA-4) is a glycoprotein transmembrane molecule which down regulate the activation and proliferation of T cells in a competitive interaction with CD28 to bind to B7.
Materials and Methods: We have studies 120 cases; patient group consist of 60 women with the experience of two or more pregnancy loss and control group consist of 60 women with at least two live births without any previous history of pregnancy loss and autoimmune diseases. Genomic DNA was extracted from whole blood using standard protocols. The CTLA-4 +49 A/G were detected using polymerase chain reaction- restriction fragment length polymorphisms assay.
Results: In this study CTLA-4 +49 A/G polymorphisms showed no significant differences between patients and controls. Polymorphic allele G showed the frequency of 39.16% among patients whereas its frequency for controls was 35.83%. The age of patient varied from 19 years to 39 years.
Conclusion: The results of the present study suggest that the CTLA-4 do not have association with recurrent miscarriage in NorthWest Iranian population.
Key words: Recurrent pregnancy loss, CTLA-4, Immunological factors.
P-150
Investigation of mitochondrial Gln tRNA molecular alterations in idiopathic repeated pregnancy loss
Vahidi Mehrjardi MY, Seyedhasani SM
Shahid Sadoghi University of Medical Sciences, Yazd, Iran.
Email: mmvahidi@gmail.com
Introduction: Recurrent pregnancy loss (RPL) is a critical medical problem in about 0.5-2% of women. The molecular genetics background for spontaneous abortion is being increasingly understood, and some polymorphisms associated with it have been reported
Materials and Methods: The nucleotide variations of threonine and proline were investigated in 96 women with idiopathic repeated pregnancy loss. The related mitochondrial area was amplified using a polymerase chain reaction (PCR). The PCR products were demonstrated by 2% agarose gel electrophoresis, and all the positive samples were purified and verified by an automated DNA sequencing method.
Results: The sequence analysis revealed 2 mutations in tRNA Gln . These mutations were 4343 in 1 cases and 4336 in 1 cases .
Conclusion: These tRNAs mutations can alter their steady state level and affect the structure of tRNAs. It results in protein synthesis defects and, in turn, mitochondrial dysfunction. The mutations of these genes may help in the assessment of RPL. Further study of an expanded series of these tRNA mutants is recommended to describe their etiologic role in idiopathic RPL.
Key words: tRNA, Mitochondrial mutation, Repeated pregnancy lo.
P-151
A novel mutation in morquio syndrome
Yavari M1, Asadollahi S2, Hasheminasab SH2, Harazi F3.
- Department of Biology, Faculty of Sciences, Yazd University, Yazd, Iran.
- Department of Biology, Faculty of Sciences, Isfahan University, Isfahan, Iran.
- Department of Biology, Faculty of Sciences, Ashkezar University, Ashkezar, Yazd, Iran.
Introduction: The Mucopolysaccharidoses (MPS) are a group of inherited metabolic disorders caused by a deficiency or malfunctioning of lysosomal enzymes which are needed to break down complex carbohydrates known as mucopolysaccharides or glycosaminoglycans (GAGs). Accumulation of GAGs causes a cascade of events leading to the progressive damage of cells, tissue and organs. Morquio disease or Mucopolysaccharidosis Type IV (MPS IV) belongs to this group and has two sub-types, A and B. Type A is also known as Morquio A, GALNS deficiency, N-acetylgalactosamine-6-sulfate sulfatase deficiency or more simply MPS IVA. This lysosomal enzyme involved in the catabolism of keratan and chondroitin sulfate. Patients who inherit two mutated GALNS gene alleles have a decreased ability to degrade the glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate, thereby causing GAG accumulation within lysosomes and consequently pleiotropic disease. The objective was to report the results of clinical characteristics, enzyme activity determination and mutation analysis of GALNS gene in an Iranian patient with mucopolysaccharidosis (MPS) type IVA (Morquio A disease).
Materials and Methods: The 10 years old Iranian boy with MPSIV was firstly diagnosed by urin GALNS determination who w:as char:actraised by multiple skeletal abnormalities and dwarfism, kyphosis, hypermobility joints, difficultly in walking to 8 years old and now he can’t walk. But his intelligence was normal. We investigated for all coding exons and adjacent intron regions of GALNS gene by PCR sequencing method.
Results: We performed urin test (MS/MS) from patient. In this test total GAGS was high and MPS was likely. So lysosomal enzymes in dried blood were checked and MPS IVA was diagnosed. Two heterozygous missens mutation as c.135 G>T (S>I) and c.510 C>A (P>H), and a homozygous mutation as c.181 A>G (y>C) in GALNS gene were detected in this patient. The mutation c.181 (y>C) is a novel variant which is not reported yet.With the method of gene analysis of new variant, the mutation c.181 (y>C) was considered to be a pathogenic mutation.
