International Journal of

Key Lectures (Alphabetic order)
 
9^th Yazd International Congress and Student Award on Reproductive Medicine
 
K-1
Ovarian rejuvenation through platelet-rich autologous plasma
 
Aflatoonian A.
Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: abbas_aflatoonian@yahoo.com
 
Platelet-rich plasma (PRP) is a novel method that has been successfully employed for a range of medical issues. PRP holds a high concentration of platelets found in plasma many types of protein molecules, cytokines, and growth factors. Therefore, it is considered as a reasonable method with beneficial effects on tissue regeneration, angiogenesis activation, inflammation control, and anabolism. PRP was also proposed as an acceptable and potentially successful approach for improving the fertility outcome in poor ovarian responders (PORs) as well as women with primary ovarian insufficiency (POI). However, there is still insufficient clinical data on the application of PRP in the field of ovarian infertility. Preliminary studies revealed that in women with POI, intraovarian injection of autologous PRP might be an alternative experimental treatment option. Furthermore, it has been shown that PRP injection could be positively applied as an effective and safe approach in PORs before the IVF procedure to increase the clinical pregnancy and live birth rates. Our recently published clinical trial showed a 47% pregnancy among PORs in response to PRP injection; of those, 50% led to healthy live births. In addition, we found menstruation recovery among 22.2% of women with POI after the PRP injection. In the end, ovarian autologous PRP injection could be a chance for conception without donor eggs in poor responder patients, along with improving the life quality of women suffering from early menopause without synthetic hormonal treatment.
 
K-2
Potential novel treatments in polycystic ovary syndrome
 
Aflatounian A^{1, 2}.

Email: aflatoonian@gmail.com
Polycystic ovary syndrome (PCOS) is a common and complex endocrine disorder, which is characterized by the presence of defining reproductive and endocrine defects. PCOS patients also suffer from metabolic features, including obesity, insulin resistance, liver steatosis, and an increased risk of type 2 diabetes. Despite the recent advances in our understanding of the underlying factors involved in the development of PCOS, the exact cause of the syndrome is still obscure. Hence, there is no cure for this condition and the current management strategies are limited to symptomatic treatment options. However, during the past decade, several novel therapeutic agents with a more mechanistic approach have displayed promising results in ameliorating defining PCOS traits. For instance, the administration of resveratrol, a sirtuin 1 activator, in a randomized controlled trial was able to reduce serum androgen levels and decreased insulin resistance in PCOS patients. Precursors of NAD⁺, which in turn can induce the activity of sirtuin 1 have also shown promising results in experimental models of PCOS by reversing both metabolic and reproductive features of PCOS. More recently, specific agents have been developed to target kisspeptin, neurokinin B, and dynorphin system, which is suggested to be one of the main role players in the pathophysiology of PCOS. Such experiments have demonstrated the amelioration of PCOS-like traits in animal models of PCOS following the treatment with agents that target the kisspeptin, neurokinin B, and dynorphin system. Additionally, the administration of a number of supplementations has been evaluated in several clinical and experimental studies. Coenzyme Q10, bioflavonoids, N-acetyl-L-cysteine, and berberine with antioxidant properties and melatonin as a key regulator of circadian rhythm have been displayed positive impacts on the clinical and experimental features of PCOS. However, further investigation is still required in order to evaluate and validate the efficacy of such novel therapeutic agents in the management of PCOS.
 
K-3
ROS and assisted reproduction
 
Agarwal A.
Center for Reproductive Medicine, Cleveland Clinic, Cleveland, Ohio, USA.
Email: agarwaa@ccf.org
 
Abstract not received.
 
K-4
Recurrent implantation failure: Update etiology, diagnosis, treatment, and future direction
 
Aghahosseini M.
Department of Obstetrics and Gynecology, Tehran University of Medical Science, Tehran, Iran.
Email: marzieh.aghahosseini@gmail.com
Implantation failure can be used for both patients who have never shown increased level of human chorionic gonadotropin, and those who have increased HCG without later evidence of gestational sac and only applicable in ART.
It is important to consider age, stage of embryo- cleavage, or blastocyst. Therefore, recurrent implantation failure (RIF) is the failure of clinical pregnancy after 4 good-quality embryo transfers at least three fresh or frozen IVF cycles in women under age 40 years. Maternal age, BMI, smoking, and stress considered as the risk factors of RIF. Pathophysiology mechanism of RIF include immunological (maternal killer cells, peripheral, uterine Th1/ th2 ratio, TNF-α level-autoantibodies, and Aps-hereditary thrombophilia), Infection, Leukemia inhibitory factor, anatomical abnormalities, endometrial thickness, and genetic. Therapeutic interventions for RIF are optimal IVF treatment (Embryo factor-transfer methods-ovulation induction protocol-progesterone support) immunotherapy such as Tacrolimus, IVIG, PBMC (peripheral blood mononuclear cell), and granulocyte colony-stimulating factor (G-CSF), treatment of infection, correction of intra uterine pathologies, salpingectomy, endometrial injury, genetic (PGS) – PGD, endometrial receptivity array, male factors, lifestyle modification, supportive treatment. The recommendation for women with RIF perhaps the best is personalized medicine.
 
K-5
Tissue engineering and regenerative medicine in Iran: Current progress of Iranian Universities
 
Ai J.
Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Email: jafar_ai@tums.ac.ir
 
Tissue engineering and regenerative medicine (TERM) is an emerging field focused on the development of alternative therapies for tissue/organ repair. This highly multidisciplinary field, in which bioengineering and medicine merge, is based on integrative approaches using scaffolds, cell populations from different sources, growth factors, nanomedicine, gene therapy, and other techniques to overcome the limitations that currently exist in the clinics. The field of TERM in Iran, dating back to early 1990s and the advent of stem cell researches. During two decades ago, Iran has exhibited a remarkable increase in scientific publication in different aspects including TERM and today, Iran is one of the privileged countries in stem cell therapy in the Middle East. The main goals in TERM are the application and fabrication of scaffolds for tissue engineering of nerve, heart, liver, bone, and cartilage tissues. Todays some of the product from engineered tissues in laboratory move to the clinic in Iran but there are some problems in the clinical application of constructs that need to be solved them.
K-6
The positive effect of kaempferol on fibroblast cell reprogramming
 
Akhound-Attar M^{1, 2}, Esmaeili-Dehaj F^{1, 2}, Aghaei Sh², Aflatoonian B², Moshtaghioun SM¹, Farashahi-Yazd E².

Email: ehsanfarashahi@gmail.com
 
The reprogramming of differentiated cells creates a suitable source of pluripotent cells, which could be achieved via using Yamanaka factors, the most common method. The low efficiency of this reprogramming is challenging. In order to increase efficiency, various methods have been tested before.
Using of small molecules, especially natural ones, for elevating of reprogramming efficiency are recently more considered. Of this group, Kaempferol, for the known epigenetic and anti-oxidant effects on stem cells used in our study.
Recombinant fibroblast cells, with inducible Yamanaka factors (FUW-tetO-hOKMS) gene cassette, exposed to different concentrations (1, 5, and 10 µM) of Kaempferol (K group) and 0.5 mM of sodium butyrate (S group) separately for 5 days, before the cassette induction until iPS cell like colonies formation. Cellular morphology changes of these samples were evaluated quantitatively and qualitatively. The molecular assays including qRT-PCR and IF of pluripotent markers will be performed ahead.
The first cell morphological changes were observed on the fifth day after tetO-hOKMS cassette induction in case (K) and control (S) groups. Quantitative and qualitative morphological examinations of cells showed that embryonic-like colonies were only formed among cells treated with 5 µM Kaempferol (two colonies) and cells treated with sodium butyrate (one colony). However, the failure in the expansion of these small numbers of colonies has made it difficult to continue research.
The low number of embryonic-like colonies compare to the results obtained from similar studies indicates the low efficiency of reprogramming likely due to our poor performance and the work requires more repetitions and removal of the reprogramming induction barriers. However, by considering the similar positive efficacy of Kaempferol to the sodium butyrate (as a common reprogramming agent) in cell reprogramming, it makes sense that more studies on Kaempferol effects on cell reprogramming.
K-7
The success of various endometrioma treatments in infertility: A systematic review and meta-analysis of prospective studies
 
Alborzi S.
Department of Obstetrics and Gynecology, Shiraz University of Medical Sciences, Shiraz, Iran.
Email: alborzisa@gmail.com
Endometriosis is seen in 0.5-5% of fertile and 25-40% of infertile women. To investigate this conflict between gynecologists that ovarian endometriomas should be removed or not before making any decision about pregnancy among infertile women, we decided to carry out a systematic review and meta-analysis to compare the effect of various available therapeutic methods and notice the impact of these options on women ́s pregnancy rate. This review is based on PRISMA recommendations with an electronic search using the following databases: Pubmed, Scopus, Google scholar, etc from 2000 and 2018, in the English language. The studies compare pregnancy rate based on four different treatment types of OMAs between infertile women: (surgery + ART, surgery + spontaneous pregnancy, aspiration ± sclerotherapy + ART, and ART alone). At least 8 prospective studies were included, in which 553 infertile women were compared in term of treatment methods of OMAs before trying to become pregnant. Treatments are usually based on the patient's clinical condition and must be individual, with the purpose of relieving pain, improving fertility, or both. We haven’t any significant difference between our 4 groups of study, however the success of surgical procedure compared to other methods was higher and the success of ART alone was the least.
 
K-8
Feto-maternal immunological cross talk
 
Alecsandro D.
Department of Immunology, IVI Madrid, Madrid, Spain.‎
Email: Diana.alecsandru@ivirma.com
 
Not only embryo aneuploidies, but also the other factors such as immune-related maternal tolerance to pregnancy might contribute to implantation failure or miscarriage.. Maternal tolerance begins at the uterine level following by successful adaptation to the semiallogeneic fetus is a complicated process. The fetal cells that come into direct contact with the mother's immune cells in the uterus are uterine natural killer (uNK) cells in trophoblast cells, the layer that surrounds the blastocyst. The key function of the materno-fetal tolerance process is the remodeling of the spiral arteries, with the destruction of the media by invading extra villous trophoblasts (EVT) cells. The EVTs that invade the maternal decidua have fetal origin, and express high levels of human leukocyte antigen-C (HLA-C) recognized by uNK killer cell immunoglobulin-like receptors (KIRs). Maternal and paternal HLA-C allotypes are expressed at the same time and at high levels on the EVT cell surface. Placentation is regulated by interactions between maternal KIRs expressed by uNKs, and fetal HLA-C molecules, expressed by EVTs. Insufficient invasion of the uterine lining by trophoblasts and vascular conversion in the decidua are thought to be the primary defect among disorders such as recurrent miscarriage, preeclampsia, and fetal growth restriction. This process is regulated by the interaction between maternal KIRs, expressed by the uNKs, and their ligand HLA-C, expressed by EVTs. Pregnancies are at increased risk of recurrent miscarriage in mothers who are homozygous for KIR haplotype A (KIR AA) when the fetus has more HLA-C2 genes than the mother does and when additional fetal HLA-C2 alleles are of paternal or oocyte donor origin. There is a lower live birth rate and an increased miscarriage rate after double embryo transfer in KIR AA patients. In addition, the live birth rate decreases significantly as the embryo HLA-C2 load increases. The maternal immune system is one of the main actors at the maternal–fetal interface, and its lack of activation but not a rejection seems to influence the placentation and pregnancies outcomes.
 
K-9
Treatment of poor responder patient
 
Aleyasin A.
Department of Obstetrics and Gynaecology, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran.
Email: aleyasin.gyn@gmail.com
 
Decrease of ovarian reserve (DOR) is one important reason that negatively affects female fertility. DOR has been found in the 8-15% of assisted reproductive technology cycle. For women over 40 years DOR is about 50%, and 10% of women may experience an early reduction in ovarian reserve regardless of age. To find a proper treatment, first, we need to define DOR or poor responder patients, which two different criteria, named Bologna or Poseidon, shall be studied. Bologna criteria includeMaternal age ≥ 40 years or any other risk factors for DOR, antral follicle count < 5-7 follicles or anti-mullerian hormone (AMH) < 0.7-1.3 (ng/ml), and a previous DOR < 3 oocytes with a conventional controlled ovarian hyperstimulation (CoH). According to the Poseidon classification patient are subdivided into four subgroups based on first, age, second, antral follicle count or AMH, third, ovarian response, and finally, expected poor responder.
There are different lab tests for diagnosis of DOR including, FSH/E2, inhibin B, AMH (in day 2-3 menstrual cycle), and more recently insulin-like growth factor-1 in day 3 menstrual cycle. Additionally, transvaginal sonography is also recommended as another diagnosis method. To reach the best outcome of treatment in poor responder patient, it is recommended to decrease the required time to cumulative live birth rate per cycle, which is mainly dependent on the number of oocytes and an euploid embryos. For optimization of the oocyte number per ovarian cycle in DOR, there are number of recommendations to follow:
- Long protocol with conventional COH as the first choice for the subgroup of Poseidon 3 or 4.
- Mild stimulation protocol
- Dual stimulation protocol
To achieve better outcome adjuvant therapy in DOR patients is recommended as following:
- Growth hormone (GH)
- DHEA and other androgens
- LH supplementation
As known before, gonadotropin is commonly used in the COH cycle for poor responder patients. Current evidence supports a maximum daily dose of 300 IU of FSH, contrary to the previous recommendation of 600 IU. As mentioned before, the best outcome in poor responder patients is to obtain a euploid embryo. Because the availability of at least one euploid embryo increases the rate of pregnancy in poor responder patients up to approximately 60%, no matter the age. However, new evidence from basic science studies provides a biological explanation for the age-related effect on aneuploidy. This is due to impaired mitochondrial function, increased granulosa cell apoptosis, and increased level of oxidative stress in germline cells.
 
K-10
Development of synthetic protein-free chemically defined, safe and efficacious media products for human ART: Compliance with safety, regulatory, and cultural norms
 
Ali J.
Department of Obstetrics and Gynaecology, Women's and Children's Health Complex, University of Malaya Medical Centre, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
Email: jaffarali@um.edu.my
 
This presentation describes the continuous ongoing research efforts to develop the SYNBIOS MEDIA which are a series of synthetic and chemically defined formulations of protein-free embryo culture, handling and cryopreservation media developed following >20 years of systematic research (first communicated in 1997 at the Fertility Society of Australia Scientific Conference - Ali, 1997; and published in 2000 - Ali et al, 2000, Human Reprod 15:145-156) specifically to address the safety, and compliance with both regulatory issues and cultural norms. Conventional gamete and embryo culture, handling and cryopreservation media utilize donor serum protein supplements, which carry (i) a theoretical risk of disease transmission through protein-bound pathogenic agents; (ii) harmful undeclared protein contaminants; and (iii) donor micro DNA/RNA strands, and (iv) is prone to batch to batch variation in their composition affecting the quality of embryos generated between batches. Proteins cannot be sterilized with absolute certainty. The synthetic SYNBIOS MEDIA are devoid of added serum proteins making it among the safest media for both human and animal application with no (i) no pathogenic agents and thus no disease transmission, (ii) no undeclared harmful proteins contaminants, (iii) no donor micro DNA/RNA contaminants preserving the genetic purity of the embryo, and (iv) with no batch to batch variation; the latter ensures the quality of embryos generated between batches are maintained. It also complies with the cultural norms and lifestyle of many religions and beliefs. The synthetic SYNBIOS MEDIA products do not contain donor DNA/RNA because it is devoid of donor serum proteins. This attribute eliminates the risk of crossover of the embryonic genome with third party genetic material ensuring that embryos generated during ART treatment retain their two-parent genetic constitution thereby preserving the purity of lineage of the progeny, making it Halal-compliant and Halal-certified human embryo media. The efficacy of synthetic SYNBIOS MEDIA is similar to currently available media. The synthetic SYNBIOS MEDIA protein-free embryo culture media can be frozen-stored for up to 24 months without loss of efficacy. Freezing enables longer shelf life making SYNBIOS MEDIA embryo culture and handling media are thus less wasteful and economical for users.
 
K-11
Current insights in in vitro maturation of oocyte
 
Amidi F^{1, 2}.

1. Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
2. Department of Infertility, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran.

Email: famidi@sina.tums.ac.ir
 
Oocyte in vitro maturation (IVM) is an assisted reproductive technology in which oocytes are retrieved from the antral follicles of unstimulated or minimally stimulated ovaries. IVM involves retrieval of immature germinal vesicle stage oocytes and culture of intact cumulus-oocyte complexes in vitro until the metaphase II stage. Maturation of oocytes includes the nuclear and cytoplasmic maturation of oocytes. Only the oocytes whose nucleus and cytoplasm are matured simultaneously can have adequate fertility and the potential for embryo development.
The in vitro maturation of oocytes is mainly affected by culture conditions. Since the metabolic dynamics and required nutrients are not entirely the same in different stages of follicular development, optimization of each step is needed to achieve a higher maturation rate and better oocyte quality, based on the sequential culture system.
The enrichment of culture media, standardization of the stimulation protocols and management of cytoplasmic maturity are strongly recommended for improved IVM cycles. With the development of IVM technology, the combination of natural cycle IVF with the IVM of immature oocytes can be used as an attractive regimen to promote IVM treatment.
 
K-12
AMH and ovarian surgery
 
Asefjah H.
Laleh Hospital, Tehran, Iran.
Email: hossein.asefjah@gmail.com
 
Ovarian reserve is defined as the number and quality of follicles in the ovary at a set time. The hypothesis that endometrioma can impact ovarian reserve was established when histology-related research hypothesized that growing cysts, stretching the cortical tissue, might cause structural variations and circulation impairment, possibly leading to a decrease in the primordial follicle cohort in affected ovaries. The effects of ovarian endometrioma (OMA) (without previous surgery for OMA) on the ovarian reserve remain to be elucidated. The most reliable and extensively used ovarian reserve marker has been the level of anti-Mullerian hormone (AMH) due to its consistency throughout the menstrual cycle and following hormonal variations or treatments. AMH levels were noticeably lower in females with endometrioma in contrast to control groups (healthy ovaries and/or benign ovarian cysts). As has been detailed in previous publications, the presence of OMA is correlated with a decrease in AMH levels and adversely affects the ovarian reserve. However, numerous studies call into question the adverse influence of endometrioma on the ovarian reserve. AMH levels were downregulated only in subjects having undergone surgery independently of the presence of current endometriomas. Nieweglowska and co-workers reported that significantly decreased AMH levels were observed only in females with bilateral endometrioma, rather than in those with unilateral endometrioma. Similarly, Esinler reported that endometriomas with ≤ 3 cm in diameter did not impact the ovarian reserve. Of note, data from females with unilateral endometrioma are poorly informative, since the contralateral intact ovary compensates for ovarian function and fertility potential. Since larger cysts may be associated with lower levels of AMH, a relatively small cyst may not cause AMH levels to be significantly altered. women with endometrioma exhibited a progressive decline in serum AMH levels, faster than that in age-matched healthy females. It is emphasized that even experienced surgeons and accurate techniques cannot avoid operative ovarian reserve damage. AMH concentrations decreased noticeably after one year in patients with bilateral endometriomas, in individuals with cyst size >7 cm and in stage IV groups.
AMH hormone is a glycoprotein, a substance produced by granulosa cells in ovarian follicles. It is first made in primary follicles that advance from the primordial follicle stage. At this stage, follicles are microscopic and cannot be seen by ultrasound. An AMH test is often used to check a woman's ability to produce eggs that can be fertilized for pregnancy. A woman's ovaries can make thousands of eggs during her childbearing years. The number declines as a woman gets older. AMH levels help show how many potential egg cells a woman has left.
Unlike FSH, which may vary day to day and month to month, AMH is more consistent and, when combined, AMH and FSH provide the best insights compared to FSH alone. AMH is produced by the granulosa cells that line the tiny follicles within the ovaries. AMH serum levels differ widely according to genotype. Very low serum AMH concentration is characteristic of AMH mutations. However low AMH concentration in newborns or in boys undergoing puberty is physiological (Grinspon et al., 2011), and should not be interpreted as a sign of AMH mutation. Measurement of serum AMH is even more sensitive and specific than the AFC as it also reflects pre-antral and small antral follicles (< 2 mm), which are hardly seen in the ultrasound. Serum AMH is therefore a deeper “probe” for the growing follicular pool than the AFC (Dewailly et al., 2014a).
The level of AMH in the blood can help doctors estimate the number of follicles inside the ovaries, and therefore, the woman's egg count. A typical AMH level for a fertile woman is 1.0-4.0 ng/ml; under 1.0 ng/ml is considered low and indicative of a diminished ovarian reserve. To encourage your body to naturally raise levels of AMH is to choose a diet that is rich in necessary nutrients, such as vitamin D. Many women with a low AMH get pregnant naturally, though it's less likely as the score falls below “low." AMH levels vary from month to month, and a lower level doesn't say with absolute certainty that you can't get pregnant.
AMH levels below 1.6 ng/mL predict a smaller number of eggs retrieved with IVF. Levels below 0.4 ng/mL are severely low and are not compatible with successful IVF. Women with very low (< 0.5 ng/ml) AMH levels undergoing IVF still have reasonable chances of achieving a pregnancy, but their prognosis is significantly affected by chronological age. Women with PCOS often have elevated AMH levels, likely to be due to the high levels of follicles they have in the early stage of development. Studies showed that stress exposure was related to reproductive failure. In this study, we found that there was a significant correlation between psychological stress and decreased AMH levels for infertile women.
Endometrioma is the cystic lesion of ovaries originating from endometrial glands and stroma; it is identified in 17-44% of patients with endometriosis. Numerous existing studies have reported the association between endometrioma and infertility. Surgical excision has commonly been considered to avoid further ovarian damage. However, the potential adverse effect of this surgery on the ovarian reserve has recently become a focal point. Whether or not surgical excision can facilitate subsequent conception in young women planning to be pregnant is controversial. Whether non endometriotic ovarian cystectomy or unilateral oophorectomy can decrease ovarian reserve will be discussed during the presentation, and few techniques of laparoscopic cystectomy will be demonstrated.
K-13
Regenerative medicine in liver and pancreas
 
Azarpira N.
Shiraz Transplant Research Center, Shiraz Institute for Stem Cell and Regenerative Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
Email: negarazarpira@yahoo.com, azarpiran@sums.ac.ir
Many deaths in the world occur every year due to chronic liver and cirrhosis diseases. Liver transplant is the gold standard treatment but organ shortage is a great obstacle. There are several cell sources for liver regeneration such as primary hepatocytes, Mesenchymal (MSCs), embryonic stem cells and induced pluripotent stem cells (iPSCs). Nowadays, tissue-engineering strategies in liver regenerative medicine is an attractive issue; like cell sheet, liver organoids, recellularized liver, and bioartificial liver organs. Regarding diabetes mellitus, a whole pancreas transplant is the treatment of choice. Allogeneic islet transplantation in the liver of carefully selected diabetic recipients via portal vein infusion is an alternative cell-based strategy. Other stem cell sources like chemically induced islet-like cells, embryonic stem cells & iPSCs are alternative sources. The application of ECM scaffold functionalization to present bioactive motifs is an important issue in tissue engineering strategies for the pancreas. Currently, macroencapsulation and microencapsulation systems are used in several clinical studies. However, there are many challenges in both primary and stem cell therapy for liver and pancreas disease such as ethical problem, immune rejection, and best cell number and rout of transplantation.
 
K-14
Biosensors for cancers of the reproductive system: Novel ideas in detection
 
Azimzadeh M^{1, 2, 3}.

1. Medical Nanotechnology and Tissue Engineering Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
3. Department of Advanced Medical Sciences and Technologies, School of Paramedicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: mos.azimzadeh@gmail.com
 
Cancer still is the most challenging disease globally, despite fascinating advances in medicine. Those types that assault reproductive-related organs are among cancers with a higher death rate, and the numbers are overgrowing. Therefore, early detection of cancers is the best way to a most functional treatment and/or inhibition of the progress. Current cancer detection strategies predominantly suffer from low sensitivity and specificity and need expensive instruments with an expert operator. However, biosensors have shown a promising ability to detect/quantify molecular biomarkers associated with Cancer's early stages over the past decades. They can be very sensitive and selective, especially biosensors using nanomaterials and nanostructures, called nanobiosensors. Biosensors are categorized based on their transducer types, and they can be in various working platforms from electrode and lab tubes to papers and chips. Later called lab-on-a-chip devices, a biosensor is integrated into a microfluidics chip and can do sample handling and manipulation before detection. Here, I introduce biosensors, their types and platforms, and their applications to detect reproductive-related cancers.
K-15
Microfluidics in stem cells studies: Applications in reproductive sciences
 
Azimzadeh M.

1. Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Medical Nanotechnology and Tissue Engineering Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
3. Department of Advanced Medical Sciences and Technologies, School of Paramedicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: mos.azimzadeh@gmail.com
 
Microfluidics is revolutionizing biology and medicine and over the past two decades, their applications are constantly growing. Stem cell studies are one of the most important applications of microfluidics since they can have the variety of advantages over conventional stem cell studies. Microfluidics devices are enabling us to closely monitor the stem cells even in single-cell studies to analyse their metabolism and secretions. In addition, it is possible to stimulate cells with chemical, biological, physical, and mechanical simulates and evaluating the differentiation, reprogramming and performing physiological, immunological, and morphological studies. Moreover, they can be used to co-culture different cells together yet in two separate chambers, study organoids, and other 3D culture applications. Plus, the chance of contamination and cross-contamination is very low because they are sealed in the microfluidics channels and chambers. Cell sorting applications of microfluidics are also a great opportunity for scientists to separate specific stem cells from human sample cells. Last but not least, a very innovative application of microfluidics in medicine named organ-on-a-chip devices that can mimic a human organ can benefit from using stem cells.
 
K-16
Derivation of pluripotent cells from mouse spermatogonial stem cells (SSCs)
 
Azizi H.
Faculty of Biotechnology, Amol University of Special Modern Technologies, Amol, Iran.
Email: hosseinazizi1358@gmail.com
 
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. Here we describe, that only during a special time window (41 until 125 days) after initiation of germline 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. 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 spermatogonial stem cells (SSCs) in vivo. The generated gPS cells expressed pluripotency marker, in-vitro differentiated into all three germ layer lineages, formed complex teratoma after transplantation in SCID mice and produced chimeric mice. 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.
 
K-17
Mesenchymal stem cell-derived extracellular vesicle transplantation in animal model of premature ovarian failure
 
Baharvand H^{1, 2}.

1. Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
2. Department of Developmental Biology, School of Basic Sciences and Advanced Technologies in Biology, University of Science and Culture, Tehran, Iran.

 
Email: Baharvand50@yahoo.com
 
Abstract not received.
 
K-18
Human endometrial organoids
 
Baharvand H^{1, 2}.

1. Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
2. Department of Developmental Biology, School of Basic Sciences and Advanced Technologies in Biology, University of Science and Culture, Tehran, Iran.

Email: Baharvand50@yahoo.com
 
Abstract not received.
 
K-19
Three-dimensional scaffolds and differentiation of embryonic stem cells into oocyte-like cells, mimic cells –matrix interaction model
 
Bahmanpour S.
Department of Reproductive Biology and Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
Email: bahmans@sums.ac.ir
 
Stem cells are undifferentiated cells that present in the embryonic, fetal, and adult stages of life and give rise to differentiated cells that make up the tissue and organs. Due to their unlimited source and high differentiation potential, stem cells are considered as potentially new therapeutic agents for the treatment of infertility. Stem cells could be stimulated in vitro to develop various numbers of specialized cells including male and female gametes suggesting their potential use in reproductive medicine. During the past few years, considerable progress in the derivation of germ cells from pluripotent stem cells has been made. In addition, stem cell-based strategies for ovarian regeneration and oocyte production have been proposed as future clinical therapies for treating infertility in women. Three-dimensional (3D) culture matrices is a new technology in stem cells differentiation mimicking the tissue microenvironment. Based on biomaterials and porous substrates that can support cell proliferation, differentiation and regeneration, using scaffold can make tremendous progress in this field. Some growth factors, such as epidermal growth factor (EGF) also facilitate normal meiosis during oocyte maturation in vivo. A 3D culture system along with scaffolds can apply the induction of oocyte differentiation from embryonic stem cells. Therefore, embryonic stem cells can be induced to differentiate into oocyte-like cells using embryoid body protocol along with the three-dimensional microenvironment in vitro. For the effective differentiation of oocyte-like cells can employ the presence growth factor such as EGF and assessed the markers of germ cell differentiation, meiotic progression and oocyte maturation. In our study, EGF exposed cells in the three-dimensional microenvironment, upregulated the gene expression levels of premeiotic (oct4, Mvh), meiotic (SCP1, SCP3, Stra8, Rec8) and oocyte maturation (GDF9, CX37, ZP2) were significant. The high efficiency of differentiation into oocyte-like cells from embryonic stem cells reflects the culture method employed in the 3D co-culture system. These results showed that this culture system along with EGF improved the rate of in vitro oocyte differentiation.
 
K-20
Modern and future therapies in endometriosis
 
Chaichian Sh.
Pars Advanced and Minimally Invasive Medical Manners Research Center, Pars Hospital, Iran University of Medical Sciences, Tehran, Iran.
Email: shchaichian@gmail.com
 
Endometriosis is estimated to affect 7-15% of reproductive-aged women. It has been considered one of the most important and debatable gynecological diseases, however, selecting therapeutic strategies in this field is even more challenging. Even if it’s believed that the gold standard for treatment is surgery by some authorities, one should keep in mind that medical treatment is the cornerstone modality in this regard. There is an ongoing need for safe, effective, cheap medical therapies for endometriosis patients, both in conjunction with and independent of surgical interventions. Most conventional therapies for endometriosis work by a similar mechanism, and efficacy is variable. In recent years, there has been increased interest in the development and testing of novel pharmacotherapies for endometriosis.
In the present presentation, we discuss both conventional and emerging treatments for endometriosis, with different presentations across the lifespan and discuss how emerging therapies might fit into future medical management of endometriosis. Conventional therapies include nonsteroidal anti–inflammatory drugs, combined oral contraceptives, progestins, GnRH agonists/antagonists, and aromatase inhibitors. Emerging therapies are focused on disease-specific targets such as endothelial growth factor receptors, etc. It seems that the field of medical treatment of endometriosis is now moving toward modifying the immune and inflammatory responses in endometrial implants. If these drugs show efficacy in clinical trials, combining them with current medical treatment is expected to result in a profound impact on symptom and disease burden for patients who suffer from endometriosis worldwide.
 
K-21
The reproductive microbiome
 
Dashti S.
Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: saeideh_dashti@yahoo.com
 
The microbiota is defined as all of the micro-organisms that live in the human organs. Five distinct microbiota are determined in different anatomical locations of human body. Lactobacillus species are dominant healthy microbiota in the lower reproductive tract. These micro-organisms provide asuitable environment with low PH in the vagina that inhibits the growth of most pathogenic bacteria. Recently we know that the upper reproductive tract is not sterile and the endometrium has some own microbiota. However, the role of these micro-organisms in reproductive outcomes is less investigated. Researches confirmed lactobacillus- dominant vagina can lead to better outcomes with ART. Some studies showed endometrial dysbiosis can disrupt endometrial receptivity and lead to infertility. It seems non-lactobacillus-dominant microbiota in the reproductive tract can cause the lower chance of successful implantation and the high miscarriage rate. In recent years some oral and vaginal supplements are introduced as probiotic products and claimed that may recover vaginal environments with better reproductive outcomes. Some studies recommended that pre-conception counselling and lifestyle modification can impact reproductive outcomes but more investigations are needed. It is important to know what factors can disrupt normal reproductive tract microbiota and how we can determine them.
 
K-22
Sperm selection using Zeta potential method
 
Dehghan Marvast L.
Andrology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: laleh.dm236@gmail.com
 
In routine ICSI, the selection is based on morphology and viability, which does not necessarily preclude the chance injection of DNA-damaged or apoptotic sperm into the oocyte. Sperm with a high negative surface electrical charge, named “Zeta potential”, are mature and more likely to have intact chromatin. In this procedure, sperm is selected based on the presence of a negative charge on sperm membrane. Thus, the aim of our work is the comparison between pure gradient and Zeta method, to select spermatozoa with normal chromatin. Our aim is to develop a simple Zeta potential method for sperm isolation; and to analyze the sperm maturity, morphology, and DNA parameters.
 
K-23
The use of time-lapse technology for embryo culture, is it necessary?
 
Faramarzi A.
Fertility and Infertility Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran.
Email: a.faramarzi90@gmail.com
 
Setting up efficient criteria and reproducible approach to identify the best embryo is an important challenge in in vitro fertilization laboratory. Also, embryo culture in optimal conditions is a crucial factor for assisted reproductive techniques success. Conventional incubators and morphological microscopy assessment are routinely used to culture and select embryos with the highest developmental potential to transfer. Conventional microscopy analysis requires embryo removal from the stable condition of the incubator. So, it exposes the embryos to temperature, pH and oxygen level changes. However, the morphological analysis may include discrete data of blastomere size, number and symmetry, fragmentation, the appearance of inner-cell mass, and trophectoderm of the blastocyst.
In recent years, new incubators and culture medium have been improved which provide better development of embryos. Time-lapse technology provides continuous culture and observation of embryos. It eliminates the need for embryo removal from the stable condition of the incubator. Also, time-lapse technology allows embryologists to assess the exact developmental events of embryos. The embryologists are allowed to access and register the embryo development events from extrusion of the second polar body to blastocyst formation. Time-lapse technology has spread rapidly and a large number of in-vitro fertilization labs produced considerable data. Although, there is no consensus on which morphokinetics parameters, or combination of them, should have a main role in the selection of an embryo. Several confounding factors including patient characteristics and clinical procedures have been seen to influence the development of embryos.
However, there is not sufficient research of difference in clinical pregnancy, live birth, miscarriage rates between Time-lapse technology and conventional incubation. The application of this technology is quickly growing, becoming increasingly more accurate. Studies contain deep-learning models, artificial intelligence, and embryo morphokinetics are currently increasing. It enhances hopes for time-lapse technology for clinical use in the near future.
 
K-24
The language of life
 
Fazeli AR^{1, 2, 3}.

1. Institute of Veterinary Medicine and Animal Sciences, Estonian University of Life Sciences, Kreutzwaldi Tartu, Estonia.
2. Department of Pathophysiology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia.
3. Academic Unit of Reproductive and Developmental Medicine, Department of Oncology and Metabolism, The Medical School, University of Sheffield, Sheffield, UK.

Email: a.fazeli@sheffield.ac.uk
 
Language is a means of communication between individuals. Languages can have different forms. The majority of languages are spoken with words but other forms of language using signs and chemical codes as means of communication do exist too. These means of communication may vary between individuals of different species and even of the same species. One can even extend the concept of language and communication, to cells signalling each other and communicating between each other. Extracellular Vesicles (EVs) are nanoparticles that vary in size from around 50 nanometer (nm) to 1000 nm. These vesicles are found in all biofluids such as blood, saliva and semen. EVs are made of bio-membranes and carry a cargo consisting of mRNAs, microRNA, long non-coding RNAs, proteins and even DNA. Based on their size and route of their biogenesis, EVs are regarded as exosomes, microvesicles and apoptotic bodies. EVs are exceedingly regarded as a universal means of communication between different cells and cell types within one or between different species. In recent years, scientific literature has pointed to the role that EVs play in communication between the embryo and the maternal tract. This communication prepares the mother for the process of implantation and may have important implications for the success of the implantation process. Due to this capacity, EVs provide an excellent opportunity for biomarker discovery and understanding the events involved during the implantation process. During my presentation, I will discuss the recent research conducted in my laboratory to establish sensitivity and specificity of EV’s as biomarkers for predicting the success of the implantation process in IVF clinics.
K-25
Realtime and multilevel digital monitoring in ART: Are we ready to move IVF to the next level?
 
Findikli N.
Bahceci Health Group, Fulya IVF Center, Hakki Yeten Cad, 11 Kat 3, Terrace Fulya Istanbul 34365, Istanbul, Turkey.
Email: nfindikli@bahceci.com
 
Although tremendous improvements have been made in both clinical and laboratory approaches in recent years, current success rates in assisted reproductive technology are far below the expected levels. Among many, the most pronounced reasons include suboptimal diagnosis as well as treatment techniques or models, limits of morphology-based gamete and embryo selection, operator- and infrastructure-dependent variations in culture environment and manipulations as well as patient-specific characteristics that require personalized approaches. Recent studies indicate that electronic or AI-based novel approaches in coping with each limitation/obstacle can potentially show improvements in the outcome per se. However, promising reports usually suffer from either being enforced to fit in a certain isolated patient population or having problems associated with reproducibility in different clinical and laboratory settings. There is no doubt that such approaches will be used in routine applications in the future of assisted reproductive technology. However, overestimating the possible benefits and/or premature use of such approaches/technologies can also carry a risk of creating loss of interest among peers. To minimize such obstacles and upgrade the system for the complete digital transformation, we have developed in our group a customized, multilevel digital security, data management, and process monitoring system that can effectively be integrated and used real-time in different departments (clinical, laboratory, and genetics), simultaneously. Its gradual implementation has resulted in numerous positive and beneficial outcomes while we have also found out several key areas that are needed further improvements, as well as areas that created new risks associated with digitalization. In general, the transition from paper-based to digital data management and tracking platform usually come with certain infrastructural costs and risks that should be individually assessed for each clinic. Besides investing and planning for a dedicated and skillful IT team, management and training of the critical personnel are found to be one of the most important parameters for a successful implementation of such transformation. Furthermore, digital transformation should also be considered as a continuous process. Our customized digital monitoring system has now been effectively communicating and interacting with not only the medical and laboratory departments but also with other departments such as patient relations and accounting. With this opportunity, we are now in the process of developing AI-based applications that can help the key stakeholders (doctors, embryologists, and managers) to decide which is the best for the patient at any given time during his/her treatment. In this talk, together with the authors’ personal experiences, current approaches, and status of digital transformation in reproductive medicine will be summarized and discussed.
K-26
Microfluidics in embryo culture system
 
Ghazali Sh, Halvaei I, Khalili MA.

1. Department of Midwifery, School of Medical Sciences, Sanandaj Branch, Islamic Azad University, Sanandaj, Iran.
2. Department of Anatomical Sciences, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
3. Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: sh_ghazali@yahoo.com
 
Microfluidics is a multidisciplinary field of study that is rapidly growing in reproductive biology and assisted reproductive technology, based on its scientific and technical aspects. Using sub-microliter amounts of fluid in the microfluidic device instead of conventional micro-droplets might help to emerge a new horizon in assisted reproductive technology to investigate fluid behaviour, embryo culture, and embryo selection. Mechanical and biochemical features of microfluidics can improve all steps conducting in IVF laboratories from sperm selection and oocyte preparation to embryo culture and embryo assessment. Main studies have been focused on the application of microfluidics in rodents, nonetheless, recently some of them have turned to clinical usage.
In order to achieve success in in vitro embryo culture and selection, mimicking in vivo environment is logically necessary. Therefore, in microfluidic devices, embryo surrounding microenvironment, dynamic fluid environment, and chemical components should be considered. There are some challenges in investigating the microfluidic novel systems for embryo culture which include; disturbance of fluid flows, media evaporation, osmolality alteration, design and structure of devices, and possible toxicity of polymers used in 3D printed devices. Due to eliminating some of them, setting up computer-controlled Braille platform, and, using PDMS-parylene-PDMS hybrid membrane or polystyrene (PS) had been suggested. In this regards, dynamic fluid condition (microfunnel-pulsatile) could show a greater number of cells per blastocyst compared with static culture. In conclusion, despite some pros and cons, microfluidics has been rapidly noticed in the embryo culture system. Owing to complexities and multi-parametric issue of embryo culture, comprehensive and comparative investigations are suggested.
 
K-27
The effect of supporting the one carbon cycle on in vitro and in vivo fertilization outcomes
 
Jafarpour F, Nasr-Esfahani MH.
Department of Animal Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
Email: nasr.royan@gmail.com
 
One-carbon cycle (1-CC) is comprised of a series of metabolic pathways that can be categorized into folate and methionine cycles, transsulfuration pathway and recently formaldehyde cycle. These metabolic pathways are central to various important cellular functions that provide one-carbon units for essential biosynthetic reactions and for writing epigenetics marks. Folate is one of the most important promoters of 1-CC. It is well recognized that serious deficiency of folate during pregnancy is associated with adverse outcomes such as neural tube defects in the child. However, there is a growing concern about the potential adverse effects of high dose folate supplementation before and during pregnancy. Regard to this, there are few studies which report that excess folate consumption can cause developmental abnormalities and cognitive abnormalities in offsprings in mice. Moreover, two common inherited human mutations are the 677C > T and 1298A > C mutations in the gene encoding methylenetetrahydrofolate reductase, which converts 5, 10-me-THF to 5-methyl-THF (m-THF, the active form of folate). These mutations may increase the un-metabolized form of folate and decrease m-THF which may explain to some extent why these mutations might be more prone to infertility and certain chronic illnesses. Therefore, monitoring of pregnant women for adequate dietary folate intake regarding the presence or absence of these mutations is required.
In addition, despite extensive improvements in assisted reproductive techniques (ARTs), the pre-implantation and post-implantation outcomes of ARTs have remained low. One of the main causes of these insufficiencies may be related to the in vitro manipulations and composition of the culture medium. Therefore, any changes that can bring these conditions closer to the in vivo situations can probably have a significant impact on ARTs. One of the important metabolic pathways during the process of folliculogenesis, oocyte maturation, and embryonic development is 1-CC. So, adding the substrates and cofactors of 1-CC may improve ARTs outcomes. In this presentation, first, we discuss the effect of folate deficiency and also the excessive dose of folate on fertility outcomes and then discuss the importance of various micronutrients involved in 1-CC during in vitro conditions.
 
K-28
PGT-A in ART anno 2021: An update
 
Jaroudi S, Fatemi HM.
ART Fertility Clinics and Genomics, Abu Dhabi, United Arab Emirates.
Email: Souraya.Jaroudi@artfertilityclinics.com
 
Preimplantation genetic testing for aneuploidy (PGT-A) is increasingly employed in assisted reproductive technology (ART). By sifting out embryos with abnormal chromosome numbers (aneuploid), PGT-A should theoretically improve pregnancy success. However, earlier versions of PGT-A were ineffective, and in some cases, detrimental, due to biopsy-induced trauma and because the technology at the time could analyse only a fraction of all chromosomes. More recently, the emergence of technologies enabling all chromosomes to be analysed and a switch to less traumatic blastocyst-stage biopsy have seen widespread uptake of PGT-A. Assessing the full impact of blastocyst biopsy PGT-A requires consideration of multiple factors, including embryonic mosaicism, the sensitivity of the technological platform used, embryo loss during long-term in vitro culture, embryo cryopreservation, and inter-clinic variability in expertise. PGT-A has been shown to increase the success of in vitro fertilization (IVF), by reducing the risk of miscarriage, shortening the time to pregnancy, and allowing more confident single embryo transfers without compromising outcomes. While it is widely accepted that chromosome aneuploidy, a common feature of human embryos, is a major cause of IVF failure, miscarriage and congenital defects, controversy remains around the routine implementation of PGT-A in clinical practice. The possibility of bypassing biopsy with non-invasive PGT-A (niPGT-A) is becoming highly attractive. niPGT-A via the analysis of cell-free DNA (cfDNA) present in the spent culture media is currently an area of active development. Furthermore, next-generation sequencing increased the resolution and sensitivity of PGT-A allowing the identification of chromosomal mosaicism and the detection of segmental copy number variations (CNVs), both areas of disagreement due to their uncertain clinical significance. The concordance rates between PGT-A results from the original trophectoderms samples and re-biopsies have been shown to be reduced for mosaic or segmental CNVs compared to euploid and whole chromosomal aneuploidy results. Nonetheless, segmental errors are believed to be responsible for 6% of clinical miscarriages and can result in genetic conditions, such as Cri-du-chat, accounting for approximately 0.05% of births. Variable mosaicism rates based on single TE biopsies (2-13%) are influenced by the next-generation sequencing assay adopted, the stimulation protocols and the IVF laboratory culture conditions. As biopsies are not necessarily representative of the whole embryos, the true prevalence and clinical implications of blastocyst mosaicism are still under question. CNVs are increasingly recognised as natural events in the preimplantation embryo. Furthermore, observations of developmental competence in a subset of mosaic blastocysts reaching healthy live births lead to the suggestion of corrective mechanisms. However, there is no direct evidence to support this theory at present. The effects of low-moderate level mosaicism on ongoing pregnancy rates remain unclear, with studies examining the transfer of such embryos reporting conflicting data. Elucidating the uncertainties around mosaicism and segmental CNVs within well designed studies would lead towards improved PGT-A and patient management in ART.
 
K-29
Endometrial scratching: What is the end of role?
 
Karimzadeh MA.
Madar Hospital, Yazd, Iran.
Email: Makarimzadeh@yahoo.com
 
Embryo implantation is one of the most important factors influencing pregnancy in assisted reproductive technology cycles and is usually attributed to a lack of uterine receptivity. Although some studies suggest that endometrial scratching may double the chances of getting pregnant, conclusive scientific evidence on its benefits and knowledge of the underlying mechanisms is limited. It is not clear exactly how endometrial scratching works, but it is thought that scratching the uterine lining may induce an inflammatory response. The subsequent repair process may improve the chances of implantation by:

• The release of growth factors, hormones, and proinflammatory cytokines, which make the newly-formed lining more receptive to an implanting embryo
• Activating genes that are important for the preparation of the endometrium, which may not otherwise be turned on, at the time of attempted implantation.

Mechanical disruption to the endometrium has been shown to modulate the genetic expression of factors important for implantation, including laminin alpha 4, integrin alpha 6, matrix metalloproteinase 1, and glycodelin A. The optimum time for endometrial scratching is controversial and should be discussed in the presentation. A Cochrane review of nine randomized trials including 1512 women with unexplained subfertility suggested an overall benefit from endometrial scratching. However, the review also stated significant limitations to the included studies, and cautioned against drawing confident conclusions from these findings. The authors also emphasized the importance of balancing the possible benefits against the potential risks. Although Olesen in 2019 was shown that the pregnancy rate is higher after endometrial scratching but other studies showed differences in live birth and clinical pregnancy rates among the endometrial scratch and control groups were not significant. But, recently in 2020 results of the SCRaTCH trial that was a non-blinded randomised controlled trial in women with one unsuccessful IVF/ICSI cycle would lead to a higher live birth rate after the subsequent IVF/ICSI treatment compared to no scratch. According to available data, more research is needed in order to determine whether endometrial scratching significantly improves the chances of pregnancy with IVF. Finally, this procedure should not be offered in daily practice, and its use in patients undergoing IVF for the first time is not supported.
K-30
ART and male infertility
 
Kazemeyni SM.
Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran.
Email: kazemeyni@yahoo.com
 
I had the opportunity that my clinical practice has begun when the assisted reproductive technology (ART) has begun in Iran. In fact, I was a member of the early team of Yazd Infertility Center.We have witnessed dramatic advances and success of this high technology how to treat male infertility. Before the ART era, there were many limitations for the treatment of infertile men, especially those who were azoospermic or even oligospermic. But in spite of this progress, some side effects have grown. Most harm is to ignore men's health, while male fertility and health are closely interrelated. Another problem is paying less attention to the best and near most natural therapy process and the trend to bypass it with ART.
Unfortunately, as some recent data, almost one third of men in infertile couples were never evaluated. Therefore, due to both major reasons as men’s health and the best treatment of infertility, the andrology department is essential for any ART centers.
 
K-31
In vitro spermatogenesis in artificial testis for male infertility
 
Koruji M^{1, 2}, Bashiri Z^{1, 2}, Asgari HR².

1. Stem Cell and Regenerative Medicine Research Center, Iran University of Medical Sciences, Tehran, Iran.
2. Department of Anatomical Sciences, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.

Email: koruji.m@iums.ac.ir
 
Increasing male infertility rates have led to a greater need for new artificial testicular systems in order to preserve fertility. Laboratory studies have shown that preservation, proliferation, differentiation, and transplantation of spermatogonial stem cells or testicular tissue could be ways to maintain the fertility of childhood cancer patients and azoospermic men in the future. In this regard, tissue and cell culture, supplements and 3D scaffolds have created a new perspective on the differentiation of stem cells in vitro, which could improve the outcomes of male infertility. The 3D matrix appears to allow for the formation of colonies and the proper arrangement of testicular cells, although differentiation has not yet been fully obtained. Therefore, in the future, new systems will be needed so that they can cause proliferation and maturation of germ cells in laboratory conditions and ultimately produce functional sperm by emphasizing regeneration of the germ cell microenvironment.
 
K-32
New horizons in onco-fertility
 
Ledger W.
Director of Reproductive Medicine, Royal Hospital for Women, University of New South Wales, Sydney, Australia.
Email: ledger@unsw.edu.au
 
Improvements in the management of many common cancers in young people have led to significant growth in the number of long-term survivors of cancer in people of reproductive age. However, survival comes at a price, with many young people suffering significant side effects from chemotherapy and radiotherapy. These include significant damage to reproduction due to the gonadotoxicity of cancer treatments. Recognition of the importance of future fertility to cancer survivors has led to increasing interest in the subspecialty of “oncofertility” the preservation of reproductive options for young men and women before cancer treatment. Fertility preservation is relatively straightforward for most post-pubertal males and cryopreservation of semen has been performed for over half a century for this group of patients. More recently, technological developments in cryopreservation of oocytes and embryos have allowed realistic chances of future fertility of female patients who preserve gametes or embryos before treatment. In younger patients or those who do not have time to undertake a stimulated IVF cycle to obtain oocytes, ovarian tissue cryopreservation is an increasingly used option. Testicular tissue is also frozen for pre-pubertal boys, but cannot currently be used to reinitiate spermatogenesis. Future prospects include novel means of ovarian protection providing improvement on the largely ineffective use of gonadotropin-releasing hormone agonists, and increasingly, in vitro maturation of immature oocytes to reduce the time taken to perform fertility preservation before cancer treatment can be started.
 
K-33
Sperm selection using motile sperm organelle morphology examination (MSOME) in ART
 
Mangoli E.
Andrology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: es.mangoli@gmail.com
 
The ultimate aim of any sperm selection method is to provide the best-quality sperm possible so as to maximize the outcome of whatever assisted reproductive technology (ART) procedures are to be undertaken. Gamete micromanipulation, such as intracytoplasmic sperm. injection (ICSI), is very useful for treating couples with compromised sperm parameters. An alternative method of sperm selection has been described; the spermatozoa are selected under high magnification (over 6000x) with sperm organelle morphology examination (MSOME) criteria and used for ICSI. This technique, named intracytoplasmic morphologically selected sperm injection (IMSI), has a theoretical potential to improve reproductive outcomes among couples undergoing assisted reproduction technology (ART). According to the majority of studies, it is not recommended to use MSOME/IMSI routinely in the ART program. The couples with repeated implantation failures, patients with severe male factor infertility, sperm DNA damage, advanced male, and maternal ages are the populations who will have higher chances to conceive from this technique. It is also recommended that diagnostic morphological evaluation of semen samples with MSOME is done before ICSI/IMSI procedure. The effectiveness of IMSI is still controversial mainly due to differences in inclusion criteria, stimulation protocols, seminal and oocyte qualities, and many other confounding variables within the ART program. However, there is no doubt that the use of MSOME/IMSI techniques can be helpful for some infertile couples to have a baby.
 
K-34
The indication of surgery in endometriosis
 
Mehdizadeh A.
Endometriosis Research Center, Iran University of Medical Sciences, Tehran, Iran.
Email: amehdizadahkashi@yahoo.com
 
Endometriosis is highly prevalent, yet compared to equally prevalent conditions it is poorly understood and a challenge to manage. It has been estimated that more than 176 million women worldwide suffer from endometriosis and its associated symptoms including infertility, cyclical and non-cyclical abdominal pain, dysmenorrhea, dyspareunia, dysuria, and dyschezia. Endometriosis may be categorized into three entities: peritoneal endometriosis, ovarian endometriotic cysts (endometrioma), and deep endometriosis (DE) (previously known as deep infiltrating endometriosis or DIE). Indications for endoscopic diagnosis and treatment in endometriosis are as follows: Pain, Organ destruction and/or Infertility. Surgical removal of the lesions is considered the “gold standard” for symptom control. Surgery is an important treatment option for women with DE.
However, like medical intervention, surgery is not always successful and is also associated with clinically relevant risks (Chapron et al., 1998; Becker et al., 2017). Surgical treatment failure can be partially attributed to the heterogeneity of endometriosis but it is also correlated with factors such as surgical experience, the complexity of each case, and anatomical locations of the disease. Surgeons must have significant knowledge of pelvic anatomy in order to have an approach to a grossly distorted surgical field. Thus, pelvic anatomical landmarks represent essential points of reference to start procedures such as mobilization of the pelvic viscera, wide peritoneal resections, or the identification of further anatomical structures to be preserved, such as bowel, ureter, vessels, and parasympathetic and orthosympathetic pelvic neural fibres in nerve-sparing procedures (Ceccaroni et al., 2018). The principles for identifying and treating deep endometriotic lesions and the good practice recommendations in the text aim to support clinicians and surgeons in counselling and treating (or referring) women presenting with DE.
 
K-35
Sharing beliefs-triangle of sexuality, religion, and infertility: Argumentative review
 
Merghati Khoei E^{1, 2}, Merghati A³, Merghati khoei T².

1. The Family and Sexual Health Division, BASIR Neuroscience Institution, Tehran University of Medical Sciences, Tehran, Iran.
2. Department of Private and Islamic Law, Faculty of Law and Political Sciences, University of Tehran, Tehran, Iran.
3. Faculty of Theology, Farabi College, University of Tehran University, Qom, Iran.

Email: effat_mer@yahoo.com
 
The influences of culture are present in different areas of sexual and reproductive behaviours as well as help-seeking behaviours in the case of infertility. The conflict between religious teachings, sexuality, and infertility related interventions is of increasing importance in traditional societies today. In other words, the clash between religious tradition, sexuality, and infertility can be a controversial scientific issue in clinical settings. This article explores these issues and draws insights from Iranian’s religiosity, where this challenging triangle is considerable in the context of competing sexuality and infertility related help-seeking behaviours. It raises far-reaching issues concerning the distinction between belief and practice, as well as the role of religious teaching in the sexual and reproductive sphere.
In this article, we have attempted to find a way forward which not only recognizes cultural limitations in terms of the religiosity and sexuality dichotomy but also its strengths in terms of the need for acknowledgement and civility in the assisted reproduction technology (ART) sphere. The authors also advocate that Muslim countries with advanced ART should adopt a fact-specific approach that is sensitive to the sexuality issues in the contexts with specific religious interpretation (Tafsir), and which focuses upon the values of religiosity, tolerance and mutual respect to one’s sexuality.
In sum, we argue that ART policies in Iran may lend themselves to a model of accommodation and compromise which avoids stubbornness and instead seeks out common ground in “religious teachings, sexuality educations, and infertility management. Although the task may be challenging, the consequences of failure, to our mind, justify the effort.
 