Conclusion: The MPS IVA patient showed severe multiple skeletal deformities, normal intelligence, muscle weakness, short stature, who carries homozygous mutations c.181 A>G (y>C). The bioinformatics analysis in POLYPHEN predicted this mutation as being probably pathogenic.
Key words: Mucopolysaccharidoses (MPS), Morquio disease, GALNS gene.
P-152
A newborn with ambiguous genitalia and a complex X;Y rearrangement
Dehghani MR1, Hashemian Z2, Zuffardi O3.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of medical Sciences, Yazd, Iran.
- Shahid Sadoughi University of medical Sciences, Yazd, Iran.
- University of Pavia, Pavia, Italy.
Introduction: In most mammals, sex is determined at the beginning of gestation by the constitution of the sex chromosomes, XY in males and XX in females.
Materials and Methods: Array Comparative Genomic Hybridization (Array-CGH) analysis was performed by using oligonucleotide aCGH platforms (180K SurePrint G3 Human Kit, Agilent Technologies, Santa Clara, CA), as reported elsewhere. Changes in DNA copy number at a specific locus were observed as the deviation of the log2 ratio value from 0 of at least three consecutive probes, by using Genomic Workbench v. 5.0.14 software (Agilent, ADM-2 algorithm with a threshold of 5). Oligomer positions refer to the Human Genome GRCh37 (hg19) assembly.
Results: Array-CGH revealed an unbalanced rearrangement resulting in the deletion of the distal Xp and the duplication of the proximal Xp contiguous region with presence of the Y chromosome from Ypter to Yq11. Fluorescent in situ hybridization (FISH) showed that this portion of the Y was translocated to the tip of the abnormal X and that the duplicated portion of chromosome X was inverted. Altogether, the abnormal chromosome was a dicentric one with the centromere of the Y chromosome apparently inactivated.
Conclusion: The presence within the translocated Y chromosome of the SRY gene explains the devolopment of testes although it is not clear the reason for the genitalia ambiguity.
Key words: Ambiguous genitalia, 46, XX testicular DSD, Inverted duplication and Xp terminal Deletion (Invdup del), Rearrangement, Array Comparative Genomic Hybridization, FISH.
P-153
Effect of genetic variation of beta defensin 126 on ICSI and IVF outcome in unexplained infertile men
Abedini M, Mohseni Meybodi A, Sabbaghian M.
Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: maria.abedini@gmail.com
Introduction: Despite improved methods for evaluation of sperm quality, infertility remains unexplained in about 20% of affected couples. During sperm maturation, a Cysteine-rich secretory glycoprotein α-defensin 126 secreted by the epididymal epithelium adsorbed to the entire sperm surface. It remains on the sperm until sperm become capacitated in the female reproductive tract. Its removal from over the head of sperm is required for sperm zona recognition. A cytosine dinucleotide deletion in the open reading frame of second exon of DEFB126 gene generates an abnormal mRNA. Men homozygous for this mutation have reduced chances of successful fertilization.
Materials and Methods: Genomic DNA from the peripheral blood of 92 male partners of unexplained couples who underwent ICSI (n=74) and IVF (n=18), were extracted. PCR was performed and molecular genotyping for the DEFB126 variant was done by single strand conformational polymorphism (SSCP), tetra PCR and DNA sequencing. ELISA and immunocytochemistry by indirect immunofluorescence antibody performed for the assessment of this protein expression on sperm cells.
Results: In our study this allele frequency in Iranian men was 0.54. Statistical analysis shows, no significant differences were found between homozygote mutation and wild type carriers in fertilization rates, implantation rates and clinical pregnancy of IVF and ICSI. Our results by ELISA and immunocytochemistry showed that the protein expression was less in men with del/del genotype in comparing to other genotypes (p<0.005).
Conclusion: Although previous studies found that DEFB126 variation would affect sperm function and male fertility rate, in the present study, no significant differences were found between homozygote mutation and wild type carriers in fertilization rates, implantation rates and clinical pregnancy. Further confirmation in a larger scale study is needed.
Key words: Unexplained male infertility, Deletion, DEFB126, IVF, ICSI.
P-154
Association of TNFR1 36 A/G polymorphism with azoospermia in Iranian infertile males
Ashrafzadeh Mehrjardi HR1, 4, Dehghan Tezerjani M2, Khademi M1, Ghasemi-Esmailabad S2, Mossadegh AM3, Ghasemi N4.
- Department of Biology, Faculty of Science, Islamic Azad University, Ashkezar Branch, Yazd, Iran.
- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Department of Pharmacy, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
- Recurrent Abortion Research Center, Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Introduction: Infertility is the failure to achieve a pregnancy after one year of regular sexual intercourse without contraception. Half of the cases of infertility are due to male factors and nearly 60-75% of reasons of male infertility are unknown, due to unknown mechanisms of molecular defect. Tumor Necrosis Factor α (TNF-α) is a multi-functional cytokine that regulates cellular processes related to spermatogenesis. Tumor Necrosis Factor Receptor 1 (TNFR1) mediate TNF-α activity and changes in its structure can affect TNF-α activity. Studies show that variation in the TNFR1 gene may be associated with male infertility.