K-36
Potential regenerative effects of amniotic-fluid derived exosomes on the rat model of azoospermia
 
Mobarak H¹, Heidarpour M¹, Rahbarghazi R^{2, 3}, Nouri M^{2, 4}, Mahdipour M^{2, 4}.

1. Department of Clinical Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran.
2. Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
3. Department of Applied Cell Sciences, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
4. Department of Reproductive Biology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.

Email: mahdi.mahdipour@gmail.com
 
Any defect during the spermatogenesis process may cause temporary or permanent male infertility. Cell-free therapies and by-products such as exosomes have been used as alternative modalities for the treatment of tissue injuries. There is no data on the use of extracellular vesicles to restore male fertility. This study aimed to explore the therapeutic effects of amniotic fluid-derived extracellular vesicles including exosomes (AF-Exos) on the recovery of sperm production capacity in a rat model of azoospermia. Exosomes were isolated from amniotic fluid samples via ultracentrifugation and characterized by scanning and transmission electron microscopy (SEM and TEM), dynamic light scattering (DLS), and western blotting techniques. The induction of non-obstructive azoospermia (NOA) in rats was performed by intratesticular administration of 5 mg/kg/testes Busulfan. Azoospermia was confirmed with histological and spermiogram analysis. AF-Exos samples (10 and 40 µg exosomal protein) were injected into the testes of NOA rats. Two months after intervention, the spermatogenesis rejuvenation was evaluated via histopathology (H & E staining), spermiogram, and hormonal analysis. The expression level of a regeneration marker (OCT-3/4) was also studied via immunohistochemistry staining and the number of spermatogonial progenitors was as well evaluated. AF-derived Exos showed sphere-shaped morphology with 50 ± 7.521 nm mean diameter and -7.16 mV zeta potential, and are positive in specific surface markers (CD63, CD9, and CD81). Histopathological and spermiogram data revealed that the spermatogenesis index and sperm parameters were significantly improved after AF-Exos injection compared to azoospermic groups. Also, after AF-Exos injection the OCT-3/4+ cells were increased in NOA rats exhibited spermatogenesis restoration. Both doses of exosome (10 and 40 µg) restored the testicular function in NOA rats. Except in a high dose of AF-Exos (40 µg) for testosterone and FSH, no statistically significant differences were found regarding hormonal levels post-exosome injection. Our study demonstrated that AF-Exos have the potential capacity to facilitate regeneration in the spermatogenesis process and improve sperm quality through paracrine effects via releasing potential restoratives factors into the site of injury. This study provides a novel therapeutic insight on the NOA treatment.
 
K-37
Cycle regimens for frozen-thawed embryo transfer
 
Moini A^{1, 2}.

1. Department of Endocrinology and Female Infertility, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
2. Department of Obstetrics and Gynaecology, Tehran University of Medical Sciences, Tehran, Iran.

Email: a_moini@royaninstitute.org
 
Frozen–thawed embryo transfer (FET) enables the excess embryos generated by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to be stored and utilized at a later date. This reduces wastage after IVF and increases the chance of conceiving after one cycle of ovarian stimulation and oocyte retrieval. In recent years, the number of FET cycles performed has increased dramatically due to the trend towards transferring fewer embryos after a fresh IVF cycle, and as a result of improved laboratory techniques. In contrast to the complex stimulation protocols employed to stimulate multiple follicular growth for IVF, frozen embryo transfer (FET) protocols are simpler, with the primary aim limited to adequate preparation of the endometrium to receive the thawed, transferred embryo(s). However, despite the growing importance of FET in the treatment of subfertility, there is little consensus on the best method for endometrium preparation in ovulatory women. In order to optimize pregnancy rates, the development of embryos and endometrium should be synchronized. This can be achieved in various ways. The simplest method of endometrium preparation is represented by natural cycle FET (NC-FET), in which the endocrine preparation of the endometrium is achieved by endogenous sex steroid production from a developing follicle. Timing of embryo transfer is determined by detecting the spontaneous luteinizing hormone surge or by administering human chorionic gonadotropin  to initiate luteinization. A frequently employed alternative approach is represented by ‘artificial cycle protocols’ in which exogenous estrogens and progesterones are administered, with or without co-treatment with gonadotropin-releasing hormone agonists. In artificial cycle FET (AC-FET), estrogen and progesterone are administered in a sequential regimen that aims to mimick the endocrine exposure of the endometrium in the normal cycle. Initially, estradiol is given in order to cause proliferation of the endometrium, while suppressing the development of the dominant follicle. This is continued until the endometrium is observed to be 7-9 mm thick on ultrasound, at which time progesterone is added to initiate secretory changes. The physiological mid-cycle shift from estrogen to progesterone is thus emulated. The timing of embryo thawing and transfer is planned according to the moment of progesterone supplementation.
In conclusion, natural cycle treatment has a higher chance of live birth and lower risks of PIH, PPH and VPTB than AC for endometrial preparation in women receiving FET cycles. Ovarian stimulation with Gn/FSH or AI may be promising, but the evidence is scare and needs to be evaluated in future studiespregnancies after NCFET have a more favourable outcome compared with AC-FET, with lower rates of HDP, preeclampsia, LGA and macrosomia. The development of gestational hypertension in FET cycles seems not to be influenced by the mode of endometrial preparation. This is valuable information, as the number of FET cycles has increased, including the ‘freeze-all’ strategy. Future studies are required to clarify the underlying biologic mechanisms of our findings, and further randomized controlled trials are needed to improve the quality of evidence.
K-38
Ex vivo spermatogenesis: Static or dynamic culture system
 
Movahedin M.
Anatomical Sciences Depatment, Medical Sciences Faculty, Tarbiat Modares University, Tehran, Iran.
Email: movahed.m@modares.ac.ir
 
Infertility is one of the most important problems in human societies, today. This issue can change the social life of infertile couples and has nothing to do with the cause of infertility. However, it should be noted that about 50% of infertility cases are related to men. In vitro germ cell maturation and enrichment transfer techniques could potentially help to preserve fertility, especially in pubertal males without mature germ cells. In addition, this technique could also be potentially used for the treatment and the maintenance of biological paternity of oligozoospermic or azoospermic patients. Today, with advances in reproductive biotechnology, it is possible to produce in vitro male haploid cells. This matter can help a large group of infertile patients. To achieve this goal, many researchers have studied different culture systems and other factors involved in stimulating ex vivo spermatogenesis. Various methods have been proposed, including organ culture system, two/three-dimensional culture and isolated cell culture method or adding the required supplements of tissue or cell in the culture medium. In order to bring the culture system closer to the in vivo conditions with the aim of spermatogenesis, major changes are necessary. One of these changes is the use of dynamite culture instead of static culture. Recently, bioreactor, in which biological or biochemical processes are developed under a closely monitored and tightly controlled environment, is one of the latest approaches that often used to culture cell and tissue in-vitro. It is suggested that the organotypic culture of testicular tissue or fragments is capable of maintaining the architecture and viability of germ cells, and induction of in-vitro spermatogenesis. Moreover, the addition of a mini-bioreactor or microfluidic device has shown the potential to improve organotypic culture systems, as it can lead to long-term ex vivo maintenance of testis tissues which is required for producing sperm. Although these techniques have only been applied in lab animals, there is reproductive technology advancement hope for the near future that these methods will also give surprising results in humans.
K-39
Sperm selection using magnetic activated cell sorting (MACS) in assisted reproduction
 
Nabi A.
Andrology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: ali.nabi67@yahoo.com
 
Magnetic separation has been successfully applied to many aspects of both biomedical and biological research and also in clinical areas like cellular therapies for human autoimmune disease, like rheumatoid arthritis, diabetes, multiple sclerosis, and SLE.  Infertile men with poor sperm motility and morphology were found to have increased sperm DNA fragmentation compared with individuals with normal semen analysis may also have a high degree of sperm DNA fragmentation, which can be a major cause of unexplained infertility, and sperm DNA fragmentation. Aberrant chromatin packaging during spermatogenesis, defective apoptosis before ejaculation, or excessive production of reactive oxygen species (ROS) in the ejaculate. Exposures to environmental or industrial toxins, genetics, and lifestyle are also known factors that may cause sperm DNA fragmentation and infertility. Although the factors present in the paternal genome that may have an impact on poor reproductive outcome are still not wall defined, there is accumulating evidence linking sperm nuclear DNA abnormalities to poor reproductive we come one of the most suspected organelles in the sperm nucleus. Several studies using the magnetic activated cell sorting (MACS) technique with human spermatozoa have been published over the years. Interests in these studies were mainly the molecular efficiency of the technique and improving the post preparation sperm quality. Researchers have evaluated the percentage of sperm recovery following the use of MACS as a sperm preparation technique, and they concluded that the integration of MACs with density gradient centrifugation (DGC) is an effective sperm preparation technique that does not lead to significant cell loss and separating a distinctive population of non-apoptotic spermatozoa with intact Membranes might optimize the outcome of assisted reproduction. Reduction of apoptotic spermatozoa within the ejaculate using the MACs system results in a distinc reduction of spermatozoa with DNA fragmentation, enrichment of spermatozoa free of apoptosis, improvement of sperm viability, motility, and mitochondrial membrane integrity.
 
K-40
Sperm selection using PICSI method
 
Narimani N.
Hasheminejad Kidney Center, Iran University of Medical Sciences, Tehran, Iran.
Email: Nima_dr2001@yahoo.com
It has estimated that up to 50% of the infertility cases are predominantly or partly caused by male factors. About 10-20% of couples suffer from unexplained infertility, the male partner has sperm DNA fragmentation index (DFI) above 20%. It has been shown that increased sperm DFI may lead to decrease chance of natural pregnancy and assisted reproductive techniques success rate. In this patients, the potential underlying causes should be treated first. However, in non-responders, other strategies such as antioxidant medical treatment, sperm selection techniques and testicular sperm extraction may be useful. In this regard we want to talk about physiologic intracytoplasmic sperm injection as a probable useful sperm selection technique.
 
K-41
Regenerative medicine for female reproductive system
 
Nikukar H^{1, 2}.

1. Medical Nanotechnology and Tissue Engineering Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Department of Advanced Medical Sciences and Technologies, School of Paramedicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: habibnik@ssu.ac.ir
 
Tissue engineering and regenerative medicine make a bright future for the regeneration, repair, and replacement of various tissues and organs. In the reproductive system, most of the major acquired or congenital organ failures lead to a great functional problem, infertility. Everybody with infertility will have great social and family obstacles, often with psychological consequences for the couples.
Regenerating the absent organ or repairing and replacing the diseased tissue are the novel choices for the treatment of reproductive system diseases due to organ or tissue failure. By tissue engineering for female patients, that is using the triad of potent cells, scaffolds and growth factors could make an artificial uterus, tubal organs, ovary, and follicles. Selection of the best cells, scaffolds, and stimulation factors to make a functional tissue is the aim of many research programs around the world. Various types of stem cells, organic and inorganic biocompatible scaffolds, and different types of proteins, enzymes, and small molecules as stimulators have been used. Engineered tissues could apply as the in vitro research models and for clinical use to restore reproductive function.
Taken together, this medical technology prepares the introductory facilities for germ cell support and in vitro fetal growth and complete artificial uterus for ex vivo embryo growth and maturation (biobag). For all the possible instances, religious beliefs, law and reproductive health ethics should be considered.
K-42
Using embryo culture medium as a diagnostic factor
 
Omidi M.
Gandhi IVF Clinic, Tehran, Iran.
Email: omidi.marjan@ymail.com
 
The success rate of assisted reproduction is remained low despite performing several studies around the world. Different strategies have been used for the improvement of assisted reproduction technology (ART) outcomes. More attention has been paid for omics technology in recent decades. Metabolomics is a non-invasive technique to evaluate oocyte quality, and competence, embryo viability, and endometrial receptivity. In fact, metabolomics provides sufficient data about the oocyte, embryo, and endometrium for the treatment of patients with subfertility. Also, this method, by selecting the best embryo for transfer, can reduce the number of transferred embryos, and the risk of multiple pregnancies as well. Evaluation of oocytes based on metabolomics can replace other methods of selection with the high variability like morphology or invasive method like polar body biopsy. Amino acids turnover can predict embryo viability with high rate of implantation resulting to a live birth. Metabolomics evaluation of endometrium is also associated with the receptivity of endometrium and also for diagnosis of endometriosis. High-quality researches are needed for drawing the final conclusion about the efficacy of metabolomics on ART outcomes including live birth and miscarriage rates.
 
K-43
Sperm selection using Microfluidic method
 
Pacey A.
Department of Oncology and Metabolism, The Medical School, University of Sheffield, The Jessop Wing, Sheffield, UK.
Email: a.pacey@sheffield.ac.uk
 
Abstract not received.
 
K-44
Uterine myoma and infertility
 
Parsanezhad ME.
Infertility and Reproductive Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
Email: parsameb@gmail.com
 
Uterine myoma or fibroid is the most common benign gynecological tumors in women of reproductive age. Fibroids are hormone-dependent smooth-muscle tumors with a wide heterogeneity in composition, size, and number. Most women with fibroids are fertile; however, fibroids may affect fertility by distorting the pelvic anatomy and the intrauterine environment. The way by which myoma result in infertility remains to clearly understood. Besides anatomical distortion, the possible mechanism impairing fertility are; endometrial function alteration (increased uterine contractility and impairment of the endometrial and myometrial vascularization and blood supply, alters the local hormone balance that could affect gamete transport and/or reduce embryo implantation. Submucosal and intramural myomas with pressure effect on uterine cavity are associated with decreased pregnancy and implantation rates after ART cycles. The management method highly depends on the size, number, and location.
 
K-45
Organoids: A paradigm-shifting stem cell-based technology for drug discovery and development, companion diagnostics, and preclinical patient stratification
 
Pourfarzad F.
Hubrecht Organoid Technologty, Netherland.
Email: f.pourfarzad@huborganoids.nl
 
Hubrecht organoid technology (HUB) has developed the 3D culture system to establish and expand human and animal epithelial tissue from a variety of organs, both healthy and diseased, such as cancer. The organoid technology is based on the work of Hans Clevers that identified adult stem cells in many human tissues, including intestine, liver, pancreas, breast, and lung. Organoid cultures have the virtually unlimited expansion, genetically and phenotypically stable and retain biological and functional properties of the original tissue (Barker et al., Nature 2007; Sato et al., Nature 2009, 2011; Gastroenterology 2011; Huch et al., Nature 2013; Karthaus et al., Cell 2014; Cell 2015; Boj et al., Cell 2015). Organoids recapitulate the original tissue response to external stimuli and provide a unique and robust in vitro model for drug development, diagnostics, and patient stratification.
The HUB is collaborating with and licensing the technology to the Pharmaceutical and Biotech industry. In addition, HUB has built a comprehensive Living Biobank of well-characterized Organoids from different healthy, disease, and cancerous tissues of multiple organs. In combination with the Living Biobank and Organoid technology, HUB offers a unique platform to develop assays and provide preclinical drug discovery, toxicity, personalized medicine services.
 
K-46
Treatment of male infertility
 
Rahavian AH.
Andrology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: amirrahavian@yahoo.com
 
Some drugs and medical illnesses suppress the fertility capacity of men, so the first step of medical treatment of male infertility is identifying these drugs and withdraw them and diagnosing the diseases like hypogonadotropic hypogonadism, hyperprolactinemia, and genital tract infections and manage them.
One of the male infertility causes is excessive reactive oxygen species (ROS) production. Physiologic amount of ROS is necessary for the reproductive system, but the excessive amount of it causes cellular damage. Different sources of ROS production are smoking, varicocele, heat, etc. and eliminating these sources is necessary.
The other option for decreasing ROS in the seminal fluid is antioxidant drugs. Each of these drugs has the different effects on semen parameters and it is proven that multiple antioxidant therapy is superior to solo antioxidant therapy.
For antioxidant therapy, we should know the minimum and maximum dosage of each drug because over and under the administration of antioxidants may have the deleterious effects on semen parameters.
Not all infertile men are a good candidate for antioxidant therapy and also we cannot administered all antioxidant drugs for every patient so for precise selection of patients and antioxidant drugs, it is highly recommended to evaluate oxidative stress of semen and start antioxidant therapy based on it.
 
K-47
Anti-müllerian hormone and polycystic ovary syndrome
 
Ramezani Tehrani F.
Reproductive Endocrinology Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Email: ramezani@endocrine.ac.ir; framezan@ post.harvard.edu
 
Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive women. The prevalence of PCOS varied based on the recruitment process of the study population, the criteria used for its definition and the method used to define each criterion. Rotterdam criteria introduced in 2004 and included polycystic ovarian morphology (PCOM) as a criterion for PCOS. This definition made a lot of concern in terms of validity and reliability of its assessment and the necessity for revising its definition given using advanced high-resolution ultrasounds devices. Several efforts were made to introduce a more reliable substitute for PCOM, among all candidate options anti-Müllerian hormone (AMH) was the best marker reflecting antral follicular count (AFC) given its exclusive production by granulosa cells of the ovary. Attempt was also made to identify the optimal diagnostic threshold for AMH for substitution of PCOM in PCOS criteria, however, a universally approved threshold has not yet been introduced. Moreover, there are studies that suggested AMH as a surrogate marker for diagnosis of PCOS due to overproduction of AMH by granulosa cells in anovulatory status, cross-communication of AMH with FSH and LH which leads to hyperandrogenism. It seems that the AMH cut-off value of ~5.7 and ~3.7 ng/ml in the early and late reproductive period has adequate sensitivity and specificity for making PCOS diagnosis.
K-48
Somatic and germ line gene therapy
 
Razban V.
Molecular Medicine Department, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran.
Email: razban_vahid@yahoo.com
 
Genetic and molecular approaches are applied to diagnose or predict a disorder then the offers are dealing with the disease or termination of pregnancy. This is called the diagnostic therapeutic gap. Gene therapy, as an old dream, aims to fill this gap. It involves the introduction of genetic material into the cells to cure or prevent the inherited and non-inherited diseases and expected to be curative and durable. By developing efficient gene delivery and gene editing systems, now gene therapy is applicable. To meet the expectations, there are two alternative strategies: somatic and germ line gene therapy with their advantages and challenges.
K-49
Can in vitro culture condition influence pre-implantation embryo aneuploidy?
 
Sadeghi MR.
Reproductive Biotechnology Research Center, Avicenna Research Institute (ARI), ACERCR, Tehran, Iran.
Email: E-mail: Sadeghi@Avicenna.ac.ir
 
Natural fecundity has a low rate in humans compared to other mammals, so that the probability of pregnancy for a healthy fertile couple in each menstrual cycle is less than 25%. On the other hand, according to the available evidence, about 10-40% of pre-implantation embryos are lost after fertilization in normal healthy women. Aneuploidy is the most important cause of embryo implantation failure. Aneuploidy increases as women are aging. Some studies have reported the rate of 20 to 40 % of chromosomal abnormalities in embryos among healthy fertile women following natural conception. However, the rate of aneuploidy of in vitro fertilized embryos is much more than embryos from natural conception, and this rate increases with maternal age from 73% in women under 35 to 87% in women aged 41 or older.
The causes of aneuploidy in human embryos remain largely unknown. In addition, the level of aneuploidy in the gametes which produce these embryos is lower than the level of aneuploidy in the resulting embryos, so that approximately 20% of the retrieved oocytes in IVF cycles and about 9% of the sperm in each ejaculate have aneuploidy on their haploid chromosomes. Interestingly, about 90% of aneuploidy of human embryos originates from oocytes. In particular, the type of chromosomal abnormalities in embryos and oocytes is quite similar, mainly aneuploidy, while most chromosomal abnormalities in sperm are structural.
Furthermore, the rate of aneuploidy in embryos that underwent intracytoplasmic sperm injection (ICSI) procedure is higher compared to embryos obtained from IVF. Therefore, it seems that in vitro oocyte manipulation for ICSI has adverse effects on oocytes and leads to aneuploidy in embryos. Two hypotheses can be considered for the relatively high rate of embryo aneuploidy in IVF/ICSI cycles; controlled ovarian hyperstimulation (COH) is the first exogenous factor and the second is IVF laboratory conditions. For COH, various factors such as the type and dose of drugs, duration of ovarian stimulation, and even the aspiration from ovarian follicles can affect the integrity of the oocyte chromosomes.
However, multiple environmental variables in embryology laboratories can affect the chromosomal integrity of oocytes and embryos. These variables include the type of culture medium, culture conditions, pH, temperature, osmolality and oxygen tension, contaminants and volatile compounds, gamete manipulation, and gamete aging in terms of immaturity or post-maturity of the oocytes. Serious variations in the IVF laboratory environment may lead to mitotic spindle alterations, centrosome amplification, cell-cycle checkpoint defects, non-separation of chromatids, and telomere stability, causing defects in chromosome segregation and aneuploidy.
Studies conducted in IVF centers show as much as 40% variations in aneuploidy rates between different IVF centers; donated oocytes of young women and even the oocytes of the same donors were used in these centers by implementing a similar screening method for chromosomal abnormalities.
This represents the importance of controlling the environment of the embryology laboratory using quality assurance and quality control policy to minimize the rate of embryo aneuploidy and subsequently increase the success rate of IVF. Therefore, the aneuploidy rate of preimplantation embryos can be a key performance indicator to measure the effectiveness of an IVF laboratory and IVF center.
 
K-50
Sperm DNA fragmentation, contributing factors, and clinical setting
 
Salehi P.
Department of Infertility, Isfahan Milad Hospital, Isfahan, Iran.
Email: Dr_p_salehi@yahoo.com
 
Sperm DNA fragmentation index (DFI) along with semen parameters evaluation, is considered an important diagnostic method in assessing male fertility potential. Researches Evidence support the association between DFI with male infertility, natural conception, and assisted reproductive technique outcomes. Various factors contributed to DNA fragmentation generation in men, such as testicular dysfunction, diseases correlated with testicular such as varicocele, exposure to high-risk chemicals components, hyperthermia, and poor lifestyle and nutrition. There are various laboratory methods for assessing DFI clinically. Patients with varicocele, unexplained infertility, recurrent pregnancy loss, recurrent failure of assisted reproductive techniques, and those at risk of lifestyle/environmental exposures are recognized candidates for DFI evaluation. Although no comprehensive treatment has yet been developed to overcome sperm DNA fragmentation, physicians offer different treatments to reduce the DNA fragmentation and improve sperm DNA and chromatin integrity. It seems in the clinical setting, oral antioxidants therapy, varicocele repair, use of recurrent ejaculations alone or combined with micromanipulation-based sperm selection techniques, and the use of testicular sperm for intracytoplasmic sperm injection are suitable options for improving the quality of DNA.
 
K-51
Long life endometriosis
 
Salehpour S.
Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Email: saghar.salehpour2014@gmail.com
 
Endometriosis is defined as the presence of endometrial like tissue outside the uterus which induces a chronic, inflammatory reaction. The condition is predominantly found in reproductive age, from all ethnic and social groups. The associated symptoms can impact on general physical, mental, and social well-being in all over the life of the patient. Therefore it is crucial to give a careful note to women's complaint and history and make a precise decision. Treatment must be individualized taking the impact of the disease and the effects of treatment into account. For a pain management before any decision for surgery, a multidisciplinary clinic including pain manager, psychologist, physical medicine, nutritionist, and gynecologist must be involved. And then the first line of treatment of pain should be medical. For infertility, the first line of treatment should be IUI or IVF. Although there is some evidence that the success of infertility treatment and ART is lower in the presence of endometriosis, but the side effects of early surgery and decrease ovarian reserve and recurrence rate of 20-50% after surgery should be included into account.
 
K-52
Fetal lung cells for cell therapy of lung injury in an animal model
 
Samadi Kuchaksaraei A, Ganji F.
Department of Medical Biotechnology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran.
Email: ali.samadi@iums.ac.ir; samadikuchaksaraei@yahoo.com
 
The regeneration-inducing capacity of the cells derived from human fetal lung has not been systematically studied for cell therapy of lung injuries. We hypothesize that due to the commitment of these cells to the the respiratory system, they have a high potential to promote regeneration in respiratory system. In this study, lower respiratory tissues were isolated from 12-19 weeks human fetuses. The cells were characterized by their morphological and gene expression profiles and their ability to form organoids. The cells were then intra-tracheally delivered to rats with pulmonary injury induced by bleomycin at day 0 and 14 after induction of injury. Rats were sacrificed on day 28 after injury and their lungs were evaluated histologically. We have shown that cell therapy reduced fibrosis and collagen deposition and promoted the regeneration of alveoli. Also, cell therapy increased the expression of surfactant protein C and IL-10 and decreased the expression of aquaporin 5 and transforming growth factor beta. Here, we show that fetal human lower respiratory tract cells can significantly increase the process of lung regeneration. This finding not only introduces a potential cell source in this area but also suggests a potential phenotypic target for the derivation of regenerative cells from multipotent or pluripotent stem cells.
 
K-53
AZF_C delitions Y chromosome and male infertility
 
Sadighi Gilani MA.
Department of Urology, Tehran University of Medical Sciences, Tehran, Iran.
Email: masadighi@gmail.com
 
Male infertility is a common condition with heterogeneous causes. Genetic or epigenetic variations or both contribute up to 15%–30% of cases of male infertility. Genes control a variety of physiologic processes, such as spermatogenesis which occurs in a sequential manner with mitotic, meiotic, and postmeiotic differentiation phases and secretion of hormones. The genetic abnormalities involved in male infertility may be chromosomal (numerical and structural aberrations) or monogenic disorders, mitochondrial DNA (mtDNA) mutations, microdeletion of the Y chromosome, multifactorial disorders, imprinting disorders, or endocrine disorders of genetic origin. The Y chromosome is one of the smallest human chromosomes and microdeletions on the long arm of this chromosome (Yq) is one of the most significant causes of male infertility. Y chromosome microdeletions were present in about 5.2% -12.1% of Iranian infertile men with azoospermia and severe oligozoospermia. The azoospermia factor (AZF) region is further subdivided into 3 non-overlapping regions termed as AZFa, AZFb, and AZFc. Deletion of AZFa and A2Fb which are less common are associated with non-treatable azoospermia. Deletions of the AZFc region are most common in men with idiopathic oligozoospermia or azoospermia. Cases with AZFc deletions show a progressive deterioration in spermatogenesis and cases develop azoospermia over a period of time. The clinical outcomes of intracytoplasmic sperm injection (ICSI) for oligozoospermic patients with Y chromosome AZF microdeletion are comparable to those of infertile patients with normal Y chromosomes. For azoospermic men with AZFc deletion microdissection, TESE is recommended with a success rate of sperm retrieval about of 36.3%. In conclusion, the pregnancy and delivery in oligozoospermic patients with AZFc deletion are comparable with other studies, but despite of sperm retrieval in azoospermic patients with AZFc deletion, the chance of pregnancy or delivery in these patients is very low. More attention to surgical points for sperm retrieval and more extensive search for sperm finding and refinement of sperm freezing and ICSI procedure is needed for better results.
 
K-54
An introduction to tissue engineering: How to select the best scaffold
 
Sefat F.

1. Dpartment of Biomedical and Electronics Engineering, School of Engineering, University of Bradford, Bradford, UK.
2. Interdisciplinary Research Centre in Polymer Science and Technology (IRC Polymer), University of Bradford, Bradford, UK.