Materials and Methods: This case-control study includes 108 azoospermic men and 119 healthy controls. We investigated the association of TNFR1 36 A/G in the population with idiopathic azoospermia who referred to Yazd Institute of Reproduction Sciences. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR- RFLP) method was used to genotype the single nucleotide polymorphism in the both groups. PCR fragments digested by enzymes Mspa1I, and were separated by gel electrophoresis and then the frequency of A→G substitution in azoospermic males and fertile men were counted.
Results: According to our study, G/G genotype in the control group has higher frequency among men with azoospermia [p=0.01; OR=2.29 (1.248-4.229)]. Our findings also show that the allele frequency of G allele in azoospermic men is significantly different in comparison with control group [p<0.001; OR=2.302 (1.580-3.55)].
Conclusion: It seems that the G/G genotype and G allele are significantly associated with increased risk of idiopathic azoospermia.
Key words: Polymorphism, Male infertility, Sperm, Cytokines, Tumor necrosis factor alpha.
P-155
Epigenetic evaluation of histone methylation on HOXA1-5 genes in cumulus cells of polycystic ovary syndrome patients
Asiabi Kohneh Shahri P1, 2, Shahhoseini M2, Favaedi R2, Hassani F3, Movaghar B3, Karimian L3, Eftekhari Yazdi P3.
- Faculty of Basic Science and Advanced Technologies at University of Science and Culture, ACECR, Tehran, Iran.
- Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
- Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Introduction: Polycystic ovary syndrome (PCOS) is the major cause of an ovulatory infertility with uncertain etiology. Epigenetics and environment play critical roles in PCOS. Of important genes correlated with human reproductive system disorders are HomeoboxA (HOXA) cluster genes. Regarding the epigenetic role of histone modifications in regulation of gene expression, lysine (K) methylation of histone3 (H3K9me), as a repressive epigenetic mark, on the promoter regions of HOXA1-5 genes were evaluated in this study. For this respect, cumulus cells (CCs) which have critical roles during folliculogenesis, oocyte maturation, ovulation and fertilization, were aimed to monitor expression profile and epigenetic alterations of HOXA1-5 genes.
Materials and Methods: CCs were collected from 20 PCOS patients and 20 fertile women (18-36 year) with male infertility problems, referred to Royan Institute to have IVF-ICSI under GnRH antagonist protocol. Informed consents were obtained from the participants. After evaluation of genes expression by qRT-PCR, chromatin immune precipitation (ChIP) coupled with real-time PCR was performed to evaluate incorporation of H3K9me into regulatory regions of HOXA genes.
Results: Expression data, revealed significant decrease in mRNA level of HOXA1 (p<0.05) and significant increase in HOXA2, HOXA3, HOXA5 (p<0.05) in cumulus cells of PCOS patients vs. control group. There was no significant change in expression level of HOXA4 (p<0.05) among two studied groups .Obtained data from ChIP Real-time PCR verified well the results of gene expression; in the way that histone methylation level in regulatory region of HOXA1 does not show any alteration but there were significant decrease of H3K9me in promoters of HOXA2, HOXA3, HOXA4 and HOXA5 (p<0.05), in PCOS patients vs. control group.
Conclusion: These findings demonstrate the significant association of aberrant histone methylation with impaired oocyte maturation and confirm the functional role of epigenetics in pathogenesis of PCOS.
Key words: HOXA1-5, Histone methylation, Epigenetic, PCOS.
5 genes.
Materials and Methods: CCs were collected from 20 PCOS patients and 20 fertile women (18-36 year) with male infertility problems, referred to Royan Institute to have IVF-ICSI under GnRH antagonist protocol. Informed consents were obtained from the participants. After evaluation of genes expression by qRT-PCR, chromatin immune precipitation (ChIP) coupled with real-time PCR was performed to evaluate incorporation of H3K9me into regulatory regions of HOXA genes.
Results: Expression data, revealed significant decrease in mRNA level of HOXA1 (p<0.05) and significant increase in HOXA2, HOXA3, HOXA5 (p<0.05) in cumulus cells of PCOS patients vs. control group. There was no significant change in expression level of HOXA4 (p<0.05) among two studied groups .Obtained data from ChIP Real-time PCR verified well the results of gene expression; in the way that histone methylation level in regulatory region of HOXA1 does not show any alteration but there were significant decrease of H3K9me in promoters of HOXA2, HOXA3, HOXA4 and HOXA5 (p<0.05), in PCOS patients vs. control group.
Conclusion: These findings demonstrate the significant association of aberrant histone methylation with impaired oocyte maturation and confirm the functional role of epigenetics in pathogenesis of PCOS.
Key words: HOXA1-5, Histone methylation, Epigenetic, PCOS.
Type of Study: Congress Abstract |
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