Email: F.Sefat1@bradford.ac.uk
 
Tissue engineering and regenerative medicine are fast developing approaches in the production of new organs and body tissues. On the other hand, it is a field that seeks to replace/repair or enhance the biological function of a tissue or an organ by manipulating cells via their extra-cellular environment. The concept of directly engineering tissue was articulated in detail in 1985 by Fung co-workers and the term “tissue engineering” was first used during a meeting sponsored by the National Science Foundation in 1987. Even though everyone believes that the field of tissue engineering may be relatively new, the idea of replacing tissue with another goes as far back as the 16^th century. Over the past few decades, there has been a wide range of researches that have been conducted on the provision of tissue-engineered and regenerative medicine which lead to a significant improvement in the production of scaffolds with similar characteristics to a natural tissue/organ. These scaffolds needed due to either trauma/injury, genetic disorders and diseases where can lead to damage and degeneration of tissues in the human body, which necessitates treatments to facilitate their repair, replacement or regeneration. The aim of this talk is to talk about basic principles of tissue engineering and regenerative medicine and in particular show the path for selecting the best biomaterials known as scaffolds to complete the treatment of damaged/diseased tissue or organs.
 
K-55
Practical use of tissue engineering in treatment of ovarian cancer and vaginal infections
 
Sefat F.

1. Dpartment of Biomedical and Electronics Engineering, School of Engineering, University of Bradford, Bradford, UK.
2. Interdisciplinary Research Centre in Polymer Science and Technology (IRC Polymer), University of Bradford, Bradford, UK.

Email: F.Sefat1@bradford.ac.uk
 
The female vaginal tract has its own innate defence system that involves the natural microbiota, the vaginal epithelium and proteins that help manage a healthy microbiome. In this project, we are concerned with what happens when things go wrong with this balance. Bacterial vaginosis (BV) is a common vaginal infection with 1 in 3 pre-menopausal women in the UK suffering from BV at some point in their life. Intravaginal treatment can be impeded during menstruation and BV can often recur within a few weeks if not treated effectively. Management of the vaginal biofilm is an emerging sector in women’s health. Globally, 20-30% of women of reproductive age suffer from BV, as yet only partially understood but associated with an increased chance of preterm abortion, pelvic inflammatory disease and sexually transmitted diseases. The symptoms of discharge and offensive smell can cause considerable distress. The aim of this project is to offer women a temporary scaffold encapsulated with a drug that can manage the biofilm on the epithelial layer of the vagina.
 
K-56
Genetic aspects of ovarian reserve
 
Shelling A.
Department of Obstetrics and Gynaecology, University of Auckland, New Zealand.
Email: a.shelling@auckland.ac.nz
 
Ovarian aging exhibits no obvious signs therefore women who delay pregnancy later in life may be faced with unexpected fertility issues. The ability to accurately predict a woman’s reproductive lifespan is becoming of increasing importance. Ovarian reserve tests are typically a measure of antral follicles and roughly correlate with the number of primordial follicles remaining in the ovary. The rate of decline of the ovarian reserve is variable between women, and current ovarian reserve tests have limitations. Current techniques, such as the anti-mullerian hormone (AMH) test, lack long-term accuracy and predictability. The identification of genetic variants associated with an increased risk of accelerated ovarian aging may allow for a more accurate and predictive screening tool. To present the current literature on genetic prediction of ovarian reserve and determine whether genetics markers of the ovarian reserve may complement the best current predictors of ovarian reserve. The advantage of using genetic markers of ovarian reserve is that they are present throughout life, and analysis only needs to be done once. Our data suggest that common variants influencing age at menopause may also modify the risk of accelerated ovarian ageing. Our results extend findings from recent genome-wide association studies and may guide future research efforts in identifying further genetic biomarkers influencing ovarian reserve.
K-57
Exosome therapy in treatment of infertility
 
Soleimani M^{1, 2}.

1. Tissue Engineering and Hematology Department, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
2. Shahid Beheshti University of Medical Sciences, School of Advanced Technologies in Medicine, Tissue Engineering and Nanomedicine Research Center, Tehran, Iran.

Email: soleim_m@modares.ac.ir, soleimani.masoud@gmail.com
 
Abstract not received.
 
K-58
Etiologies of male oxidative stress
 
Talebi AR¹, Talebi AH².

1. Andrology Research Center, Yazd Reproductive Science Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Department of Language and Literature, Yazd University, Yazd, Iran.

Email: proftalebi@gmail.com
 
Reactive oxygen species (ROS) are formed during normal cellular metabolism. In the male reproductive system, they are involved in many physiological processes, including capacitation, hyper-activation, acrosome reaction and sperm-oocyte fusion. The generation of the ROS occurs via three methods in spermatozoa: (1) in the cell membranes, using nicotinamide adenine dinucleotide phosphateoxidases, and (2) in the mitochondria, using nicotinamide adenine dinucleotide phosphateoxido-reductase and finally, (3) immature spermatozoa with residual cytoplasm contains high levels of glucose-6-phosphate dehydrogenase, a cytosolic enzyme that utilizes the hexose monophosphate shunt to produce abnormally high levels of NADPH. Excessive NADPH results in a greater production of superoxide anions by NADPH oxidases. Activated leukocytes (peroxidase positive) also produce large quantities of ROS and it has been shown that leukocytes are the predominant source of ROS in raw human semen. When ROS increases beyond the physiological levels, overwhelming opposing antioxidant forces, oxidative stress (OS) results. When this occurs, ROS can lead to lipid peroxidation, DNA damage, and OS-induced apoptosis and autophagy, which can be harmful to the highly susceptible sperm cells.
In addition to several diseases like diabetes and varicocele, there are many extrinsic factors affecting human spermatozoa by elevating ROS level. Smoking is one of the most clinically relevant and preventable causes of OS. Alcohol consumption is considered as another etiological factor for ROS production.  Malnutrition and poor diet can also induce ROS production. On the other hand, obesity and over-nutrition also play a significant role in inducing OS in the reproductive system. Stress is linked to increasing in ROS in the seminal plasma and impaired sperm quality. There are many proposed mechanistic effects of medicines on ROS and its related OS. Bactericidal antibiotics can overproduce ROS and lead to mitochondrial dysfunction in mammalian cells including spermatozoa. Environmental pollutants such as nitric oxide, sulfur dioxide, carbon tetrachloride, ozone, wood dust, particulate matter, volatile organic compounds, bisphenol A, xenoestrogens, and phthalates can potentially induce OS and decrease sperm quality. Additionally, studies have shown that pesticides such as lindane, methoxychlorate and dioxin-TCDD have been related to testicular OS in animals and humans. Finally, the radiation and electromagnetic fields such as cellular phones also can cause an elevation in ROS production and decrease sperm fertility potential. 
 
K-59
Reproductive tissue engineering by marine scaffolds: Decellularization and 3D bio-printing
 
Tamadon A.
The Persian Gulf Marine Biotechnology Research Center, The Persian Gulf Biomedical Sciences Research Institute, Bushehr University of Medical Sciences, Bushehr, Iran.
Email: amintamaddon@yahoo.com
 
The body structure of marine invertebrates is made up of a variety of scaffolds of proteins, glycoproteins, carbohydrates, and bio-glass, all of which can be used to engineer human tissues, either intact or manipulated. The combination of existing scientific evidence shows that marine invertebrate scaffolds can create new capabilities in the pharmaceutical and medical industries. Production and recombination of human cell-compatible scaffolds from marine invertebrates or the removal of cells in invertebrates and the use of the natural structure of marine scaffolds are applications of marine tissue engineering to produce soft reproductive tissues. As a result, the biodiversity of marine invertebrates of the Persian Gulf with simple body scaffolds for the extraction of bio-ink compounds for 3D bio-printers and the decellularization of their tissues has provided vast research potential. In this direction, the Marine Comparative and Experimental Medical Center in the Persian Gulf Marine Biotechnology Research Center has completed various projects in the construction of marine scaffolds. Three invertebrate species of the Persian Gulf have been studied for this purpose, which are jellyfish, brown algae, and sponge. In the 3D bio-printer made by the researchers of this center, alginate compounds extracted from brown algae were used for flexible scaffold printing. Furthermore, jellyfish tissue and sponge after decellularization were used to culture human fibroblasts.
 
K-60
Challenge of cell therapy in endometriosis
 
Tanideh N^{1, 2}, Iraji A^{1, 3}.

1. Stem Cells Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
2. Department of Pharmacology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
3. Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.

Email: tanidehn@gmail.com
 
Endometriosis is a common medical problem, occurring in 10–15% of reproductive‐age women and 20 to 50% of infertile women. Endometriosis is an inflammatory, mostly estrogen-dependent condition that happened due to the development of endometrial tissue outside of the uterus. Manifestations include pelvic pain, dysmenorrhea, infertility, and in some cases ovarian cancer. Despite the high prevalence of endometriosis, the pathogenesis of this disease remains poorly understood. However, the most accepted theories are angiogenesis, cellular invasion, adhesion formation, fibrosis, neuronal infiltration, and abnormal cell growth. Also, some recent studies suggest that endometrial stem/progenitor cells function in the development of endometriosis. In other words, potential stem cells can form endometriotic implants. However, regarding the fact that endometrium becoming fibrosis during the progress of endometriosis, stem cell therapy offers the potential treatment of tissue injury and fibrosis. It is accepted that stem cell has the capacity of self-renewal and differentiation into other cells simultaneously confirming its important role in maintaining the homeostasis, repair, and renewal of endometrial fibrosis. Regarding the controversial role of stem cells, more efforts are needed to explore the specific stem cell-based therapy and make its clinical use.
 
K-61
Mimicking nature in wise strategy
 
Vaiarelli A.
GeneraLife Centers for Reproductive Medicine, Rome, Italy.
Email: alberto.vaiarelli@gmail.com
 
Controlled ovarian stimulation (COS) remains a challenging clinical step in assisted reproductive technique, especially in some specific patients group in which no evidence-based guidelines are available. Up to now, the clinical approach to the infertile women needs several decisions that reside in the clinician's hands. For this reason, COS is one of the cornerstones of in vitro fertilization (IVF). Today the measure of success in IVF must be the cumulative live birth rate per started cycle. Its purpose is to obtain an adequate response in terms of oocytes’ number and quality to improve treatments’ efficacy and efficiency by obtaining several competent embryos. The ability to predict the ovary response is the priority to obtain the right number of oocytes and to define the right individual treatment for the right patients. Many factors can be used as predictors of ovarian response such as: age, biochemical parameters, follicle-stimulating hormone (FSH), anti-müllerian hormone, and morphological parameters (antral follicular count) but also some clinical conditions like polycystic ovary syndrome and low body mass index.
Although some data suggested that recombinant-FSH and human menopausal gonadotropins (hMG) for COS in long agonist protocols perform similarly, the evidence is limited in antagonist protocols, i.e. the most commonly used at present. Therefore, the decision on which gonadotrophins should be used for COS is still uncertain, especially in patients at their first COS (naïve) and/or in freeze-all strategies. However according to the evidence already published r-FSH and hMG have a different endocrine profile, the serum levels of FSH, androgens, and estradiol were significantly higher with hMG than r-FSH in conventional COS. Moreover r-FSH significantly increases the number of oocytes retrieved and embryos obtained compared with hMG after COS. The duration of COS was significantly longer and the total amount/dose of gonadotropin was significantly higher with hMG than with r-FSH.
Finally, no difference has been reported in term of ovarian hyperstimulation syndrome risk and ovarian hyperstimulation syndrome profile between hMG and r-FSH Regarding luteinizing hormone (LH) activity during COS, LH supplementation in COS continues to be actively debated and controversial, causing some confusion between practitioners. Current evidence suggests that r-LH supplementation appears to be beneficial in i) hypo-hypo, ii) patients with hyporesponse to FSH monotherapy, iii) advanced maternal age, iv) patients with very low endogenous LH during COS.
Finally, r-FSH + r-LH combination may be effectively used to obtain COS in IVF patients without increasing the overall costs for the patients or the National Health Service in a specific setting.
 
K-62
An introduction to tissue engineering: How to select the best cells
 
Zandieh Doulabi B.
Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Amsterdam, Netherland.
Email: bzandiehdoulabi@acta.nl
 
Stem cells are special cells that are able to develop into many different cell types and or tissues, ranging from bone cells to brain cells. Stem cells are divided into embryonic stem cells and adult stem cells Subtypes. Embryonic stem cells (ESC’s) and induced pluripotent stem cells (iPSC’s) can turn into more than one type of cell from different origins. Adult stem cells can differentiate toward one tissue (such as the liver) or to multiple tissues (such as the bone, brain, and cartilage) from the same origin in the case of mesenchymal stem cells. Given their unique regenerative abilities, stem cells offer new potentials to repair or treat diseases such as bone and cartilage defects, diabetes, and heart disease.
The general principles of tissue-engineering involve combining stem cells, a natural/synthetic scaffold, and required chemical/mechanical factors to build a functionally, structurally and mechanically compatible living tissue.
An ideal tissue engineered construct needs an excellent microenvironment with the optimal cell adhesion, growth, and differentiation capacity in controlled pH, temperature, oxygen tension and be properly adapted to the mechanical force of the microenvironment. Therefore, besides the choice of stem cells, the development of such a construct requires a careful selection of three key components: 1) scaffold, 2) growth factors, 3) extracellular matrix.
For the permanent repair of damaged tissues, the following criteria are essential to consider:

• An adequate number of cells and their ability to differentiate into desired phenotypes,
• Cells must be able to adapt to the three-dimensional structure and produce their own extracellular matrix
• Tissue-engineered cells also must be structurally and mechanically compliant with the native cells and be able to integrate with native cells without the risk of immunological rejection and pose no biological risks.

The source of cells utilized in tissue engineering can be autologous (from the patient), allogenic (from a human donor but not immunologically identical), or xenogeneic (from a different species donor). Each stem cells source has its (dis) advantages in usage. For example, the autologous cells represent an excellent source for use in tissue engineering because of the low association with immune complications but their use is in general not cost-effective for batch controlled clinical use. In contrast, allogenic cells and xenogeneic stem cells offer advantages over autologous cells in terms of uniformity, standardization of procedure, quality control, and cost-effectiveness. Their disadvantage may lie in immunogenic related adverse effects.
In conclusion, the use of an appropriate multipotent or pluripotent stem cell in tissue engineering is an emerging concept. Many technical questions need to be answered and require close collaborations between scientists, clinicians, engineers, and legal and ethical regulating bodies (for example when embryonic stem cells or genome-edited iPSC’s are used) to obtain the goal of functional tissue restoration.

4^th Congress of Reproductive Genetics

K-63
Evaluating ovarian impairments in implantation failure
 
Aflatoonian A.
Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: abbas_aflatoonian@yahoo.com
 
Recurrent implantation failure (RIF) can be defined as a failure to achieve a clinical pregnancy after transfer of at least four embryo of good quality in a minimum of three fresh or frozen cycles in women under the age of 40.RIF is often a complex problem with a wide variety of etiologies and mechanisms as well as treatment option.
Recurrent miscarriage (RM) is defined as two or more consecutive pregnancy before the 20^st week of pregnancy or a fetal weight of less than 500 grams that affect approximately 5% of conceived women worldwide.
RIF and RM are multifactorial disorders with three main causes including gamete and embryo factors, factors affecting endometrial receptivity and ovarian factors affect gamete and embryo factors and include premature ovarian failure (POF) and polycystic ovarian syndrome. Primary ovarian insufficiency (POI) is a heterogeneous disease caused by a variety of mechanisms that affects ∼1-2% of under-forty years old women. The etiology of POI has been found to be genetic mutations, chromosomal, and autoimmune.
About 10% of cases of POI are related to genetic diseases and over 50 genes are known to be causally related to POI. The most frequent conditions associated with POI are Turner syndrome and fragile X pre-mutation; mutation of BRCA 1-2 genes and several other mutations and genetic syndromes that have recently been highlighted, although they rarely occur.
Fragile X mental retardation type 1 gene pre mutation has been frequently found in POF or POI. Tet (Ten-eleven translocation) Tet1 deficiency leads to POF by influencing the quality of oocytes and reduces expression of X-chromosome-linked genes, such as Fragile X mental retardation type 1. ATG7 (autophagy-related genes) and ATG9A variants have also a functional link with POI. If a diagnosis of genetic-based POI is determined before the onset of POI, counseling on currently available fertility preservation techniques is advisable.
Advanced maternal age, high follicle-stimulating hormone, low antral follicle count, and low Anti-Mullerian Hormone result in fewer number of oocytes retrieved, high number of immature oocytes, reduced fertilization and embryo utilization rate. Advanced age also cause aneuploidy, increase mitochondrial damage and decrease in mitochondrial membrane potential. Its impact on zona hardening and subsequent defective hatching as a cause for RIF has been suggested by a few studies. PCOS is the most common cause of anovulatory infertility in the developed countries and also the most commonly identified abnormality among women with recurrent miscarriage. Spontaneous loss of fetus occurs in 40% of women with PCOS and the possible causes may include obesity, hyperinsulinemia, insulin resistance, hyperandrogenemia, hyperhomocysteinemia, high levels of plasminogen activator inhibitor-1 factor, poor endometrial receptivity, and elevated levels of luteinizing hormone. Subacute Cd (cadmium) exposure disrupt the Hypothalamic–pituitary–gonadal axis function, leading to PCOS and POF features and other abnormalities in female rats.Chromosomal abnormalities of the male or female partner (deletions and duplications-dicentric and ring chromosomes-balanced and unbalanced translocation-single gene defect) and Maternal cytoplasmic factors or mutations in cell cycle control gene also can cause RIF and RM.
K-64
Genetics and pharmacogenetics aspects of PCOS
 
Afsharian P.
Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
Email: Parvaneh.Afsharian@gmail.com, p.afsharian@royan-rc.ac.ir
 
The aim of treatment of polycystic ovarian syndrome (PCOS) is restoring the ovulation; due to PCOS is the most common cause of anovulatory infertility and the most prevalent endocrine disorder in reproductive age women (5-20%) with the main symptoms including oligomenorrhea/anovulation, clinical and/or biochemical hyperandrogenism and polycystic ovaries. PCOS is a multifactorial disease including hormonal, genetic, environmental and immunological factors. Alteration in genetic factors can be related to etiology of disease are most related to its symptoms. These genes are estradiol biosynthesis genes (CYP19 or Aromatase); gonadotropin-releasing hormone, follicle-stimulating hormone receptor, luteinizing hormone, anti-müllerian hormone; and transforming growth factor beta gene family.
Commonly applied treatments in PCOS consist of clomiphene citrate, aromatase inhibitors and recombinant follicle-stimulating hormone to make ovulation induction, however outcomes of this treatment are often unpredictable. In some cases, patients are resistance to treatment. Therefore, an era of predicting drug response on the basis of one’s genome is drawing close to reality, entitled pharmacogenomics needs to be studied. We aimed to summarize the way in which genetic variability might modify effects of drug-metabolizing enzymes, transporters and receptors, thereby altering response to drugs used in ovulation induction in PCOS patients. For example CYP2D6 is responsible for CC metabolism and is mostly expressed in liver.
 
K-65
Sperm DNA damage and male infertility
 
Agarwal A.
Center for Reproductive Medicine, Cleveland Clinic, Cleveland, Ohio, 44195, USA.
Email: agarwaa@ccf.org
 
Abstract not received.
 
K-66
Pharmacogenomics in infertility
 
Al-Amri AH.
Molecular Genetics Lab, National Genetics Center, Royal Hospital, Muscat Sultanate of Oman, Oman.
Email: ahmedamri@hotmail.com
 
With 80 million couples worldwide having difficulty to conceive a child naturally, infertility sounds to be a real serious medical issue that requires a great attention. Despite the carried out studies and the great efforts to understand this problem, there is still a gap between our current knowledge/capabilities and the available options for the treatment. One possible reason for such a gap may be related to factors other than advanced maternal age, ovarian reservation, cigarette smoking, and hormonal changes. Pharmacogenetics is a relatively new approach that has been considered to fill in this gap, and utilized to develop personalized therapies. The main aim of pharmacogenetics is to study the genetic variations in order to optimize the success and minimize adverse effects of drugs. Therefore, the approach has been used to study the molecular changes in various biochemical markers involved in reproduction. It is being put forward as an alternative to the “one-size-fits-all” approach that has been ineffective in many conditions.
 
K-67
New genetic finding in PGD
 
Baldi M.
Genoma Group, Molecular Genetics Laboratory, Rome, Italy.
Email: segreteria@marinabaldi.it
 
Abstract not received.
 
K-68
What is important of male infertility environment?
 
Barratt Ch.
Head of Devision System Medicine, University of Dundee, Scotland.
Email: c.barratt@dundee.ac.uk
 
Abstract not received.
 
K-69
Covid 19, Vaccines and infertility
 
Biglari A.
Department of Genetics and Molecular Medicine, School of Medicine, Zanjan University of Medical Sciences (ZUMS), Zanjan, Iran.
Email: biglari@zums.ac.ir, biglari63@hotmail.com
 
ACE2-based changes in the testicles, hormonal changes in patients, and the possible formation of antisperm antibodies (ASA) can cause male infertility as a complication of coronavirus infection. Although some studies have shown abnormalities in spermatogenesis and sperm motility, there is still no evidence of long-term persistence of these disorders as chronic complications of the disease. Also, after the availability of the vaccine against this virus, the possibility of the vaccine affecting the fertility of men and women has been considered. In the case of female infertility, one cause for concern is the presence of a protein called syncytine 1, which is similar to part of the new corona virus spike. It is claimed that corona vaccines can produce antibodies against syncytine 1 and cause infertility in women. Further studies will be needed to conclude, which will be discussed in this article.
K-70
Chromosomal microarray methods for detection of aneuploidy and structural chromosomal abnormality in Paroxysmal nocturnal dyspnea
 
Dehghani MR.
Medical Genetics Reserch Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: dehghani.dr@gmail.com
 
Since the identification of the exact number of human chromosomes in 1956, different techniques have been developed to identify the number of chromosomes and structural chromosomal abnormalities. Some of them such as karyotyping and fluorescence in situ hybridization (FISH) are valuable tools in both research and diagnosis. Resolution limitation is one of the important limitations of these techniques. The inability to study the entire genome simultaneously was the next limiting factor. Fortunately, the advent of new technologies to help identify postnatal and prenatal chromosomal abnormalities has had a positive effect on reducing abnormal birth rates and increasing success in assisted reproductive techniques. In 1997, microarray-based comparative genomic hybridization (array CGH) was introduced. Array CGH has the high resolution of FISH and the ability to study all chromosomes simultaneously. This technique has led to great advances in medical genetics.
 
K-71
Prenatal screening and prenatal diagnosis, new challenges in Iran
 
Eslamian L.
Fetomaternal Department, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran.
Email: leslamian@tums.ac.ir
 
Approximately 3% to 5% of pregnancies are complicated by birth defects or genetic disorders. The rapid evolution of cytogenetic methods and the advancement of molecular genetics have greatly contributed to the reduction of births with genetic defects. Prenatal tests include prenatal screening tests and prenatal diagnostic tests, which aim to detect metabolic, chromosomal, and anatomical abnormalities of the fetus as soon as possible during pregnancy. In 2007 American Congress of Obstetricians and Gynecologists (ACOG) released “ACOG Practice Bulletin No. 77,” which recommended making aneuploidy screening or invasive testing available for all women, ideally at their first prenatal visit. This idea was revolutionary at the time, as previously only women who were considered to be at high risk had been offered these tests. In Iran, prenatal screening tests are considered the health care system and every pregnant woman with a positive screening is referred for prenatal diagnostic tests. But Iran is currently facing a serious challenge for PGS and PGD. The Iran parliament has decided to eliminate prenatal screening tests from PHS to prevent population decline.
K-72
Non-invasive prenatal testing (NIPT) challenges in future
 
Garshasbi M.
Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Teheran, Iran.
Email: Masoud.garshasbi@gmail.com
 
Non-invasive prenatal testing (NIPT) for select fetal trisomies became clinically available in 2011 and transformed the landscape of prenatal screening in many countries. With very high sensitivity and very low false-positive rates, this advance represented a tremendous step forward in Down syndrome detection. However in the case of the following conditions the interpretation of NIPT results might be challenging and even error-prone: mosaicism, co-twin demise/vanishing twin, maternal genetic abnormalities, maternal medical conditions (e.g. organ transplant, malignancy), copy number variation of the portion of a fetal chromosome too small to be detected by the standard cytogenetic testing.
Since it is noninvasive, safe and allows the early detection of abnormalities, NIPT expanded rapidly and the test is currently commercially available in most of the world. As NIPT is being introduced globally, its clinical implementation should consider various challenges, including financial-economic, social, and organizational/ educational challenges. Subsequently, with a higher depth of sequencing and improved bioinformatics analyses, NIPT expanded to include detection of a number of microdeletions. Genome-wide screening for copy number variants has also been reported and is now offered clinically.
Noninvasive identification of fetal single-gene disorders, and ultimately analysis of the fetal genome, has become the “next frontier” in prenatal diagnosis. By increasing the portfolio of what can be offered by NIPT it will be necessary to think ahead the challenges of interpreting the incidental findings that will come up and how to share these findings with the families in future.
 
K-73
Preimplantation genetic testing (PGT) using next generation sequencing (NGS)
 
Ghaffari SR.
Clinical Genomics and Personalized Medicine, Infertility Center, Avicenna Research Institute, Tehran, Iran.
Email: saeed@ghaffari.org
 
Abstract not received.
 
K-74
Genetic impairments of implantation failure
 
Ghasemi N.
Abortion Research Centre, Reproductive Sciences Institute, Yazd Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: Nghasemi479@gmail.com
Embryo implantation is a critical stage of pregnancy, and failure in implantation is a main factor in early pregnancy loss or assisted reproductive failure. In humans, natural pregnancy per cycle is poor (~30%), and 75% of pregnancy are terminated because of implantation failure.
A variety of cellular actions and molecular pathways implicated in embryo-uterine interaction during implantation have been recognized, which through gene expression and genetically engineered mouse models studies was found.
However, multiple molecules are engaged in the control of implantation, but their particular actions stay uncertain. Successful implantation of a good quality human embryo in a receptive endometrium needs a significant and complicated cooperation of factors. Studies on gene- and protein expression have guided to recognition of many endometrial biomarkers and genes of both successful and unsuccessful implantations.
The functions of many candidate genes stayed important because their knocked out commanded to embryonic lethality or systemic faults. Increasing numbers of studies represented that genetic factors influence invasion and angiogenesis developments and they are crucial in embryo implantation. Molecular and genetic studies specify that ovarian hormones produced signaling molecules, containing cytokines, growth factors, homeobox transcription factors, lipid mediators and morphogen genes, work by way of autocrine, paracrine and juxtacrine relations to indicate the complex process of implantation. However, the categorized environment of the molecular signaling pathways that administrate embryo-uterine interactions in early pregnancy remains to be discovered in depth. This could be so, genetic defects and even genetic polymorphisms of genes involved in the developments of implantation could control, or at least intensify effects to implantation failure.
 
K-75
NIPT application and detection of genetic diseases in PND
 
Giglio S, Provenzano A.
Medical Genetics Unit, Department of Biomedical Experimental and Clinical Sciences, Mario Serio and Medical Genetics Unit, Meyer Children's University Hospital, University of Florence, Italy.
Email: Sabrinarita.giglio@unifi.it
 
Different genetic conditions associated with congenital anomalies/intellectual disability can contribute to long-term disablement, with significant impacts on individuals, families, health-care systems, and societies. Although congenital anomalies may be the result of one or more genetic, infectious, nutritional or environmental insults, it is often difficult to identify the exact causes. Conventional karyotype, quantitative fluorescence-polymerase chain reaction (QF-PCR) and chromosomal microarray are able to detect about 40-50% of the causes of fetal anomalies, with therefore about 50-60% of cases in which it is not possible to define the etiology. In fact, 10-20% of isolated or syndromic congenital anomalies can be associated with monogenic diseases, whose diagnosis is often established based on a family anamnesis, clinical examination, and pedigree pattern and confirmed through genetic examination. Next-generation sequencing enables to sequence of the fetal exomes, furnishing a broader diagnostic capability compared to traditional and molecular cytogenetics prenatal tests. This approach adds at least an extra 10% of clinically relevant information in cases of fetuses with structural anomalies. Moreover, the same noninvasive prenatal test can now be performed for definitive diagnosis of some monogenic recessive and X-linked conditions, or also in paternally inherited dominant and de novo conditions.
We recently demonstrated that trio-whole exome sequencing (trio-WES) using fetal cell free-DNA (cff-DNA) can be analyzed with sufficient sensitivity and that an adequate strategy can identify the cause of pregnancies at risk for malformative disorders. Our work suggested that for fetuses with proven congenital malformation an extended US examination together with trio-WES on cff-DNA may be helpful in detecting an underlying congenital disease.
Furthermore, subsequent analyzes also in couples with low risk or negative anamnesis for genetic pathologies, allowed us to further demonstrate that genome-wide sequencing is an effective method that will likely be more used in the coming years as a clinical tool for diagnosis. Moreover, we are able to demonstrate that the WES analysis on cell-free DNA has a better diagnostic yield than the same test performed on DNA extracted from the amniotic fluid, proving that WES on cff-DNA is especially suitable, with an appropriate genetic counseling, in fetuses with genetic diseases.
 
K-76
Chromosome X-inactivation and infertility
 
Hozhabri H.
Medical Bioinformatician Centogene, Rostock, Germany.
Email: Hossein.hozhabri@gmail.com
 
Abstract not received.
 
K-77
Genetic aspects of male infertility
 
Kalantar SM.
Research and Clinical Center for Infertility, Yazd Reproduction Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: smkalantar@yahoo.com
 
About 15% of couples do not achieve pregnancy within one year and seek medical treatment for infertility. One in eight couples encounters problems when attempting to conceive a first child and one in six when attempting to conceive a subsequent child. Three percent of women remain involuntarily childless, while 6% of parous women are not able to have as many children as they would wish. Infertility affects both men and women. In 50% of involuntarily childless couples, a male-infertility-associated factor is found together with abnormal semen parameters. Male infertility is a common and severe health problem. Infertility not only affects one’s ability to have children, but also has emotional, psychological, family, and societal effects. The incidence may be increasing during the time of those affected, roughly 40% have idiopathic infertility. It is likely that the majority of those patients have genetic abnormalities that are the cause of their infertility. The understanding of the genes involved in spermatogenesis, sperm maturation, and normal sperm function is key, but we must also focus on better methods of accelerating advances into meaningful clinical diagnostic tests and therapies. During the past 30 years, significant improvements in technology have advanced the treatment of male infertility and Genetic evaluation as well. The primary advance has been intracytoplasmic sperm injection (ICSI) in conjunction with in vitro fertilization through ART cycles. Although this technological leap has allowed thousands of men to father a child who otherwise would have been unable to do so, the scientific study of the causes of male infertility has not kept pace. All urologists working in the field of Andrology must have an understanding of genetic abnormalities associated with infertility so that they can provide correct advice to couples seeking fertility treatment. Men with very low sperm counts can be offered a reasonable chance of paternity, using IVF, ICSI, and sperm harvesting from the testes in case of azoospermia
In fact, the clinical application of ICSI proceeded without sufficient scientific study of its safety to the offspring, or the future genetic ramifications several researchers and clinicians, and an international audience of experts in the field, reviewed the study of the genetics of male infertility, the tools available in the laboratory and clinic, the current state of knowledge, and the future of research and translation into clinical diagnostics and in this webinar the colleagues discussing the following aspects as:

• The genetics of male infertility in the era of genomics
• Important environmental factors on fertility potential
• DNA damage how can affect the fertility potential of male
• Methods and tools for the study of the genetics of male infertility
• Clinical approach for evaluation of azoospermia men
• Management of azoospermia due to microdeletions in y chromosome
• Regeneration approach as new tools for now and near future to generate spermatogenesis.

 
K-78
Personalized medicine for the embryo and the fetus
 
Modarressi MH.
Department of Medical Genetics, Tehran University of Medical Sciences, Tehran, Iran.
Email: modaresi@tums.ac.ir
Since the conception of precision medicine has been put forward in reproductive medicine, this idea has been popularized and applied in many specialties. Significant progress has been made toward personalizing the entire process, including diagnosis, treatment planning, and embryo identification, and combining large-scale genetic information data and knowledge discovery can offer better prospects in reproductive medicine. The causes of infertility are various, and many factors influence the success rates of ART which are complicated; hence, different genetic diagnostic methods of reproductive medicine for the diagnosis of infertility causes and transfer of healthy embryos, needs to be precise. During the last decay, next generation sequencing influenced reproductive medicine to personalize the diagnostic methods. Prenatal genetic diagnostics and preimplantation genetic diagnosis can and should be expanded to incorporate genetic, genomic and transcriptomic data to develop new approaches to diagnosis and fetal treatment. I would like to review recent advances in prenatal genetic diagnostics and preimplantation genetic diagnosis, the challenges associated with these new technologies such as next generation sequencing and how the information derived from them can be used to personalize and advance fetal care.
K-79
New strategies for the diagnosis of male infertility: Current developments and prospects
 
Mojarrad M.
Department of Medical Genetics, Mashhad University of Medical Sciences, Mashhad, Iran.
Email: mojaradm@mums.ac.ir, majidmojarrad11@gmail.com
 
Infertility is one of the common health issues around the world. Although Female factors are the main cause of infertility, male factor infertility involves approximately 30%- 35% of infertile couples. Genetic factors account for 15% of infertility male factors. By emerging the next generation sequencing technology, the role of male genetic factors is becoming increasingly prominent. Various whole-exome sequencing approaches such as family-based whole-exome sequencing are powerful tools for accurately identifying the novel variants responsible for infertility. In this talk, we try to outline different strategies for studying the causes of male infertility and the latest findings in this area. Eventually, it seems that male monogenic factors play a much greater role in infertility problems than previously thought. Despite the remarkable advances in male infertility, due to the high prevalence of this disease, we expect to see many more discoveries in the near coming years.
 
K-80
Azoospermia
 
Narimani N.
Hasheminejad Kindney Center, Iran University of Medical Science, Tehran, Iran.
Email: Nima_dr2001@yahoo.com
Infertility affects 15% of couples, and male factors are implicated as a cause in 50% of patients. Azoospermia is considered the most severe form of male factor causes and is defined as the absence of sperm in the semen sample. It can be further categorized into obstructive and non-obstructive azoospermia. Non-obstructive azoospermia may be due to testicular (primary testicular failure) or pre-testicular (secondary testicular failure) causes. Obstructive azoospermia could be due to congenital and/or acquired etiologies. Genetic factors may play an important role in both types of azoospermia. Before the advent of intra-cytoplasmic sperm injection (ICSI) azoospermic men considered sterile and were in need of sperm or fetus donation. In the era of new assisted reproductive technique, these male patients could have their own child. Since ICSI could surpass be natural selection, the genetic abnormalities could easily transmitted to the offspring. Therefore genetic counseling should be considered before ART.
 
K-81
Personalized ovarian stimulation for assisted reproductive technology
 
Nasr-Esfahani MH^{1, 2}.

1. Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
2. Isfahan Fertility and Infertility Center, Isfahan, Iran.

Email: Mh_nasr@med.mui.ac.ir, Nasr.royan@gmail.com
 
When one talks about personalized medicine in the field of assisted reproduction technology, the main focus is mainly on achieving the optimal number of oocytes in successful ovarian stimulation at the same time considering the safety, success, and potential of individuals to respond. Considering that more oocytes mean higher clinical live birth rate but one single protocol for stimulation does not fit all. Considering every individual has different egg reserve which can be assessed based on anti mullerian hormone or antral follicular count, therefore, amount of Follicle-stimulating hormone given for induction of stimulation should be related to one of these two parameters at the same time considering safety which here means avoiding ovarian hyperstimulation syndrome. Literature shows that around 15 oocytes are the optimal number of oocytes for achieving a live birth and avoiding ovarian hyperstimulation syndrome. Achieving this number of oocytes may not be possible in one single cycle and multiple cycles are required and a standard dose of FSH is not justified and a tailoring Follicle-stimulating hormone dose should be defined based on the individuals age, antral follicular count and previous cycle performance.
 
K-82
Treatment of infertile male causing by AZF deletion
 
Sadighi Gilani MA.
Department of Urology, Tehran University of Medical Sciences, Tehran, Iran.
Email: masadighi@gmail.com
Male infertility is a common condition with heterogeneous causes. Genetic or epigenetic variations or both contribute up to 15%-30% of cases of male infertility. Genes control a variety of physiologic processes, such as spermatogenesis which occurs in a sequential manner with mitotic, meiotic, and postmeiotic differentiation phases and secretion of hormones. The genetic abnormalities involved in male infertility may be chromosomal (numerical and structural aberrations) or monogenic disorders, mitochondrial DNA (mtDNA) mutations, microdeletion of the Y chromosome, multifactorial disorders, imprinting disorders, or endocrine disorders of genetic origin. The Y chromosome is one of the smallest human chromosomes and microdeletions on the long arm of this chromosome (Yq) is one of the most significant causes of male infertility. Y chromosome microdeletions were present in about 5.2% -12.1% of Iranian infertile men with azoospermia and severe oligozoospermia. The AZF region is further subdivided into 3 non-overlapping regions termed as AZFa, AZFb, and AZFc. Deletion of AZFa and A2Fb which are less common are associated with non-treatable azoospermia. Deletions of the AZFc region are most common in men with idiopathic oligozoospermia or azoospermia. Cases with AZFc deletions show a progressive deterioration in spermatogenesis and cases develop azoospermia over a period of time. The clinical outcomes of ICSI for oligozoospermic patients with Y chromosome AZF microdeletion are comparable to those of infertile patients with normal Y chromosomes. For azoospermic men with AZFc deletion microdissection, testicular sperm extractions is recommended with a success rate of sperm retrieval about 36.3%. In conclusion, the pregnancy and delivery in oligozoospermic patients with AZFc deletion are comparable with other studies, but despite of sperm retrieval in azoospermic patients with AZFc deletion, the chance of pregnancy or delivery in these patients is very low. More attention to surgical points for sperm retrieval and more extensive search for sperm finding and refinement of sperm freezing and ICSI procedure is needed for better results.
 
K-83
Personalized medicine in infertile men
 
Serdar C.
Intergen Genetic and Rare Diseases Diagnosis and Research Center, Ankara, Turkey.
Email: serdarceylaner@intergen.com.tr
 
As in all fields of medicine, each patient has different details in male infertility. Treatments made without taking these details into account are more likely to fail. Today, widely used approaches in the world are generally in the form of applying some close-to-standard procedures that the centers have created for them to the patients. Such approaches are effective on a certain number of patients, but other groups cannot be successful despite repetitive procedures.
Today, thanks to the developing genomic technologies, it is possible to examine thousands of genes and copy number changes in humans at the same time. Considering that hundreds of factors in infertility have entered the literature so far, conducting extensive research is an indispensable need for most patients. The genetic change detected in some of these patients changes the treatment algorithm of the patient significantly, and in some patients, diagnoses that are not possible to treat can be made. In particular, patients in this last group are exposed to repeated in vitro fertilization or other medical practices, even though they have no hope of treatment, and lose time and money.
Although genetic tests were often put at the end of the list of studies to be carried out in infertility practices in recent years, due to the high cost of genetic tests, it is possible to move forward in the examination order due to the fact that these applications have gradually lower costs. One of the most important stages of personalized medicine applications is to evaluate the patient with large genetic panels and to evaluate the effects of other diseases related or unrelated to infertility with very wide eyeglasses.
 
K-84
Genetic and epigenetic aspects of endometriosis
 
Shahhoseini M^{1, 2}.

1. Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
2. Department of Cell and Molecular Biology, School of Biology, College of Science, University of Tehran, Tehran, Iran.

Email: m.shahhoseini@royaninstitute.org
 
Endometriosis is a major gynecological disease that affects over 10% of women worldwide. It is characterized by the implantation of functional endometrial tissue at ectopic positions generally within the peritoneum. Endometriosis is recognized as a steroid-dependent disorder; however, the cause of endometriosis is unknown and there is no definite cure for it. This is mainly because of our limited knowledge about the pathophysiology of this disease at the cellular and molecular level. A PubMed search summarizes the key mediators of pain, abnormal uterine, bleeding, and infertility in endometriosis, including sex steroid hormone receptors, inflammatory molecules, extracellular matrix enzymes, growth factors, and neuroangiogenic factors. Therefore, clarifying the molecular mechanisms underlying endometriosis is essential in order to develop advanced therapies for the disease. In this regard, access to a precise genetic/epigenetic profile of endometriosis would be helpful for the diagnosis and treatment of the disease.
 
K-85
Can personalized medicine help infertility treatment?
 
Sheikhha MH.
Biotechnology Research Center, International Campus, Shahid Sadoughi University of Medical Sciences and Health Services, Yazd, Iran.
Email: sheikhha@yahoo.com
 
Different surveys showed that the rate of infertility is above 10% in many parts of the world. This means that becoming pregnant can be a stressful process for many couples, and they have to seek assisted reproductive technology (ART) and use different drugs to help them for having a baby. It is shown that there are differences in response to different drugs in these individuals. It is proved that these differences are genetically determined. Individualized or personalized medicine is defined as using genetic information to select the most appropriate choice of pharmacological therapy. Personalized medicine is based on polymorphic DNA sequence variations. If a polymorphism occurs in the coding or regulatory regions of a gene, it can alter the function, activity, or level of expression of that gene. Using the new advanced methods of genetics testing such as automated analysis of genome-wide single nucleotide polymorphisms allows identifying polymorphisms in genes involved in drug metabolism, transport and receptors. In ART, the efficiency of the protocol is a problem that needs to be solved. If we can utilize genome sequencing as a routine clinical approach to create an individual’s own pharmacogenomics profile, therefore we can provide valuable information to help infertility specialists to use the optimal drug dosage for ART. So far, significant progress has been made toward personalizing the entire ART process, including diagnosis, treatment planning, and embryo identification. In fact, reproductive medicine is among one of the first subjects that used the concept of personalized medicine even before its popularization. The application of personalized medicine in ART starts in fact when the studies showed that the causes of infertility are various, and factors influencing the success rates of ART are complicated. In fact, there are variations in different individuals regarding their oocyte and embryo grading, endometrial condition, and semen analysis. Therefore, different steps of reproductive medicine need to be personalized, such as the diagnosis of infertility causes and transfer of the healthy embryo. One of the most important areas of using personalized medicine in reproductive medicine is discovering and validating genomic, protein, and metabolite biomarkers.                To overcome the concept of "one size does not fit all" we should consider patients’ specific molecular profiles. Finally, the main difference in personalized medicine between ART and the other subjects is that in reproductive medicine, the matter is "personalized" to not only one individual but in three different individuals; the mother, father, and embryos. Another complication of ART is that we have to apply the personalization in different biological systems including; the egg, sperm, embryo, and uterus, as different systems may be involved in etiology of infertility in different subjects.
K-86
The genetics of premature ovarian failure: Current perspectives
 
Shelling A.
Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand.
Email: a.shelling@auckland.ac.nz
 
Premature ovarian failure (POF) is a common cause of infertility in women, characterised by amenorrhea, hypoestrogenism, and elevated gonadotropin levels in women under the age of 40. Many genes have been identified over the past few years that contribute to the development of POF. However, few genes have been identified that can explain a substantial proportion of cases of POF. The unbiased approaches of genome wide association studies and next generation sequencing technologies have identified several novel genes implicated in POF. As only a small proportion of genes influencing idiopathic POF have been identified thus far, it remains to be determined how many genes and molecular pathways may influence idiopathic POF development. However, due to POF’s diverse etiology and genetic heterogeneity, we expect to see the contribution of several new and novel molecular pathways that will greatly enhance our understanding of the regulation of ovarian function. Future genetic studies in large cohorts of well defined, unrelated, idiopathic POF patients will provide a great opportunity to identify the missing heritability of idiopathic POF. The identification of several causative genes may allow for early detection and would provide a better opportunity for early intervention, and furthermore, the identification of specific gene defects will help direct potential targets for future treatment.
 
K-87
Current state of art use of stem cells for regenerating tissues and possibility of using iPSCs to generate mature spermatozoa in the future
 
Zandieh Doulabi B.
Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Amsterdam, Netherland.
Email: bzandiehdoulabi@acta.nl
 
Integrated research of stem cells and tissue engineering is essential to improve health issues in the field of regenerative medicine. Tissue engineering combines various fields such as biochemistry, cell biology, materials science, transplantation and hardware engineering in an effort to repair or replace damaged tissues. Stem cells are defined by their ability to self-renew and differentiate into a variety of cell types. Stem cells are divided into 3 groups depending on multilineage differentiation capacity (Totipotent, pluripotent and multipotent). To date, in contrast with tissue-engineered bone, cartilage, muscle, nerve, and skin, tissue engineering of other tissues and organs is much less advanced. This is caused by the fact that there is a limited choice of appropriate cells, biomaterials, chemical stimuli such as hormones and physical stimuli such as mechanical loading. Infertility affects 15% of men of reproductive age worldwide. Spermatogenesis is the proliferation and differentiation of spermatogonial stem cells. For spermatogonial stem cell therapy to be a success, and cultured sperm stem cells to become mature sperm, they need the proper stimuli such as the right microenvironment. However, until now these microenvironment conditions remain the object of speculation. Besides spermatogonial stem cells, the use of adult-derived induced pluripotent stem cells (iPSCs) might be a promising cell source to generate mature sperms. These cells, like embryonic stem cells, have the potential to form eggs and sperm. Although controversy surrounds their use, iPSCs have a huge potential for biological and therapeutic applications for male infertility, provided that in vitro spermatogenesis models for iPSCs can be established, thereby providing insights into the mechanism of human spermatogenesis and its regulation. The challenge remains that the molecular mechanisms underlying human male germ cell development remain poorly understood. But the iPSCs will have extra therapeutic implications for male infertility when combined with genome editing technology in near future.

Award Winners
 
A-1
Designing and implementation of mental health intervention for oocyte donor women
 
Adibmoghaddam E¹, Kazemi A¹, Kheirabadi G²,        Ahmadi S³.

1. Department of Reproductive Health, Faculty of Nursing and Midwifery, Isfahan University of Medical Sciences, Isfahan, Iran.
2. Department of Psychiatry, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
3. Isfahan Fertility and Infertility Center, Isfahan, Iran.

Email: adibme921@gmail.com
 
Background: Oocyte donors face medical risks, socio-cultural challenges and the psychological consequences toward participating in assisted reproductive technology (ART). Developing a documentation-based intervention reduces the psychological burden from participation in this process.
Objective: The present study was conducted with the aim of designing and implementing a mental health intervention for oocyte donor women.
Materials and Methods: The present study was conducted by using mixed methods study in four stages. In the first stage, a qualitative study was conducted to explain the experiences of women who donate oocytes and providers of ART. In the second stage, the draft of mental health intervention plan for oocyte donors was designed based on qualitative study and review of literature, and a list of needs and strategies of the plan were provided to faculty members using a Delphi technique. Then the intervention was designed based on the strategies with the highest score. In the third stage, in order to review the content of the intervention, an expert panel was held and then approved by the panel members. The fourth stage of the study was a quantitative stage and was conducted as a two-group field trial with the aim of determining the effect of the intervention plan on the mental health of oocyte donors. The study sample size was 72 participants (36 participants in each group). Data collection tool was depression, anxiety and stress scale (DASS-21) questionnaire which was completed in two stages before superovulation induction and after oocyte retrieval. Also, the researched made questionnaire for measuring worry and satisfaction with participation in ART which was completed after oocyte retrieval and data analyzed using SPSS software version 19.
Results: The findings of the qualitative study led to the formation of 7 main categories including "decision challenge","donation complications","challenges of the oocyte donation process", "emotional experiences”,“donor perspective versus recipient perspective","Needs and requests" and"structural defects"resulted. In designing the draft of the plan, the intervention was designed in the form of educational pamphlets, consultations and Instagram posts according to the strategies with the highest score. The results of the intervention showed before ovulation induction, the scores of DASS-21 in the intervention group were significantly lower than the control group. After oocyte retrieval, the scores of the DASS-21 and also, worry in the intervention group were significantly lower than the control group and the score of satisfaction with the donation process was significantly higher than the control group. The mean score of the depression and stress in the control group before ovulation induction and after oocyte retrieval were not statistically significant, but in the intervention group the mean score of the depression and stress after oocyte retrieval were less before ovulation induction (p ˂ 0.001). After oocyte retrieval, the mean score of anxiety in the control group, was increased significantly (p = 0.02); while in the intervention group, there was decreased significantly(p ˂ 0.001).
Conclusion: The results of this study showed the designed intervention plan was effective on mental health in oocyte donors.Therefore,the implementation of the designed intervention in fertility centers could be useful in promoting the mental health of women who donate oocytes.
Key words: Mental health, Oocyte donation, Mixed methods study.
 
The original full text of this abstract has been published in Reproductive Health 2020; 17(1): 1-5. https://doi.org/ 10.1186/s12978-020-0864-9.
How to cite to this article: Moghaddam EA, Kazemi A, Kheirabadi G, Ahmadi SM. A mental health intervention program for the oocyte donors: protocol for a mixed methods study. Reproductive Health 2020; 17(1): 1-5.
 
A-2
Whole-exome sequencing in patients with primary ovarian insufficiency
 
Akbari A¹, Padidar K^{1, 2}, Salehi N^{1, 3}, Mashayekhi M⁴, Almadani N¹, Sadighi Gilani MA⁵, Bashambou A⁶, McElreavey K⁶, Totonchi M^{1, 7}.

1. Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
2. Department of Molecular Genetics, Faculty of Basic Sciences and Advanced Technologies in Biology, University of Science and Culture, Tehran, Iran.
3. Department of Bioinformatics, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.
4. Department of Endocrinology and Female Infertility, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
5. Department of Andrology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
6. Human Developmental Genetics Unit, Institut Pasteur, Paris, France.
7. Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.

Email: totonchimehdi@gmail.com
Background: Primary ovarian insufficiency (POI) is a complex and relatively poorly understood disorder. It affects 1% of women below the age of 40 accompanied by raised gonadotropins and estradiol deprivation. Women with POI have no antral follicles and size of ovaries are below normal.
Objective: Our objective was to identify the genetic cause of POI in affected members of a consanguineous Iranian family.
Materials and Methods: In this study, we recruited an Iranian family with 2 affected members to be studied by whole exome sequencing (WES). Validation of WES results and cosegregation analysis was performed by Sanger Sequencing. In silico analysis was used to predict the effect and pathogenicity level of the discovered variants.
Results: The proband and her affected sister tested normal for karyotype and FMR1 CGG repeats. A final list of pathogenic variants was prepared according to WES results, and one specific variant in a conserved domain of transcription factor protein was confirmed to be mutual among the affected. The discovered variant is very rare in the gnomAD database.
Conclusion: This study supports the clinical applicability of WES for cost-effective molecular diagnosis and improves the understanding of the genetic basis of female infertility and ovarian function. Therefore, our finding provides yet another piece of evidence that Loss of function variations in transcription factors with limited expression in ovaries may be the cause of POI.
Key words: Primary ovarian insufficiency, Familial exome sequencing, Causative mutation.
 
The original full text of this abstract has been published in Human Reproduction 2021; 36 (4): 1134-1145. https://doi.org/10.1093/humrep/deaa362.
How to cite to this article: Akbari A, Padidar K, Salehi N, Mashayekhi M, Almadani N, Sadighi Gilani MA, Bashambou A, McElreavey K, Totonchi M. Rare missense variant in MSH4 associated with primary gonadal failure in both 46, XX and 46, XY individuals. Human Reproduction 2021; 36(4): 1134-1145.

A-3
Does sperm DNA fragmentation have negative impact on embryo morphology and morphokinetics in IVF program?
 
Anbari F¹, Khalili MA¹, Agha-Rahimi A¹, Maleki B¹, Nabi A¹, Esfandiari N².

1. Department of Reproductive Biology, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Department of Obstetrics and Gynecology, Dartmouth Hitchcock Medical Center, Lebanon, NH, USA.

Email: Khalili59@hotmail.com
 
Background: Evaluation of sperm DNA integrity may predict the in vitro fertilization (IVF) outcomes.
Objective: The aim was to evaluate the relationship between the sperm DNA fragmentation (sDNAf) with embryo morphology and morphokinetic using time-laps monitoring and to select the best time points for normalization in IVF setting.
Materials and Methods: After evaluating the fertilization and pronuclei scoring, 328 normally fertilized oocytes were assessed to time of pronuclei fading (tPNf), time of 2 to 8 discrete cells (t2-t8) and abnormal cleavage patterns, such as multinucleation, direct cleavage, reverse ‎cleavage and fragmentation. Sperm chromatin dispersion (SCD) assay was used for assessment of prepared sperm chromatin status. SCD was categorized into 4 groups of < 6.5, 6.5-10.7, 10.7-20.1 and > 20.1.
Results: Significant differences were found in t6 (‎p = 0.012), t7 (p =‎ 0.045), t8 (p = ‎0.013‎) and s1 (p = ‎0.001‎) between 4 SCD groups. When, morphokinetic variables were normalized to tPNf, this difference was observed in t2 (p =‎ 0.003‎) and t6 (p = 0.017‎). Subsequently, the percentage of top quality embryos and Z1 scoring were dependent to the sDNAf rate.
Conclusion: In conclusion, tPNf was the best reference time point in IVF cycles. Also, we found high sDNAf rate had no negative impact on embryo morphology and morphokinetics in conventional IVF.
Key words: In vitro fertilization, Embryonic development, Time-laps monitoring.
 
The original full text of this abstract has been published in Andrologia. 2020 Dec; 52(11): e13798. https://doi.org/10.1111/and.13798.
How to cite to this article: Anbari F, Khalili MA, Agha‐Rahimi A, Maleki B, Nabi A, Esfandiari N. Does sperm DNA fragmentation have negative impact on embryo morphology and morphokinetics in IVF programme? Andrologia 2020 Dec; 52(11): e13798.

A-4
Gangliogenesis with folliculogenesis of ovary: Three-dimensional and two-dimensional analyses of Golgi-Cox-staining in mouse ovary
 
Asadi Zarch ME¹, Afshar A², Rahmanifar F³, Jafarzadeh Shirazi MR¹, Baghban M⁴, Dadpasand M¹, Mohammad Rezazadeh F¹, Khoradmehr A², Baharvand H^{5, 6}, Tamadon A².

1. Department of Animal Sciences, College of Agriculture, Shiraz University, Shiraz, Iran.
2. The Persian Gulf Marine Biotechnology Research Center, The Persian Gulf Biomedical Sciences Research Institute, Bushehr University of Medical Sciences, Bushehr, Iran.
3. Department of Basic Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran.
4. Department of Obstetrics and Gynecology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
5. Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
6. Department of Developmental Biology, University of Science and Culture, Tehran, Iran.

Email: amintamaddon@yahoo.com
 
Background: The ovarian follicular development of rodents begins at neonatal period, the stage at which primordial follicles are formed. During estrous cycle, most of the follicles undergoes atresia and some of them continue their development process. The mammalian’s ovary is regulated by some factors including hormonal factors and direct neuron effects. Previous studies have shown that the fate of follicles in this cycle are affected by hormones such as follicle-stimulating hormone and luteinizing hormone. In addition, there are two different populations of neurons in ovary, the internal and external neurons. External nervous system of mouse ovary has many roles. Several studies have shown its role in developmental process, cyclic stages, pregnancy, and aging process. These nerves and also ganglia are responsible for ovarian estradiol secretion. Some studies implied that the ganglia in ovary takes part in some functions such as hormone secretion but to best of our knowledge, their relationship with follicular and ovarian development have not fully understood.
Objective: The present study was set out to investigate two-dimensional (2D) and three-dimensional (3D) evaluations of ovarian nervous network development and the structural relationship between folliculogenesis and gangliogenesis in mouse ovary.
Materials and Methods: Adult mice ovarian tissue samples were collected from diestrus and estrus stages. In details, firstly, the cardiac perfusion was performed. The collected ovarian samples were stained by a Golgi-Cox protocol. Following staining, tissues were serially sectioned with thickness of 30 μm for each section for imaging and further analysis. Ovarian tissue serial images were evaluated with Image J software for 2D analysis and with Imaris software for 3D analysis. The images of estrus and diestrus ovaries were separately compared. In addition, the 2D and 3D data of estrus ovary were comparably analyzed. IBM SPSS Statistics 26 software was used for statistical analysis. The mean differences between follicular groups were analyzed by one-way ANOVA and post hoc Tukey test.
Results: Neural filaments and ganglia were detected in the ovaries by Golgi-Cox staining. In both 2D and 3D studies, an increase in the number and area of ganglia was seen during the follicular growth (p < 0.05). The same pattern was also seen in corpora lutea development. However, in some cases such as ratio of ganglia number to follicle area, the ratio of ganglia area to follicular area, 2D findings were different compared with the 3D results. 3D analysis of ovarian gangliogenesis showed the possible direct effect of them on folliculogenesis. Golgi-Cox staining was used in this study for 3D evaluation in non-brain tissue. The results of 3D analysis of the present study showed that, in some cases, the information provided by 2D analysis does not match the reality of ovarian neuronal function. This confirmed the importance of 3D analysis for evaluation of ovarian function.
Conclusion: It was demonstrated that there was positive relationship between gangliogenesis and folliculogenesis in mouse ovary. Ovarian ganglia, as an independent part of ovarian nervous system, is likely to have an important role in folliculogenesis and luteogenesis. Additionally, Golgi-Cox staining and 3D tissue imaging, instead of 2D imaging, are promising protocols for study of ganglia in ovarian tissue.
Key words: 3D Imaging, Ganglia, Ovarian follicle, Golgi-Cox staining, Mice.

The original full text of this abstract has been published in scientific reports 2021; 11, 5547 (2021). https://doi.org/10.1038/s41598-021-84835-0.
How to cite to this article: Asadi Zarch M.E., Afshar A., Rahmanifar F. et al. Three-dimensional and two-dimensional relationships of gangliogenesis with folliculogenesis in mature mouse ovary: a Golgi–Cox staining approach. Scientific Reports 2021; 11: 5547.
A-5
Sperm telomere length in infertile men with previous failed fertilization post- ICSI
 
Darmishonnejad Z¹, Tavalaee M¹, Izadi T¹, Tanhaei S¹, Nasr-Esfahani MH^{1, 2}.

1. Department of Animal Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
2. Isfahan Fertility and Infertility Center, Isfahan, Iran.

Email: Z.darmishoun@gmail.com
 
Background: Intracytoplasmic sperm injection (ICSI) technique is used mostly for the treatment of male infertility. However, failed fertilization was observed in a litter percentage of infertile couples post- ICSI. Several factors such as abnormal sperm quality, DNA fragmentation, low or absence of sperm factors involved in oocyte a3ctivation are associated with failed fertilization post- ICSI. Sperm telomere length is one of the sperm factor at the end of the eukaryotic chromosomes that protects chromosomes from     damage and positive significant correlation was observed between this parameter with sperm DNA fragmentation.
Objective: In the light of these considerations, we aimed to assess sperm telomere length and DNA fragmentation in infertile men with previous failed fertilization post-ICSI.
Materials and Methods: In this study, semen and blood sample were obtained from 10 infertile men with a failure in ICSI fertilization and 10 fertile as a control group. Telomere length was evaluated both in sperm and        blood samples by Realtime-PCR. Finally, Independent t test, and the correlation coefficient were used for analysis of data.
Results: Mean of sperm and blood telomere length were significantly shorter in infertile with previous failed fertilization compared to fertile men (p < 0.05). Moreover, in infertile men, percentage of sperm DNA      fragmentation was significantly higher than fertile men (p = 0.01). In addition, we observed a significant correlation between sperm telomere length with fertilization rate (p < 0.05).
Conclusion: In this study, for the first time, we showed in infertile men with previous failed fertilization, sperm telomere length was low. Therefore, the reduction of sperm telomere length as one of sperm factors could associated with low fertilization potential.
Key words: Sperm telomere, DNA fragmentation, ICSI, Failed fertilization.

The original full texts of this abstract have been published in

• Reproductive Biomedicine Online 2019; 38(4): 579-587. https://doi.org/10.1016/j.rbmo.2018.12.022. Andrologia 2020; 52(5): e13546. https://doi.org/10.1111/and.13546.

How to cite to these articles:

• Darmishonnejad Z, Tavalaee M, Izadi T, Tanhaei S, Nasr-Esfahani MH. Evaluation of sperm telomere length in infertile men with failed/low fertilization after intracytoplasmic sperm injection. Reproductive Biomedicine Online 2019; 38(4): 579-587.
• Darmishonnejad Z, Zarei‐Kheirabadi F, Tavalaee M, Zarei‐Kheirabadi M, Zohrabi D, Nasr‐Esfahani MH. Relationship between sperm telomere length and sperm quality in infertile men. Andrologia 2020; 52(5): e13546.

 
A-6
Follicular reconstruction in artificial ovary made by human isolated ovarian cells from chemotherapy-induced POF patient seeded into human ovarian decellularized ECM after xenotransplantation
 
Khaleghi S¹, Eivazkhani F², Moini A³, Ghaffari Novin M¹, Nazarian H¹, Fathi R².

1. Biology and Anatomical Sciences Department, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
2. Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
3. Department of Endocrinology and Female Infertility, Royan Institute of Reproductive Biomedicine, ACECR, Tehran, Iran.

Email: rfathi79@royaninstitute.org
 
Background: Depletion of ovarian reserve due to dismiss of follicular growth in chemotherapy induced premature ovarian failure (Chemo- POF) is the main concern for oncofertility researchers who try to find a practical way to restore the ovarian function.
Objective: Artificial ovary is preparing a new niche for ovarian cells and reconstruction of follicular activity may be developed to aid infertility treatment by transplantation of engineered ovary.
Materials and Methods: Ovarian tissues were taken from 8 Chemo- POF women and 15 transsexuals. The medulla was carefully removed and the cortical tissue was cut into 5×5×5 mm3 strips and then cryopreserved. Ovarian cells were removed with NaOH as main detergent from human ovarian pieces of transsexual patients and then decellularized cortical tissue (DCT) was evaluated by DNA content analysis, hematoxylin & eosin and DAPI staining techniques. Human ovarian cortical cells (HOCCS) were finely minced and enzymatically isolated and characterized by real time PCR for IFITM3, vimentin, FSH-R and KI67 genes and immunostaining of vimentin, Inhibin-α and IFITM3 markers. Then the isolated HOCCS (2 × 10⁶ cells) from both Chemo- POF and transsexual ovaries were seeded into DCT by injection and spinner flask culturing for one wk (AO; artificial ovary). Also, MTT assay was performed to measure the in vitro cytocompatibility of ovarian scaffold. Then AO was xenotransplanted to NMRI mice beneath the abdominal sub-serosal fascia for two months. Finally, H&E, hormonal tests (FSH, AMH and E2), real time PCR (GDF-9, ZP3, VEGF, CD34 and KI67) and immunohistochemistry (IHC) (GDF-9) assessments were applied for the calculation of transplantation outcomes.
Results: H&E, DAPI and DNA content confirmed over 95% decellularization. Immunofluorescence showed that isolated HOCCS from transsexual and Chemo- POF ovarian tissues included 80-85% stromal, 5-10% granulosa and < 5% oogonial stem cells by expressing the vimentin, Inhibin-α and IFITM3 markers in passage one. Expression patterns of the mentioned proteins in passage two were appraised 70-75%, 5-10% and > 10%, respectively. Also, HOCCS well expressed Vimentin, FSH-R, FRAGILIS, DDX4, STELLA and KI67 genes in real time PCR technique. One wk culture of AO in spinner flask indicated the HOCCs could penetrate not only into the exterior surfaces also to the depth of the ovarian scaffold (H&E). Histological study and quantitative evaluation of Estradiol, FSH AND AMH production after two months of AO xenotransplantation confirmed the presence of morphologically health and secretory active reconstructed human ovarian primordial and primary follicles. IHC for GDF9 confirmed the paracrine activity of oocytes within the follicles. To approve existing active follicles within AO, the real time PCR demonstrated a good expression of the follicle-related genes like GDF9 and ZP3 in both groups. Furthermore, the angiogenesis genes VEGF and CD34, in both transplanted groups showed high expression. At the end, the expression of kI67, cell proliferation and survival factor, approved the cellular multiplication and health in AO of the both groups.
Conclusion: Our results approved that ovarian follicular reconstruction and function is possible in the case of ovarian insufficiency through xenotransplantation of AO made by DCT seeded by Chemo-POF ovarian isolated cells.
Key words: Human artificial ovary, Ovarian follicular reconstruction, Oogonial stem cells, Spinner flask.
 
A-7
Four hours or more preincubated oocytes in the simple medium provide low transcript levels of maternal effect genes for the embryos
 
Mohammadi F¹, Ashrafi M², Zandieh Z³, Nikniaz H³, Haghighi M³.

1. Nahal Salamat Infertility Clinic, Tabriz, Iran.
2. Shahid Akbar Abadi Clinical Research Development Unit (ShACRDU), Iran University of Medical Sciences (IUMS), Tehran, Iran.
3. Anatomy Department, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.

Email: F.mohammadi87@gmail.com
 
Background: Preincubation is the temporary cultivation of oocytes at 37°C and 5-6% of CO2 before ART procedures. There is not any explanation regarding a standard preincubation time in ART laboratory guidelines and it is dependent completely on the laboratory workload. Myo-inositol as the most important form of inositol, is involved in several systemic processes and its antioxidant action has been suggested recently. The study aimed to evaluate the effect of oocyte preincubation time and also myo-inositol as a medium supplement on the oocyte Zar1, Nlrp5, Npm2 transcript levels as well as the fertilization and first cleavage rates.
Objective: The study aimed to evaluate the effect of oocyte preincubation time and also myo-inositol as a medium supplement on the oocyte Zar1, Nlrp5, Npm2 transcript levels as well as the fertilization and first cleavage rates.
Materials and Methods: Cumulus Oocyte Complexes which were retrieved from superovulated NMRI mices were divided randomly in five experimental groups: (1) control (2) 4 hours preincubation in simple medium (3) 4 hours preincubation in 20 mmol/l of myo-inositol supplemented medium (4) 8 hours preincubation in simple medium (5) 8 hours preincubation in 20 mmol/l of myo-inositol supplemented medium. COCs were denuded and transcript levels of Zar1, Nlrp5 and Npm2, selected by bioinformatics, were assessed by real time qPCR method. 2PN and 2-cell rates were analyzed following oocytes and sperms co-incubation. One-way ANOVA and Kruskal-Wallis were respectively used for parametric and nonparametric variables. Statistical significance was defined as P-value ≤ 0.05.
Results: Zar1 (1-fold vs 0.4-fold) and Npm2 (1-fold vs 0.2-fold) transcript levels, as well as  2PN (84.64 ± 4.02 vs 78.90 ± 1.11) and 2-cell rates (79.58 ± 1.45 vs 59.85 ± 9.44) were reduced after 4 h of preincubation time in the simple medium compared to the control group. While Nlrp5 transcript level (1-fold vs 0.07-fold) was significantly decreased following 8 h of preincubation time in the simple medium (p ˂ 0.001).  Addition of myo-inositol to the culture medium could ameliorate maternal effect genes levels and fertilization and first cleavage rates in the oocytes preincubated for 4 and 8 hours (p ˂ 0.001).
Conclusion: However it has not found a clear boundary between optimal and non-optimal oocyte preincubation time, our findings addressed that 4 h or more preincubation time can influence the oocyte mRNA storage and ultimately leads to reduce oocyte fertilization and first cleavage rates. Besides, medium supplementation with myo-inositol could preserve the mRNAs inherited to the embryos and consequently improve fertilization and first cleavage developmental rates.
Key words: Oocyte preincubation time, Maternal effect genes, Fertilization potential, First cleavage rate, Myo-inositol supplement.

The original full text of this abstract has been published in Journal of Reproduction and Infertility 2020; 21(4): 259-268. http://dx.doi.org/10.18502/jri.v21i4.4330.
How to cite to this article: Mohammadi F, Ashrafi M, Zandieh Z, Najafi M, Niknafs B, Amjadi FS, Haghighi M. The effect of preincubation time and myo-inositol supplementation on the quality of mouse MII oocytes. Journal of Reproduction and Infertility 2020; 21(4): 259-268.

A-8
Melatonin prevents wistar rats testes from bleomycin, etoposide, and cisplatin (BEP) chemotherapy-induced reproductive toxicity: A biochemical, immunohistochemical and apoptosis-related genes based evidence
 
Moradi M¹, Goodarzi N², Faramarzi A^{3, 4}, Cheraghi H¹, Hashemian AH^{5, 6}, Jalili C^{4, 7}.
1.             Department of Clinical Sciences, Faculty of Veterinary Medicine, Razi University, Kermanshah, Iran.
2.             Department of Basic and Pathobiological Sciences, Faculty of Veterinary Medicine, Razi Universtiy, Kermanshah, Iran.
3.             Fertility and Infertility Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran.
4.             Department of Anatomical Sciences, Medical School, Kermanshah University of Medical Sciences, Kermanshah, Iran.
5.             Research Center for Environmental Determinants of Health (RCEDH), Health Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran.
6.             Department of Biostatistics, School of Health, Kermanshah University of Medical Sciences, Kermanshah, Iran.
7.             Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran.
Email: cjalili@kums.ac.ir
 
Background: Recently, the prevalence of testicular cancer, accounting for the most common cancer among young people of reproductive age (15-40 yr), has risen internationally. Bleomycin, Etoposide, and Cisplatin (BEP) chemotherapy has increased the 5-year survival rate of patients with testicular cancer at all stages of testicular germ cell tumors to 90-95%. However, nowadays there is growing concern that some cytotoxic regimens for cancer like BEP create a high incidence of male infertility and even long-term genotoxic effects, which emerge as a critical health issue. Melatonin is a well-known potent antioxidant with widespread clinical applications that recently has been giving increasing attention to its role in male sub/infertility.
Objective: In order to investigate the protective and alleviative effects of melatonin following BEPchemotherapy exposure on sperm characteristics and parameters, nitro-oxidative status, as well as histopathological, inflammatory, and apoptotic alternations in testes. Moreover, to elucidate whether exogenous melatonin attenuates BEP-induced damage in testicular cells and spermatogenesis in a dose-dependent manner?
Materials and Methods: 60 adult male Wistar rats (n = 10/group) were treated with one cycle of 21 days of 0.33 therapeutically relevant dose levels of BEP (0.5 mg/kg Bleomycin, 5 mg/kg Etoposide and 1 mg/kg Cisplatin) with or without melatonin. At the end of the study (day 35), body weight, testes weight, sperm parameters, testosterone hormone level, testicular histopathology, stereological parameters, testicular level of malondialdehyde, nitric oxide and total antioxidant capacity, the expression of apoptosis-associated genes such as Bcl2, Bax, Caspase3, p53 (Real-time PCR and immunohistochemistry), and the expression of pro-inflammatory cytokine TNF-α (immunohistochemistry) were evaluated.
Results: Our findings showed that melatonin restores the BEP-induced reduction in the body and testes weight (p < 0.05). The evaluation of quantitative analysis of the testes stereological procedures, QRT-PCR examination, and immunohistochemical  staining revealed that melatonin reversed the BEP-induced impaired spermatogenesis (p < 0.05). Furthermore, melatonin rectified BEP-induced disturbance on sperm count, motility, viability, and morphology. Additionally, co-administration of 10 and 20 mg/kg of melatonin could restore BEP-induced alteration in serum testosterone level. Moreover, melatonin enhanced the antioxidant status of the testis by elevating total antioxidant capacity and ameliorating malondialdehyde and nitric oxide levels. More notably, QRT-PCR examination indicated that melatonin therapy suppressed BEP-induced apoptosis by modulating apoptosis-associated genes such as Bcl-2, Bax, Caspase-3, p53 in the testis (p < 0.01). In this continuum, the co-administration of 10 and 20 mg/kg of melatonin with the BEP regimen decreased significantly the population of p53 and TNF-α positive cells by comparison to the BEP group. Also, melatonin with low and high doses could enhance the expression of Bcl-2 protein in spermatogenic cells line compared to the BEP-treated group.
Conclusion: This study demonstrated that melatonin protected testes against BEP-induced damage by preventing and ameliorating histopathological and stereological alterations, spermatotoxicity, nitro-oxidative stress, inflammation, and apoptosis. These findings can draw attention to the clinical application of melatonin and also suggest that melatonin may be an attractive agent for attenuating chemotherapy-associated male sub/infertility. This indolamine may also shorten the fertility recovery period in patients undergoing chemotherapy with the BEP regimen.
Key words: BEP chemotherapy, Melatonin, Sperm, Male infertility, Apoptosis.

The original full text of this abstract has been published in Biomedicine and Pharmacotherapy 2021; 138: 111481. https://doi.org/10.1016/j.biopha.2021.111481.
How to cite to this article: Moradi M, Goodarzi N, Faramarzi A, Cheraghi H, Hashemian AH, Jalili C. Melatonin protects rats testes against bleomycin, etoposide, and cisplatin-induced toxicity via mitigating nitro-oxidative stress and apoptosis. Biomedicine and Pharmacotherapy 2021 Jun 1; 138: 111481. 
 
A-9
Suppression of transforming growth factor-beta signaling enhances spermatogonial proliferation and spermatogenesis recovery following chemotherapy
 
Moraveji SF¹, Esfandiari F¹, Taleahmad S¹, Nikeghbalian S², Sayahpour FA¹, Masoudi NS³, Shahverdi A⁴, Baharvand H^{1, 5}.

1. Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
2. Shiraz Transplant Center, Namazi Hospital, Shiraz University of Medical Sciences, Shiraz, Iran.
3. Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
4. Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
5. Department of Developmental Biology, University of Science and Culture, Tehran, Iran.

Email: hossein.baharvand@gmail.com
 
Background: Spermatogonial stem cells hold great promise for fertility preservation in prepubertal boys diagnosed with cancer. However, the low number of Spermatogonial stem cells limits their clinical applications. Could small molecules (SM) are chemically synthesized molecules that diffuse across the cell membrane to specifically target proteins involved in signaling pathways, and studies have reported their ability to increase the proliferation or differentiation of germ cells.
Objective: SM which target (or modify) signaling pathways lead to increased proliferation of undifferentiated spermatogonia following chemotherapy?
Materials and Methods: In our experimental study, spermatogonia were collected from four brain-dead individuals and used for SM screening in vitro. For in vivo assessments, busulfan-treated mice were treated with the selected SM (or vehicle, the control) and assayed after 2 (three mice per group) and 5 weeks (two mice per group). We investigated the effect of six SM on the proliferation of human undifferentiated spermatogonia in vitro using a top-bottom approach for screening. We used histological, hormonal and gene-expression analyses to assess the effect of selected SM on mouse spermatogenesis. All experiments were performed at least in triplicate and were statistically evaluated by Student's t-test and/or one-way ANOVA followed by Scheffe's or Tukey's post-hoc.
Results: We found that administration of SB431542, as a specific inhibitor of the TGFb1 receptor (TGFbR1), leads to a two-fold increase in mouse and human undifferentiated spermatogonia proliferation. Furthermore, injection of SB to busulfan-treated mice accelerated spermatogenesis recovery as revealed by increased testicular size, weight and serum level of inhibin B. Moreover, SB administration accelerated both the onset and completion of spermatogenesis. We demonstrated that SB promotes proliferation in testicular tissue by regulating the cyclin-dependent kinase inhibitors 4Ebp1 and P57 (proliferation inhibitor genes) and up-regulating Cdc25a and Cdk4 (cell cycle promoting genes).
Conclusion: This is the first study to report acceleration of spermatogenesis recovery following chemotherapy by administration of a single SM. Our findings suggest that SB is a promising SM and should be assessed in future clinical trials for preservation of fertility in men diagnosed with cancer or in certain infertility cases (e.g. oligospermia).
Key words: Spermatogonial stem cells, Small molecules, Fertility preservation.

The original full text of this abstract has been published in Human Reproduction 2019; 34(12): 2430-2442. https://doi.org/ 10.1093/humrep/dez196.
How to cite to this article: Moraveji SF, Esfandiari F, Taleahmad S, Nikeghbalian S, Sayahpour FA, Masoudi NS, Shahverdi A, Baharvand H. Suppression of transforming growth factor-beta signaling enhances spermatogonial proliferation and spermatogenesis recovery following chemotherapy. Human Reproduction 2019; 34(12): 2430-242.
A-10
Mimicking the ovarian extracellular matrix, the role of natural polymrrs
 
Moshrefi M^{1, 2}, Jalali A¹, Ghasemi-Esmailabad S^{2, 3}, Samiei M^{1, 4}, Mollahoseini H^{1, 5}, Razban V^{6, 7}, Nikukar H^{1, 8}.

1. Medical Nanotechnology and Tissue Engineering Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
3. Abortion Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
4. Department of Marine Biology, Sciences and Research Branch, Islamic Azad University, Tehran, Iran.
5. Yazd Science and Technology Park, Medisa Polymer Ariya Company, Yazd, Iran.
6. Department of Molecular Medicine, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran.
7. Stem Cells Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
8. Department of Advanced Medical Sciences and Technologies, School of Paramedicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: habibnik@ssu.ac.ir
 
Background: Female infertility treatment has been entered into a new world, constructed by Regenerative Medicine, which tries to get mature female gamete by help of the Tissue Engineering. So, ovarian follicles growth, ovarian in vitro activation, and ovarian follicle & tissue culture are the processes that can develop under the progress of tissue engineering. Development of ovarian follicles depends on endocrine and paracrine signals, the follicles micro-environment and 3-dimensional architecture. So, mimicking the ovarian extra cellular matrix by ovarian tissue engineering is a possible approach in fertility treatment for patients with premature ovarian failure and onco-fertility patients who cryopreserved the ovarian cortical tissue.
Objective: The current study aimed to assemble the electrospinning scaffolds by natural polymers, for comparison with collagen, as the natural ovarian tissue polymer.
Materials and Methods: After Ethical Committee permission, a full thickness section of human ovary from surface to hilum, was prepared. After chopping and enzymatic digestion by collagenase, the isolated cells were cultured. Besides, the electrospinning scaffolds were fabricated, using the natural polymers including agarose, human serum albumin, chitosan, collagen, silk fibroin, gelatin and a synthetic polymer (poly lactic acid (PLA)). Electrospun blended scaffolds of natural polymers with PLA in ratio of 30/70; 50/50 and 70/30 were prepared. Chemical properties of manufactured scaffolds were characterized by Fourier-Transform Infrared Spectroscopy analysis. Also, water contact angle was measured to quantify the surface wettability of the prepared scaffolds. Scanning electron microscope images analyses were performed before and after cell culture and the porosity and the average of fiber diameter distribution was calculated by ImageJ software. Cytotoxicity was evaluated by MTT assay after 14 days cell culture. Also, cell morphology and growth pattern was followed by hematoxylin and eosin staining.
Results: The blend of all natural polymers with PLA led to the fiber formation in electrospinning process, except for chitosan 70% + PLA 30%. Also, electrospinning for all the polymers separately led to fiber formation except chitosan and albumin. Therefore, 21 variables of electrospun scaffolds were assembled and Fourier-Transform Infrared Spectroscopy results confirmed the polymer accuracy. The results of fiber diameter diversity showed that the thickest fibers were related to the blended electrospun scaffold (50% agarose + 50% PLA) followed by (70% collagen + 30% PLA) (≥ 200 nm). The other scaffolds fibers were below ≤ 150 nm. Compared to dish culture plate, MTT assay test after 4, 8 and 14 days cell culture on the scaffolds showed that the blends of (70% gelatin + 30% PLA) followed by (70% collagen + 30% PLA) led to the highest cell proliferation and the lowest cells toxicity, respectively.
Conclusion: Gelatin can be replaced by collagen, as the native extra cellular matrix of the ovary. Our results showed that gelatin, as an accessible natural polymer provided higher cell proliferation and lower fiber diameter than collagen. It is more accessible and cost effective with lower cell toxicity which makes it an optimized polymer for ovarian tissue engineering instead of collagen.
Key words: Artificial ovary, Extracellular matrix, Natural polymers, Scaffolds, Poly lactic acid.
 
A-11
Transcriptomic alternation of chemokines secreted from fallopian tube epithelial cells in response to spermatozoa
 
Mousavi SO¹, Mohammadi R¹, Amjadi F², Eslami M³, Aflatoonian R¹.

1. Department of Endocrinology and Female Infertility, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
2. Shahid Akbarabadi Clinical Research Development Unit (ShACRDU), Iran University of Medical Science, Tehran, Iran.
3. Applied Biotechnology Research Center, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran.

Email: r.aflatoonian@gmail.com
 
Background: Immunological response of female reproductive system to the allogenic spermatozoa is very essential for a successful fertilization and pregnancy. Fallopian tubes are the focus of this investigation since they are places in which spermatozoa are stored for a period until oocytes are ready for fertilization. Chemokines have been shown to play important roles in reproductive immunology by their chemo-attraction potential and inflammatory function. Previous studies also showed that specific chemokines can increase the survival chance of spermatozoa by modulating female immune system.
Objective: For a better understanding of how chemokines contribute with the maternal immune system in the presence of spermatozoa, we evaluated transcriptional changes of different chemokines from fallopian tube cell line in the presence of spermatozoa by PCR-array.
Materials and Methods: Semen samples were collected from 10 healthy donors who had at least one child. Samples were analyzed according to the WHO standard. To investigate the impact of spermatozoa on chemokines’ expression from epithelial cells, the fallopian tube cell line (OE-E6/E7) was co-cultured with the spermatozoa for 24 hr. The cell line without any spermatozoon was analyzed as the control group. After the co-incubation period, RNA extraction was done from washed cells. cDNA was synthetized and chemokines’ expression were evaluated by PCR-array. Finally, Independent sample t test was applied to compare differences between the groups. Furthermore, IL-8 which had the most expression compared to other chemokines is evaluated in the culture medium by ELISA.
Results: Data analysis indicated that the spermatozoa resulted in down regulation of chemokines. It has been shown that CX3CL1 which involves in T cell migration, and inflammatory chemokines such as IL-8, CXCL9 and CXCL13 were significantly decreased in the presence of the spermatozoa. IL-8 concentration in the case group was also lower than the control. Furthermore, CCL chemokines with the role in migration of inflammatory cells to the target tissue were significantly down regulated. These chemokines alternation can cause higher survival chance of the spermatozoa by preparing an anti-inflammatory condition in the fallopian tubes.
Conclusion: Compatible with previous studies our results have shown that spermatozoa can adapt to the immune system of female reproductive tract by regulating specific chemokines expression. Chemokines are appeared to be essential for preparing a safe microenvironment in the fallopian tubes for the spermatozoa.
Key words: Chemokine, Fallopian tube, Spermatozoa, Fertilization, PCR array.
 
A-12
The study of embryonic causes of recurrent pregnancy loss in couples with consanguineous marriage and normal karyotype with a specific approach to determine the importance of single genes
 
Nazari T¹, Ghasemi N², Ebrahimie E³, Ebrahimi A⁴, Talebi S⁵, Tavakkoly Bazzaz J¹, Kalantar SM⁶.

1. Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
2. Abortion Research Centre, Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
3. Department of Genetics and Evolution, School of Biological Sciences, The University of Adelaide, Adelaide, Australia.
4. Genia Medical Genetic Lab, Tehran, Iran.
5. Department of Medical Genetics, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
6. Department of Medical Genetics, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: smkalantar@yahoo.com
 
Background: Pregnancy loss is a significant health concern, especially in developing countries. Nearly 3-5% of couples trying to have children experience recurrent pregnancy loss (RPL). Unfortunately, advancing maternal age is highly associated with miscarriage while the available reproductive years are shortened, therefore determining why miscarriage occurs and how to prevent further miscarriages has become a major clinical and research focus. It has been estimated that the cause remains unexplained in more than 50% of cases, which strongly suggests that genetic factors may contribute towards the phenotype. This might be considered the fact that hundreds of genes and several critical pathways are involved in each physiological step necessary for guaranteeing reproductive success.
Objective: Mendelian forms of embryonic lethality offer a window into the essential genetic components of early embryonic development in humans. The study hypothesis was that exome sequencing can identify genetic causes of idiopathic recurrent pregnancy loss (RPL), which will further our understanding of human development at a molecular level.
Materials and Methods: This study involved 10 consanguineous couples having suffered at least 3 consecutive embryonic losses. Patients having risk factors such as abnormal karyotype, infectious disease during pregnancy, metabolic, autoimmune, endocrine disease, and uterine anomalies were excluded from the study. DNA was extracted from the tissue sample of ten aborted fetuses (probands) from ten different families with a history of idiopathic recurrent pregnancy loss. Parental peripheral blood samples were collected for confirmatory analysis and follow-up testing. Whole exome sequencing (WES) was performed using illumine HiSeq 2000 platform. Cytoscape 3.7.2 and BINGO were used for pathway and biological enrichment analysis of putatively pathogenic variants in miscarriages. All variants identified by exome sequencing were verified by Sanger sequencing in all parents.
Results: We were able to identify 32 variants (7 pathogenic, 9 likely pathogenic, and 16 VOUS) of which three genes are already known to be involved in lethal recessive disorders. These genes were compatible with the clinical phenotypes. In cases 1, 2,4 pathogenic variants were identified in know genes (CHRNG, RYR1) responsible for disorders with a clinical description that correlated with the phenotypic description. Of fetuses, in case 3,8,6 novel variants were identified in MYH3, ERBB3, FRAS1 responsible for muscular disorders and Fraser syndrome. Bioinformatics analysis of genes with mutation showed enrichment in biological processes of importance for embryonic development e.g. Fibrin clot formation complement, coagulation cascades, and Striated muscle contraction/muscle contraction, actin-myosin filament sliding. Variants with potential diagnostic value were reported to the patients referring physicians for genetic counseling and further diagnostic and reproductive action.
Conclusion: Next-generation sequencing (NGS) has been reported as being a useful tool for identifying variants in genes related to rare disorders leading to RPL. It has also helped to identify variants related to fetal molecular pathways in pregnancies having unexplained embryonic lethality or unexplained fetal malformations. This approach thus helps, genetic counseling regarding lethal fetal disorders of high-risk families and preimplantation genetic diagnosis. Future efforts should be directed towards increasing the number of sequencing families with RPL.
Key words: Multiple pterygium syndromes, Whole-exome sequencing, Recurrent pregnancy loss, Thrombophilia.
 
A-13
Design and fabrication of a microfluidics device for sperm sorting application
 
Rafizadeh Tafti S¹, Azimzadeh M¹, Nabi A².

1. Medical Nanotechnology and Tissue Engineering Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: m.azimzadeh@ssu.ac.ir
 
Background: In vitro sperm selection has been of great interest in order to optimize the selection process based on biological parameters of sperm. On the other hand, microfluidics has recently cast light on how deep human being can sort and manipulate cells and fluids, respectively. Hence, using microfluidic technology to design and fabricate a bio-chip for sperm selection seems to be a promising bet. The insane biocompatibility of the microfluidic polymers as well as a wide customizability enriches us with a variety of design and mechanism selections.
Objective: Hereby, we discuss the design, optimization, fabrication of a microfluidic biochip based on rheotaxical capability of sperms, as well as their attraction to electrostatic charge. Our chip will enable us to have a controlled flow of motile sperms attracted to electrostatic charge and guiding the cells towards groups of selection.
Materials and Methods: The mold for the chip was in Silicon and fabricated using e-beam layer deposition, photolithography and reactive ion etching. Then, polydimethylsiloxane fabricated chips were made and patched by plasma to glass to build up the chip. The system is then connected to a syringe pump and monitored by an inverted microscope.
Results: Our chip, showed high biocompatibility and was able to selects a sub population of vital sperms with a greatly higher motility profile. We also provided evidence, that electrostatic charge has the capability to stimulate the sperm cells towards target. The suggested design provides great quality sperm selection in a quicker time manner with high throughput, besides being feasible in costs.
Conclusion: Based on the results, the fabricated chip showed promise to be used in future medical applications, after taking necessary tests.
Key words: Sperm, Sorting, Microfluidics.
 
A-14
Cumulus cells conditioned medium facilitates germ cell development from human embryonic stem cells
 
Tahajjodi S¹, Farashahi-Yazd E¹, Agha-Rahimi A², Akyash F¹, Hajizadeh Tafti F¹, Aflatoonian R³, Aflatoonian B¹.

1. Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
3. Department of Endocrinology and Female Infertility, Reproductive Biomedicine Research Centre, Royan Institute for Reproductive Biomedicine, Tehran, Iran.

Email: b.aflatoonian@ssu.ac.ir
 
Background: Human embryonic stem cells (hESCs) can differentiate to germ cells as confirmed using gene expression assessments. Cumulus cells are physically closest cells to the developing oocyte and they have positive effects on oocytes maturation by secreting some factors that have a crucial role in the process of oogenesis. Thus, in vitro differentiation of embryonic stem cells may also be affected of cumulus-secreted factors.
Objective: The aim of this study is assessment the effect of the cumulus cells conditioned medium (CCCM) on differentiation of hESCs to female germ cells.
Materials and Methods: Embryoid bodies (EBs) from Yazd4-hESCs were formed and cultured for 14 days into 4 different conditions: 1) spontaneously differentiation in EB medium (SD-EB), 2) treated with 40% CCCM (CCCM-EB), 3) spontaneously differentiation in 40% DMEM+20% FBS (SD-DM), and 4) treated with 40% CCCM in DMEM+20% FBS (CCCM-DM). Expression of pluripotency and germ cells genes were examined in EBs from each group by RT-qPCR at the time of days 0, 4, 7 and 14. In addition, immunofluorescent (IF) staining was done for pluripotency and germ cells markers.
Results: RT-qPCR data revealed that CCCM-EB and CCCM-DM had a significant increase in differentiation of female germ cell from hESCs than SD-EB and SD-DM. On the other hand, comparison between basal media revealed that EB medium is a better medium than DMEM+20% FBS for female germ cell development from hESCs. Localization of the germ cells within the cultures was detected using IF for TRA-2-49, SSEA1 and VASA antibodies in all groups.
Conclusion: Cumulus cells conditioned medium supports female germ cell development from hESCs assessed by gene expression profile. Also, EB medium as basal medium has better impact on differentiation induction.
Key words: Cumulus cells, Conditioned medium, In vitro gametogenesis, Human embryonic stem cells, Female germ cells.

The original full text of this abstract has been published in Int J Reprod BioMed 2020; 18: 1-10. https://doi.org/10.18502/ijrm.v18i1.6189. How to cite to this article: Tahajjodi SS, Farashahi Yazd E, Agha-Rahimi A, Aflatoonian R, Khalili MA, Mohammadi M, Aflatoonian B. “Biological and physiological characteristics of human cumulus cells in adherent culture condition. Int J Reprod BioMed 2020; 18: 1-10.

Oral Presentations
 
9^th Yazd International Congress and Student Award on Reproductive Medicine
 
O-1
The relationship between coronavirus disease 2019 in pregnancy with maternal and fetal outcomes: An analytical cohort study
 
Samadi P¹, Alipour Z², Eskandari N², Ghaedrahmati M³, Vahedian M⁴, Khalajinia Z², Mastani JA⁵.

1. Department of Midwifery and Reproductive Health, School of Nursing and Midwifery, Tehran University of Medical Sciences, Tehran, Iran.
2. Department of Midwifery, School of Nursing and Midwifery, Qom University of Medical Sciences. Qom, Iran. 
3. Narges Social Security Organization, Dorood, Lorestan.
4. Faculty of Medical Sciences, Qom University of Medical Sciences, Qom, Iran.
5. Student Research Committee, School of Nursing and Midwifery, Qom University of Medical Sciences, Qom, Iran.

Email: kanom_alipour@yahoo.com
 
Background: Coronavirus disease 2019 (COVID-19) is a type of pneumonia, which is rapidly increased reports of death and confirmed complications. Limited data were available about COVID-19 during pregnancy.
Objective: To assess the relationship between epidemiological and clinical features of coronavirus disease 2019 in pregnancy with maternal and fetal outcomes.
Materials and Methods: This analytical and retrospective cohort study, conducted on all pregnant women who confirmed cases of COVID-19 in Nekouei-Hedayati-Forghani Hospital in Qom, from February 1, 2019, to September 15, 2020. All epidemiological and clinical features collected from pregnant women’s medical records. A logistic regression model used to determine covid-19 in pregnancy associated with maternal and neonatal outcomes.
Results: The most common symptoms reported by pregnant women with COVID-19 were shortness of breath 60%, dry cough 59% and fever 42%. After adjusting adjusted by the potential confounding factors, COVID-19 in pregnancy was associated with a significantly higher risk of admission to the intensive care unit (OR = 6.16, 95% CI = 1.23-31), cesarean section (OR = 0.45, 95 CI = 0.25-1.03); preterm birth (OR = 3.01, 95% CI = 1.4-6.54), fetal distress (OR = 5.7, 95% CI = 2.13-15.59), and the neonatal intensive care unit admissions(OR = 3.04, 95% CI = 1.21-7.70).
Conclusion: The results show that COVID-19 associated with adverse maternal and fetal outcomes such as admission to the Intensive care unit, cesarean section, fetal distress, preterm birth and neonatal intensive care unit admissions.
Key words: Coronavirus pneumonia, Epidemiological characteristics, Maternal outcomes, Fetal outcomes, Retrospective study.
O-2
Evaluation of the effects of human bone marrow mesenchymal stem cells conditioned medium on growth and maturation of mouse ovarian follicle after vitrification
 
Tork S, Molaeeghaleh N, Abdi S, Movassaghi S, Sharifi Z.
Department of Anatomical Sciences and Cognitive Neuroscience, Faculty of Medicine, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran.
Email: sh.abdi@iautmu.ac.ir
 
Background: In vitro culture of isolated follicles of cryopreserved ovaries can be proposed as a fertility preservation method. MSCs secreting various levels of growth factors and are an appropriate option for enriching the follicle culture media.
Objective: The purpose of this study was to evaluate the effect of human bone marrow mesenchymal stem cells derived conditioned medium (hBMSCs-CM) on growth and maturation of mouse ovarian follicle, and embryonic development after vitrification.
Materials and Methods: hBMSCs were cultured and the collected conditioned medium was concentrated and stored. The collected ovaries from Two-wk-old mice were divided randomly into vitrified and non-vitrified groups. Preantral follicles of both groups were isolated and cultured in α-MEM enriched with ITS and FBS supplemented with different concentrations of CM (2.5, 5, and 7.5%) for 12 days. During the culture period, survival rate, follicular maturation, follicular diameter, and levels of 17 βestradiol secretion were evaluated. In vitro fertilization and embryonic development were observed after culture.
Results: The survival rate, antrum formation, and oocyte maturation were higher in 7.5% CM subgroups than 2.5 and 5% CM in both vitrified and non-vitrified groups. Also, the follicle diameter in 7.5% CM was higher than other subgroups of both groups on day 4. Higher percentages of fertilization and embryo development were seen in 7.5% CM subgroups of the non-vitrified and vitrified group. Also, higher hormone secretion was observed in 7.5% CM subgroup in both vitrified and non-vitrified groups.
Conclusion: The present study suggests that the addition of 7.5% CM to mice ovarian preantral follicle culture media enhances follicle growth and oocyte maturation.
Key words: Vitrification, Human bone marrow mesenchymal stem cells, Conditioned medium, Follicle.
 
O-3
The effect of Diazinon on cell proliferation and apoptosis in testicular tissue of rats and the protective effect of vitamin E
 
Rahimi Anbarkeh F¹, Nikravesh MR¹, Jalali M¹, Sadeghnia HR², Sargazi Z³.

1. Department of Anatomy and Cell Biology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
2. Department of Pharmacology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
3. Neuroscience Research Center, Torbat Heydariyeh University of Medical sciences, Torbat Heydariyeh, Iran.

Email: nikraveshmr@mums.ac.ir
 
Background: Diazinon (DZN) is an organophosphate pesticide, and nowadays this pesticide is mostly used in agriculture. In this study, we analyzed the effects of DZN and vitamin E (Vit E) on apoptosis and the proliferation of germ cells in rat testis.
Objective: This study aimed to examine the effect of diazinon on apoptosis and the proliferation of germ cells in rat testis and the protective effect of vit E.
Materials and Methods: In this experimental study, 30 male Wistar rats were divided into five groups (n = 6 per group) consisting of control, sham (received olive oil), experimental group I (60 mg/kg DZN), experimental group II (60 mg/kg DZN and 200 mg/kg Vit E), and experimental group III (200 mg/kg Vit E). After six wks, left testis of rats was removed for the detection of proliferative cell nuclear antigen (PCNA) and terminal deoxynucleotidyl transferase end-labeling (TUNEL).
Results: Compared with the control group, DZN in the experimental group I decreased the number of PCNA positive cells and increased the number of TUNEL-positive cells (p < 0.001). Vit E improved detrimental changes by the decrease in the rate of apoptosis and the increase in the proliferation of testicular germ cells (p < 0.001).
Conclusion: Vit E can decrease the number of TUNEL-positive cells and increase the number of PCNA-positive cells by the neutralization of the toxicity caused by DZN in the testicular tissue.
Key words: Apoptosis, Diazinon, Proliferation, Testis, Vitamin E.

The original full text of this abstract has been published in Int J Fertil Steril 2019; 13(2): 154-160. doi: https://doi.org/10.22074/ijfs.2019.5612.
How to cite to this article: Rahimi Anbarkeh F, Nikravesh MR, Jalali M, Sadeghnia HR, Sargazi Z. The effect of diazinon on cell proliferation and apoptosis in testicular tissue of rats and the protective effect of vitamin E. Int J Fertil Steril 2019; 13(2): 154-160.

O-4
The effect of platelet lysate on mouse ovarian tissue following auto- transplantation: A biochemical analysis
 
Sanamiri K¹, Soleimani Mehranjani M¹, Shahhoseini M², Shariatzadeh S¹.

1. Department of Biology, Faculty of Science, Arak University, Arak, Iran.
2. Department of Genetics, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

Email: m-Soleimani@araku.ac.ir
 
Background: Platelet lysate (PL) has an increasing role in tissue engineering and regenerative medicine. During the early stages of ovarian transplantation, free radical production and inflammation can cause the loss of large numbers of immature follicles. PL as a condensed collection of platelet growth factors and cytokines, is obtained by lysing the platelet through temperature-shock and contains antioxidant and anti-inflammatory factors that are useful for improving ovarian graft survival.
Objective: We investigated the effect of intraperitoneal injection of PL on the transplanted mouse ovarian tissue.
Materials and Methods: 18 Naval Medical Research Institute (NMRI) mice (4-5 wk old) were divided into 3 groups (n = 6): Control, autograft and autograft + PL (5 ml/kg daily intrapritoneal injections). After 7 days, serum concentrations of total antioxidant capacity, malondialdehyde (MDA), tumor necrosis factor alpha (TNF-α), IL-6, and IL-10 were evaluated. Data were analyzed using one-way analysis of variance (ANOVA) and Tukey’s test, differences were considered significant at p < 0.05.
Results: The serum concentrations of IL-6, TNF-α and MDA increased significantly in the autograft group compared to the control group whereas these parameters reduced significantly in the autograft + PL group. Total antioxidant capacity and the serum level of IL-10 also reduced significantly in the autograft group when compared to the control while it significantly increased in the autograft + PL group.
Conclusion: Our study provides the first evidence that treatment with PL induces protective responses through reducing oxidative stress and inflammation after transplantation of mouse ovarian tissue.
Key words: Platelet lysate, Ovarian tissue, Transplantation, Oxidative stress, Inflammation.
 
O-5
Genetics and transcriptome profile of cryopreserved human sperm associated antigens (SPAGs)
 
Faraji S^{1, 2}, Rashki Ghaleno L², Hezavehei M², Sharafi M³, Totonchi M^{2, 4}, Shahverdi A^{1, 2}, Fathi R².

1. Department of Molecular and Cellular Biology, Faculty of Basic Science and Advanced Technologies in Biology, University of Science and Culture, ACECR, Tehran, Iran.
2. Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
3. Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
4. Reproductive Epidemiology Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

Email: rfathi79@royaninstitute.org
 
Background: Human sperm associated antigens (SPAGs), formerly known as sperm membrane protein, are eighteen-type proteins that some types of them, have a momentous role in various biological functions especially fertility outcome. The molecular weight range allotted to SPAG proteins is between 24-71 k Da. The role of 8 types of these SPAGs (SPAG 1, 2, 6, 8, 9, 12, 13, 15) has been confirmed in infertility. Thus, any damage in quoted SPAGs can lead to infertility. In spite of favorable aspect of cryopreserved sperm for assisted reproductive technology, detrimental impact of freezing on cells has been quoted in many studies. In this regard, cryopreservation has an unfavorable effect on sperm quality perhaps via perturbation of SPAGs expression.
Objective: This study aimed to appraise the impact of rapid freezing on the gene expression of SPAGs in normal human spermatozoa.
Materials and Methods: The semen samples were collected from 12 normospermic individuals. All twelve normo-ejaculated samples were prepared via density gradient centrifuge and thereupon, the aliquots of motile sperms were divided into two fresh and freeze groups. Afterwards, sperm samples were mixed (1:0.7) with spermfreeze® cryoprotectant for 10 minutes. Then the mixture was loaded into cryotube and freezed with rapid freezing procedure. After three days of freezing at -196°C, the specimen were thawed in tap water for 5 min and incubated for 2 hr at recovery time in a CO₂ incubator. RNA from sperm was extracted with TRIzol. After synthesis of cDNA, SPAGs gene expression analysis was performed using Real-time PCR.
Results: The results of statistical analysis showed a decrease in the gene expression of SPAG5, SPAG7, SPAG12 (SNU13/ NHP2L1) during rapid freezing procedure. The results are significant at the p ≤ 0.05 level. No significant reduction in the expression level between fresh and freeze group was found in remained SPAGs.
Conclusion: The results pointed out that cryopreservation procedure could negatively affect gene expression of some SPAGs in human spermatozoa. Hence, alteration of SPAGs expression could offer new suggestions to evaluate probable molecular correlations between freezing and increased failure rate of in vitro fertilization and intracytoplasmic sperm injection.
Key words: Antigen, Cryopreservation, Fertility, Human, Sperm.
 
O-6
Production of recombinant human leukemia inhibitory factor protein and its immunologic and anti-fertility impacts as a contraceptive candidate vaccine in mice model
 
Zare F¹, Amiri M², Hadinedoushan H¹, Dehghan-Manshadi M¹, Mansouri F², Fesahat F¹, Mirzaei F³, Saboor Yaraghi A².

1. Reproductive Immunology Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.
3. Department of Laboratory Sciences, School of Paramedicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: asaboor@tums.ac.ir
 
Background: Contraceptive vaccines are one of the methods studied to prevent fertility in mammals. Various factors are involved in the establishment and maintenance of the pregnancy and can be targeted for antifertility vaccine design. The human leukemia inhibitory factor (hLIF) is considered as a cytokine of the interleukin-6 family. LIF is also involved in the embryo implantation process.
Objective: The production and functional competence of the LIF, as the immunocontraceptive vaccine in Balb/c mice, was investigated in this experimental study.
Materials and Methods: Recombinant hLIF (rhLIF) was generated in a variety of host-vectors system. The protein expression rate and functional activity of rhLIF were assessed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and tetrazolium reduction assay, respectively. The production and characterization of Rabbit polyclonal antibody (pAb) to rhLIF was performed applying enzyme-linked immunosorbent assay and western blot techniques. The Balb/c mice were classified into two study groups. In group 1, each mouse was intraperitoneally inoculated by purified rabbit anti-rhLIF in 3^th day and day 4 following vaginal plaque observation; after sacrificing on day 7, the number of implantation sites was quantified. Mice in second group were subdivided into two vaccinated and controls groups The rhLIF protein as well as phosphate buffer saline was emulsified with Freund's adjuvant and injected into both vaccinated and control groups, respectively. The inhibitory rate of implantation was investigated in the uterine of mice. The secreted levels of interferon-γ and interleukin-4 were determined in cultured splenocyte of mice induced by rhLIF. Also, the mRNA levels of immune responsive gene 1 (IRG-1), cochlin (COCH), amphiregulin (Ar), and heparin-binding EGF-like growth factor (HB-EGF) genes were evaluated. The inhibition of fertility after delivery, reversibility of immune response against rhLIF, and survival rate of mice were assessed.
Results: Our data showed that pET32b/hLIF and pColdI/hLIF vectors could successfully express rhLIF in all hosts. The produced rhLIF was functionally active and the produced anti-rhLIF pAb could specifically bind to commercial rhLIF and native LIF extracted from mouse uterus. Passive immunization outcomes indicated that anti-rhLIF antibody entirely inhibited the fertility potential in all vaccinated mice compared to controls. Active immunization of Balb/c mice with rhLIF led to the implantation and fertility reduction rate up to 80.49% and 75%, respectively. All mice produced a high amount of anti-rhLIF antibodies in both serums and vaginal fluids wash after 16 weeks; while, these antibodies were disappeared from vaginal fluid washes six months later. The findings of splenocyte stimulation with hLIF demonstrated a significant increased level of both cytokines in vaccinated mice compared to the controls. A significant decreased gene expression of IRG-1, Ar, and HB-EGF was observed in vaccinated group compared to control group; however, the mRNA level of COCH gene showed no significant change.
Conclusion: rhLIF could inhibit pregnancy in a high rate of female mice. The immunization of female Balb/c mice with rhLIF prevented fertility and the gene expression associated with rhLIF. To investigate the side effects of this vaccine in a wide range, further studies are needed.
Key words: Leukemia inhibitory factor, Contraception, Vaccine, Active immunization, Mice.

The original full texts of this abstract have been published in:

• Protein Expression and Purification 2020; 174: 105684. https://doi.org/10.1016/j.pep.2020.105684.
• Journal of Reproductive Immunology 2020 Nov 1; 142: 103195. https://doi.org/10.1016/j.jri.2020.103195.

How to cite to thess articles:

• Zare F, Saboor-Yaraghi AA, Hadinedoushan H, Dehghan-Manshadi M, Mirzaei F, Mansouri F, Amiri MM. Production and characterization of recombinant human leukemia inhibitory factor and evaluation of anti-fertility effects of rabbit anti-rhLIF in Balb/c mice. Protein Expression and Purification 2020; 174: 105684.
• Zare F, Amiri MM, Hadinedoushan H, Dehghan-Manshadi M, Mansouri F, Fesahat F, Saboor-Yaraghi AA. Contraceptive and molecular function of a novel recombinant vaccine based human leukemia inhibitory factor on Balb/c mice: An experimental in vivo study. Journal of Reproductive Immunology 2020 Nov 1; 142: 103195.

 
O-7
Vitamin C and E supplementation effects on secretory and molecular aspects of vascular endothelial growth factor derived from peritoneal fluids of patients with endometriosis
 
Ansariniya H^{1, 2}, Hadinedoushan H², Javaheri A³, Zare F².

1. Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
2. Reproductive Immunology Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
3. Department of Obstetrics and Gynecology, Faculty of Medicine, Shahid Sadughi University of Medical Sciences, Yazd, Iran.

Email: hhadin@ssu.ac.ir
 
Background: Endometriosis is an extremely heterogeneous disease and affects about 10% of the female population during their reproductive years. Recent studies showed that endometriosis is an angiogenesis-dependent disease. Peritoneal macrophages are a well-characterised source of vascular endothelial growth factor (VEGF).
Objective: The aim of this study was to determine the VEGF gene expression and production in peritoneal macrophages of patients with endometriosis under the effects of vitamins C and E in comparison with control.
Materials and Methods: The lab trial study carried out on 50 patients undergoing laparoscopy and peritoneal fluid samples were collected from them. We compared the VEGF gene expression and production in peritoneal macrophages among groups by using real-time polymerase chain reaction and enzyme-linked immunosorbent assay methods, respectively.
Results: Our results showed that gene expressions influenced by vitamin C increased in different concentrations and incubation times, except for the incubation time after 48 h. In the case of vitamin E, this was evident with the exception of vitamin E 50 μM after 24 h and vitamin E 100 μM after 48 h.
Conclusion: Our findings indicated that vitamin C and E in different concentrations and incubation times altered VEGF gene expression in the peritoneal macrophages but they had not affected on VEGF productions.
Key words: Endometriosis, Vascular endothelial growth factor, Vitamin C, Vitamin E.

The original full text of this abstract has been published in Journal of Obstetrics and Gynaecology 2019; 39(8): 1137-1142. https://doi.org/10.1080/01443615.2019.1601167.
How to cite to this article: Ansariniya H, Hadinedoushan H, Javaheri A, Zare F. Vitamin C and E supplementation effects on secretory and molecular aspects of vascular endothelial growth factor derived from peritoneal fluids of patients with endometriosis. Journal of Obstetrics and Gynaecology 2019; 39(8): 1137-1142.

O-8
LncRNA XIST/ miR-132/ HMGA2 axis modulate Insulin Resistance in PCOS: A molecular signature for prediction
 
Rafat M, Malekzadeh K.
Department of Medical Genetics, Faculty of Medicine, Hormozgan University of Medical Sciences, Bandar Abbas, Iran.
Email: keyanoosh@gmail.com
 
Background: Polycystic ovary syndrome (PCOS) is a common heterogeneous endocrine disorder. Studies showed that insulin resistance (IR) appeared in only 60% women with PCOS and seems to be independent of obesity.
Objective: It was hypothesized that dysregulation of HMGA2/miR-132-3p/ lncRNA XIST axis may correlate with IR.
Materials and Methods: In this case-control study, four groups participated including 20 healthy controls, 30 having only PCOS, 20 only IR+ and 30 PCOS+/IR+. None of them suffered from any syndroms, no pregnancy and no history of hormonal therapy. Real‐Time PCR, ELISA and chemilumenisce recruited to assess the level of studied factors.
Results: The 87% and 63% reduction in level of lncRNA XIST and HMGA2 observed in IR+, but interestingly, both showed significant increase more than 3.3 fold in groups with PCOS+. Conversely, miR-132 expression levels increased about 3.3 and 4.0 fold in groups of PCO+/IR+ and IR+, respectively. The expression of miR-132 in PCOS+ group was significantly reduced by 98% compared to the normal group. HMGA2 is post-trancrptionally targeted and controlled by miR-132, which can explain down-regulation HMGA2 in IR. In the other side HMGA2 function in cell proliferation and its over-expresion can justify in PCOS. Taken together, these results introduced another molecular mechanism involved in onset of IR in PCOS. ROC curve analysis showed that HMGA2/miR-132/lnc-XIST have 100% sensitivity and specificity to predicate IR in PCOS patients.
Conclusion: lncRNA XIST/miR-132/HMGA2 axis can be candidated as a panel of differentiative signature to predict IR not only in women with PCOS, but also could be applicable even in healthy individuals.
Key words: PCOS, IR, HMGA2, miR-132, lncRNA XIST.
 
O-9
The correlation between varicocele and unfolded protein response occurred in ER stress
 
Hosseini M¹, Shaygannia E¹, Rahmani M¹, Eskandari A¹, Ahmadzadeh Golsefid A¹, Tavalaee M¹, Gharagozloo P², Drevet JR³, Nasr-Esfahani MH¹.

1. Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
2. Celloxess LLC, 830 Bear Tavern Road, Ewing, NJ 08628, USA.
3. Université Clermont Auvergne, CNRS, Inserm, GReD, F-63000 Clermont-Ferrand, France.

Email: mahshid.hosseini92@gmail.com
 
Background: Excessive reactive oxygen species generation plays a crucial role in male infertility, especially varicocele. One of the most cardinal pathways that defend cells against this destructive situation is the unfolded protein response (the so-called UPR/ER stress response). The UPR/ER is triggered by aggregation of unfolded/misfolded proteins in the Endoplasmic Reticulum (ER) lumen, leading to detach ER chaperons from ER membrane including inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), and the PKR-like endoplasmic reticulum kinase (PERK). In the face of stress conditions, BiP is detached from membrane sensors and the three mentioned proteins are transiently activated to modify cell survival signals. Eventually, should the stress condition prolong, apoptosis is prompted by specific inducers such as the Jun-kinase/caspase-3 pathway.
Objective: The assessment of UPR/ER pathways in a VCL-induced rat model to find out the plausible role of UPR/ER stress response in varicocele condition.
Materials and Methods: Varicocele induction was surgically performed on ten 8-wk-old adult male Wistar rats, as varicocele group, and ten rats were considered as a control-sham group. After conducting sperm function tests, the expression of BiP, Caspase-3, Bax, Bak, Bim, Bcl2, XBP1, and NRF2 using Real-time PCR, and expression of p-JNK, CHOP, and NRF2 using Western blot were assessed. The data between the two groups were compared with the Independent t test, and p-value lower than 0.05 was considered statistically significant between the two groups.
Results: To assess the activation of UPR/ER pathways in VCL testis, the BiP/GRP78/HSAP5 protein level was evaluated, and no difference in the expression of BiP in VCL testis tissue compared with control group was indicated. By prolonging UPR response, IRE1 pathway induces apoptosis by activation of ER-associated protein degradation pathway (ERAD), which is accomplished by XBP1s, and stimulation of JNK/p-JNK pathway by downregulation of Bcl2 and upregulation of Bax and Bak, leading to activation of Caspase-3. Increased level of XBP1s mRNA, phospho-JNK (p = 0.04) and caspase-3 transcript (4.84 ± 0.64 versus 1.14 ± 0.14, p = 0:03) in the VCL testis tissue, was a sign of activation of the JNK pathway.
Conclusion: Ample evidence has shown that in the UPR/ER stress response, the first pathway to be activated is PERK, then ATF6, and finally IRE1. As CHOP and NRF2 protein content were no higher in VCL testicular extracts compared to control testis, it is clear that late apoptosis pathway, PERK/ATF4/NRF2/CHOP, has not activated. Activation of the p-JNK-induced Caspase-3 apoptotic signal is also suggested that we are in the late stages of the UPR/ER stress response. The UPR/ER response is certainly activated in the VCL testis by activation of the IRE1/JNK pathway.
Key words: Varicocele, Endoplasmic reticulum stress, Unfolded protein response, ROS.

The original full text of this abstract has been published in Oxidative Medicine and Cellular Longevity 2020. https://doi.org/10.1155/2020/5909306.
How to cite to this article: Hosseini M, Shaygannia E, Rahmani M, Eskandari A, Golsefid AA, Tavalaee M, Gharagozloo P, Drevet JR, Nasr-Esfahani MH. Endoplasmic reticulum stress (ER stress) and unfolded protein response (UPR) occur in a rat varicocele testis model. Oxidative Medicine and Cellular Longevity 2020; Article ID 5909306.

O-10
A detailed study in adenomyosis and endometriosis; evaluation of the rate of coexistence between uterine adenomyosis and DIE according to imaging and histopathology findings
 
Alborzi S¹, Askary A², Khorami F³, Poordast T^{2, 4}, Abdulwahid Hashim Alkhalidi B³, Hamedi M³, Alborzi S⁵, Raeisi Shahraki H⁶.

1. Department of Obstetrics and Gynecology, School of Medicine, Laparoscopy Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
2. Department of Obstetrics and Gynecology, School of Medicine, Infertility Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
3. Department of Obstetrics and Gynecology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
4. Obstetrics and Gynecology Ward, Faghihi Hospital, Zand Blvd, Shiraz, Iran.
5. School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
6. Department of Epidemiology and Biostatistics, Faculty of Health, Shahrekord University of Medical Sciences, Shahrekord, Iran.

Email: ta.poordast@yahoo.com
Background: Endometriosis and adenomayosis are common gynecological disorders and in this work we want to evaluate the co-existence of these two diseases.
Objective: The current study was designed to evaluate the relationship between adenomyosis and its subtypes with endometriotic lesions ovarian endometrioma (OMAs) along with the posterior deep infiltrative endometriosis (DIE). We also examined the the accuracy, sensitivity, and specificity of both transvaginal sonography (TVS) and magnetic resonance imaging (MRI) in diagnosis of adenomyotic uterus.
Materials and Methods: In this retrospective cross-sectional study, we selected 154 women with coexistence of endometriosis and adenomyosis according to their imaging, intra operative or pathological findings who were nominated for laparoscopic surgery. Eighty-six patients undergoing DIE resection without laparoscopic hysterectomy (group 1), and 68 patients with laparoscopic hysterectomy plus DIE resection (group 2).
Results: The accuracy, sensitivity and specificity of ultrasonographic and MRI findings for adenomyosis diagnosis were 72.1%, 77.6%, 40.0% and 49.2%, 41.5%, 90.0% respectively. Therefore TVS was more sensitive diagnostic tool for detecting adenomyosis, however, MRI was more specific than TVS in diagnosis of diffuse adenomyosis especially with simultaneous presence of uterine leiomyoma. Regarding the association of different types of adenomyosis (focal and diffuse) with different endometriosis lesions (OMA and posterior compartment DIE), we found that diffuse type of adenomyosis is more frequent in the absence of rectal and rectovaginal septum DIE (p ≤ 0.05).
Conclusion: In addition to the questionable different nature of rectal and rectovaginal septum DIE lesion, there is no relationship between adenomyosis subtypes and endometriotic lesions.
Key words: Adenomyosis, Endometriosis, MRI.

The original full text of this abstract has been published in Reproductive Sciences. 2021. https://doi.org/10.1007/s43032-021-00527-0.
How to cite to this article: Alborzi S, Askary E, Khorami F. et al. A detailed study in adenomyosis and endometriosis: Evaluation of the rate of coexistence between uterine adenomyosis and DIE according to imaging and histopathology findings. Reprod Sci 2021.

O-11
Testing the vulnerability-stress-adaptation model of marriage in infertile couples
 
Chehreh R¹, Ozgoli G², Abolmaali K³, Nasiri M⁴.

1. School of Midwifery and Nursing, Ilam University of Medical Sciences, Ilam, Iran.
2. Midwifery and Reproductive Health Department, School of Nursing and Midwifery, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
3. Psychology Department, Islamic Azad University of Roudehen, Tehran, Iran.
4. School of Nursing and Midwifery, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

Email: g.ozgoli@gmail.com
Background: In all cultures, achieving parental role is a fundamental condition of individual perfection and sexual identity. Inability to have a child is stressful and causes disturbances in marital quality, satisfaction and stability of couples.
Objective: The purpose of this study was to test the vulnerability-stress model of marriage in infertile couples referring infertility centers in Tehran.
Materials and Methods: The present cross-sectional study was performed in two stages on 200 infertile couples (400 individual) in infertility centers in Tehran. In the first step, based on the questionnaires and the relationships between endogenous, exogenous variables; a conceptual model was designed. The predictor variables such as personality trait, infertility related stress and coping strategies, quality of life as a mediator variable and marital stability as an outcme variable, were tested. Data were collected using Norton Questionnaire, ENRICH marital satisfaction, Neo personality traits, Newton fertility problem inventory, marital instability scale and Rahim's conflict resolution strategies questionnaire. The conceptual model was tested after evaluating the data and analyzing with LISREL software.
Results: Marital satisfaction and marital quality affected marital stability in both men and women directly, while the infertility-related stress affected marital stability only in women indirectly. In men, coping strategies directly affected marital quality. Also, in both men and women, marital satisfaction directly affected the quality of marriage. In women, coping strategies directly affected marital satisfaction. Infertility-related stress in men and women directly affected marital satisfaction. Also, after examining the fit indices, the conceptual model tested in infertile couples had a good fit.
Conclusion: Infertility-related stress, coping strategies, and marital satisfaction; are the important predictors of marital quality. Also, marital satisfaction and marital quality are the predictors of marital stability.
Key words: Marriage, Couples, Infertility, Model.
 
O-12
Effect of crocin and metformin on the reproductive system dysfunction of diabetic male mice induced by methylglyoxal
 
Kheirollahi M¹, Ahangarpour A², Khorsandi L³.

1. Department of Physiology, Student Research Committee, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
2. Physiology Research Center, Department of Physiology, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
3. Department of Anatomical Sciences, Cellular and Molecular Research Center, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.

Email: akramahangarpour@gmail.com
 
Background: Diabetes has recently been a serious problem in the world. Sexual and reproductive disorders are one of the most important secondary complications in patients with diabetes.
Objective: The effect of crocin on methylglyoxal (MGO)-induced diabetes in the male reproductive system has not been studied yet; so this study performed on MGO-induced diabetic male mice.
Materials and Methods: 70 male NMRI mice, one-month-old, weighing 20-25 g were divided into 7 groups (n = 10): sham, MGO (600 mg/Kg/d), MGO+crocin15, 30 and 60 mg/kg/d, MGO+Metformin (200 mg/kg/d), and crocin 60 mg/kg/d. Methylglyoxal administered orally in 30 days. In 14^st day, after proving hyperglycemia, Metformin and crocin administered orally. On the 31^st day of the study, plasma and tissue samples prepared for experimental assessments.
Results: Blood glucose and insulin levels in the MGO group are higher than the sham group (p < 0.001), and decreased with Metformin (p < 0.001) and crocin treatment (not in all doses). Testis width and volume decreased in the MGO receiving mice, and improved in crocin treated mice (p < 0.05), but not in the metformin group. Superoxide dismutase decreased in diabetic mice (p < 0.05) and Malondialdehyde enhanced (p < 0.001). Crocin and Metformin improved MDA and SOD. Testosterone (p < 0.001), and sperm count (p < 0.05) decreased in diabetic mice, treatment in all doses recovered these variables. Luteinizing hormone increased in diabetic mice (p < 0.001) and crocin treatment (but not metformin) decreased it. Seminiferous diameter and height decreased in diabetic mice and increased in treatment groups. Vacuoles and ruptures have been seen in diabetic testicular tissue, crocin improved testicular morphology (p < 0.01).
Conclusion: MGO increases oxidative stress, reduces sex hormones, and induces histological problems in male reproductive organ. Crocin and metformin improved the reproductive damage caused by MGO induced diabetes.
Key words: Crocin, Diabetes mellitus, Methylglyoxal, Oxidative stress, Reproductive system.
 
O-13
Evaluation the effect of human sperm incubation time in polyvinylpyrrolidone on sperm structure reactive oxyen species, acrosome reaction, and mitochondorial membrane potential
 
Sabour M¹, Agha-Rahimi A², Dehghani Ashkezari M¹, Seifati SM¹, Kalantar SM³, Anbari F², Nabi A².

1. Medical Biotechnology Research Center, Ashkezar Branch, Islamic Azad University, Ashkezar, Yazd, Iran.
2. Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
3. Abortion Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: 63rahimi@gmail.com
 
Background: Polyvinylpyrrolidone (PVP) is a chemical used in intracytoplasmic sperm injection for sperm immobilization. In human sperm, PVP has been shown to damage sperm membranes, DNA integrity, mitochondrial membrane, and destroy axonal tubules and fibrous sheaths.
Objective: The aim of this study was to investigate the ideal time that sperm can be safely incubated in PVP with less possible damage.
Materials and Methods: Twenty-five normospermic samples were used. Sperm samples were prepared by swim-up method. Sperm samples incubated in 10% PVP at different time intervals (0, 15, 30, and 60 min). The effect of PVP was assessed on sperm structure, reactive oxyen species, acrosome reaction, Mitochondorial Membrane potential at different time intervals.
Results: Sperm parameters, DNA integrity and chromatin quality in 15, 30 and 60 min after incubation sperm with PVP were significantly changed compared to the 0 min. Moreover, in 30 and 60 min after incubation with PVP, above parameters were significantly changed compared to the 15 min. 60 min after incubation sperm with PVP, these parameters were significantly changed compared to the 30 min.
Conclusion: Sperm samples could be incubated with PVP for 15 min with less possible damage. While, prolonged incubation may damage the sperm parameters, DNA integrity and chromatin quality significantly.
Key words: Polyvinylpyrrolidone, Sperm mithokondria, Sperm ROS.
 
O-14
What is the accurate culture system for in vitro culture of cryopreserved human ovarian tissue?
 
Ghezelayagh Z^{1, 2}, Abtahi NS¹, Ebrahimi B¹.

1. Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
2. Department of Developmental Biology, University of Science and Culture, Tehran, Iran.

Email: bitaebrahimi@gmail.com
 
Background: Nowadays ovarian tissue banks have been set up in many countries to improve chances of child bearing for cancer patients. As transplantation of cryopreserved ovary intensifies the possibility of malignant cells reintroduction, researchers are focusing more on ovarian tissue in-vitro culture methods.
Objective: In this study we pursue three goals to achieve the accurate culture system for in-vitro culture of cryopreserved human ovarian tissue.
Materials and Methods: First, comparing agar as a cultivation substrate with matrigel-coated insert in order to attain a suitable culture substrate. Afterwards, investigating the effect of basic fibroblast growth factor (bFGF) and/or kit ligand (KL) in the culture medium. Third, evaluating the effect of Phosphatase and TENsin homolog (PTEN) inhibitor (Bpv (HOpic)) and/or mTOR activators, phosphatidic acid (PA) and propranolol (PP), on the activation and subsequent development of in-situ culture of human primordial follicles. All 7-day cultures were performed with slow frozen-thawed human ovarian cortical tissues obtained from transsexual women. At first the ovarian fragments were cultured on either matrigel-coated inserts or agar-soaked substrates. In the second phase, four different groups were examined: 1) control (base medium; BM), 2) KL (BM+100 ng/ml KL), 3) bFGF (BM+100 ng/ml bFGF) and 4) bFGF+KL (BM+100 ng/ml KL+100 ng/ml bFGF). In the third phase, control (without stimulators), Bpv (100 µM BpV (HOpic)), PA (200 µM), PA+PP (50 µM), and Bpv+PA+PP groups were compared. The incubation of ovarian cortical fragments was conducted for 24 hours with different stimulators and then for 6 days without stimulators. Follicular growth, proliferative, apoptotic and developmental gene expression, hormone secretion and PI3K/mTOR pathway protein expression were evaluated.
Results: In the first phase, no significant difference was found for follicular growth. The apoptotic index was lower in the agar cultured group and Ki67 gene expression showed a significantly higher expression in agar cultured group. In the second phase, the proportion of growing follicles had no significant difference between cultured groups. The level of estradiol hormone had significantly increased in the bFGF+KL group. The expression of Ki67 gene indicated a significant increase in the bFGF+KL group. In the third phase, the proportion of primordial and growing follicles were not significantly different after 24 hours of incubation among experimental groups. Western blot analyses indicated a significant reduction of FOXO3a in the PA+PP or Bpv+PA+PP groups compared to the control group. After 7-days of culture, the proportion of transitional follicles were significantly higher in the PA group compared to other groups. The estradiol level was significantly higher at the last day of culture compared to day 1, except for the Bpv group. Hormonal secretion was significantly higher in the PA and PA+PP groups and lower in the Bpv and Bpv+PA+PP groups compared to the control group.
Conclusion: Agar is similar to matrigel-coated inserts for culturing human ovarian tissue and it is an inexpensive substrate too. The combination of KL and bFGF positively influences steroidogenesis in the granulosa cells without increasing the total number of growing follicles. Temporary treatment of human ovarian tissue with mTOR activators, enhance the initiation of primordial follicle development and positively influence steroidogenesis, while Bpv (HOpic) has a potentially negative effect on follicular activation and function.
Key words: In-situ ovarian culture, Agar substrate, Kit ligand, Basic fibroblast growth factor, mTOR, PI3K pathways.
 
O-15
Investigation of signaling pathways to understanding Carob function for inducing spermatogenesis in an in-vitro platform
 
Hayaei Tehrani RS¹, Ghorbaninejad Z¹, Sayahpour F¹, Eghbali A¹, Moravej S¹, Eftekhari-Yazdi P², Esfandiari F¹.

1. Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
2. Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

Email: fereshtehesfandiari@royaninstitute.org
 
Background: Impairment in the spermatogenesis process is the main cause of male infertility. Recently, scientists tried to improve the efficiency of male fertility treatment through the use of herbal nutraceuticals extract. Carob is being traditionally used for male infertility treatments. However, there is no scientific evidence for the principal mechanism effect of Carob on spermatogenesis-related signaling pathways.
Objective: Herein we evaluate 3 main spermatogenesis-related signaling pathways in mouse testicular cells-enrich for spermatogonial stem cells following treatment with Carob whole extract.
Materials and Methods: To evaluate the spermatogenesis-related TGF-β, BMP4, GDNF (MEK related) Signaling Pathways following treatment with Carob whole extract, after finding non-toxically Carob concentration for testicular cell culture by pi staining (2 mg/ml), isolated cells are treated by the medium containing Carob extract and one of the following small molecules: SB431542, LDN193189 and PD0325901 respectively. Cells were collected for gene expression analysis after 9 days of treatment.
Results: Our primary results suggested that by inhibiting the BMP4 signaling pathway using LDN193189 at the presence of Carob, all of the examined genes (Plzf, Gfr-α1, Bcl-6b, Dazl, Stra8) were significantly decreased compared to Carob treat. Gene expression profiles had different patterns on inhibition of other signaling pathways.
Conclusion: It seems that the BMP4 signaling pathway is the master effector upon Carob function. Activation of this signaling pathway, directly and indirectly, effect on differentiation and self-renewal of spermatogonial stem cells to promote spermatogenesis. However, the carob contains a set of effective compounds that promote spermatogenesis by the effect on most spermatogenesis related signaling pathways.
Key words: Spermatogenesis, Spermatogonial stem cells, Carob, Signaling pathways.
 
O-16
Beneficial effects of minocycline on the ovary of polycystic ovary syndrome mouse model: Molecular docking analysis and evaluation of TNF-α, TNFR2, TLR-4 gene expression
 
Khajouei A¹, Hosseini E², Abdizadeh T³, Kian M¹, Ghasemi S¹.

1. Cellular and Molecular Research Center, Shahrekord University of Medical Sciences, Shahrekord, Iran.
2. Department of Obstetrics and Gynecology, IVF Clinic, Mousavi Hospital, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran.
3. Clinical Biochemistry Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord, Iran.

Email: sorayya.ghasemi@gmail.com
 
Background: Polycystic ovary syndrome (PCOS) is the most common cause of ovulatory infertility. Inflammation may be involved in the pathogenesis and development of PCOS.
Objective: We investigated the anti-inflammatory effect of minocycline on tumor necrosis factor-α (TNF-α), tumor necrosis factor receptor 2 (TNFR2), and toll-like receptor 4 (TLR4) expression levels and the key features of PCOS in a mouse model.
Materials and Methods: Molecular docking was performed by Molecular Operating Environment software. PCOS was induced by estradiol valerate injection (2 mg/kg/day) in 40 mice. After 28 days, the mice were divided into five groups, including control, PCOS, minocycline control, minocycline PCOS model (50 mg/kg), and letrozole PCOS (0.5 mg/kg). The Levels of follicle-stimulating hormone, luteinizing hormone, estradiol )E2), and testosterone were determined by ELISA. H&E staining was used for histological analysis in the ovarian tissues.
Results: Docking scores were -10.35, -10.57, and -12.45 kcal/mol for TNFα, TLR-4, and TNFR2, respectively. The expression levels of TNF-α, TNFR2, and TLR4 were detected by Real-Time PCR. PCOS models exhibited acyclicity, a significant increase in E2 levels (p < 0.01), and no difference in follicle-stimulating hormone, luteinizing hormone, and testosterone. The expression levels of TNF-α, TNFR2, and TLR-4 significantly increased in PCOS (2.70, 7.90, and 14.83-fold, respectively). Estradiol valerate treatment significantly increased graafian follicles (p < 0.001) and decreased corpus luteum (CL) (p < 0.01). Minocycline treatment in PCOS led to a significant decrease in E2 (p < 0.01) and graafian follicles (p < 0.001) and a significant increase in the CL numbers (p < 0.05).
Conclusion: Our findings showed the positive effects of minocycline on E2 level, CL and graafian follicles counts, suggesting that minocycline might inhibit these proteins and improve ovulation in our mouse model of PCOS.
Key words: PCOS, Minocycline, TNF-α, TNFR2, TLR-4.

The original full text of this abstract has been published in Journal of Reproductive Immunology 2021; 144: 103289. https://doi.org/ https://doi.org/10.1016/j.jri.2021.103289.
How to cite to this article: Khajouei A, Hosseini E, Abdizadeh T, Kian M, Ghasemi S. Beneficial effects of minocycline on the ovary of polycystic ovary syndrome mouse model: Molecular docking analysis and evaluation of TNF-α, TNFR2, TLR-4 gene expression. Journal of Reproductive Immunology 2021; 144: 103289.

O-17
Expression of CALM1, PSMD6, and AK124742 LncRNA genes in cumulus cells of infertile PCO women: A good predictor of successful fertilization
 
Akbari A¹, Kazemi M², Aboutorabi R¹, Mostafavi F¹.

1. Department of Anatomical Sciences, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
2. Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

Email: fs.mostafavi@gmail.com
 
Background: In human assisted reproductive technology (ART), selection of high-quality embryos to transfer usually is based on morphological criteria but it cannot be always a good predictor of successful fertilization. Analyzing gene expression of cumulus cells (CCs) might lead to some important molecular information about the embryo quality. Calmodulin 1 (CALM1), Proteasome 26S Subunit, Non-ATPase 6 (PSMD6), and AK124742 expression in the CCs of pregnant patients were more significant compared to the non-pregnant ones. One of the well-known causes of female infertility is polycystic ovary syndrome (PCOS) and the number of retrieved oocytes with a higher implantation potential is limited, so the process of selecting good embryos in PCOS patients is very important.
Objective: The aim of this study was to compare the expression of CALM1, PSMD6, and AK124742 genes in the CCs of infertile PCO patients with control fertile group.
Materials and Methods: Samples were the CCs from 33 fertile egg donor women and 33 infertile PCO women. They undergo ART and the CCs were collected and frizzed till real time PCR (RT-PCR) was performed. The expression of CALM1, PSMD6, and AK124742 genes was detected by RT-PCR. Chemical pregnancy rates were used to assess the success of ART.
Results: Clinical pregnancy was observed in 38 of the 66 patients. Expression of all three genes CALM1, PSMD6, and AK124742 in the pregnant group were higher than the non-pregnant group. This increase was not significant for the CALM1 gene but for two genes PSMD6 (p < 0.001) and AK124742 (p < 0.05) were significant. The expression of CALM1 and ak124274 gene increased significantly and the expression of psmd6 decreased significantly in PCOs group compared to the control group (p < 0.05).
Conclusion: All three genes are proper markers for predicting embryo competence due to increased expression levels in pregnant groups.
Key words: CALM1, Infertility, lncRNA, PCO, PSMD6.
 
O-18
Culture in perfusion mini bioreactor can enhance in vitro spermatogenesis
 
Amirkhani Z¹, Movahedin M¹, Baheiraei N², Ghiaseddin A³.

1. Department of Anatomical Sciences, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
2. Tissue Engineering and Applied Cell Sciences Division, Department of Anatomical Sciences, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
3. Adjunct Research Associate Professor at Chemistry Department, Michigan State University, Lansing, MI, USA.

Email: movahed.m@modares.ac.ir
Background: In vitro spermatogenesis is one of the main aim of male infertility treatment, which proves critical in cancer patients who undergo treatment with gonadotoxic drugs and methods. Conventional methods of culture cannot support organs or tissues removed from body for a long time. A biomimetic system is achieved by a bioreactor capable of culturing tissues under in vivo-like conditions. Overall, the controlled parameters are fluid flow, pH, temperature, waste removal, nutrition flow. Application of a perfusion flow is for mimicking native testicular microenvironment.
Objective: In this study, we intend to evaluate the progression of spermatogenesis after in vitro transplantation (IVT) of spermatogonial stem cells (SSCs) isolated from mouse fresh testis tissue in mini perfusion bioreactor.
Materials and Methods: Adult mouse azoospermia model was used to remove testis tissue. SSCs isolation was carried out using two enzymatic digestion methods. The cell identification was confirmed via detection of promyelocytic leukaemia zinc finger (PLZF) protein. After being labeled with DiI, the cells were transplanted into azoospermic adult mice. After being fragmented, host testes were incubated for two and eight weeks in a bioreactor. Histological, molecular and immunohistochemical assessments were done after two and eight weeks. Data were statitically analyzed using analysis of variance (ANOVA) test and significancy was considered at (p < 0.05).
Results: Histological analysis suggested successful maintenance of spermatogenesis in host testis tissues grown in the bioreactor. Molecular analysis indicated that PLZF, Tekt1 and Tnp1 genes were expressed and that their expression in the experimental IVT group was significantly more than the control group (without transplantation) and 0-day cell suspension (p < 0.05). Immunohistochemical evaluation of host testis fragments in the experimental group showed that PLZF, synaptonemal complex protein (SCP3) and acrosin binding protein (ACRBP) proteins were expressed in spermatogonial cells, spermatocytes and spermatozoa, respectively.
Conclusion: Dynamic culturing methods appear to be capable of enhancing spermatogenesis in vitro. Such three- dimensional (3D) culturing system is able to give rise to haploid cells and can offer conditions similar to those of native like physiological microenvironment of testicular tissue. The current bioreactor, thus, can potentially provide an enhanced culturing system for testicular organ culture. Our findings reveal that following two weeks of SSCs transplantation in vitro, and 3D dynamic organ culture, these cells had migrated to basement membrane of seminiferous tubules and settled down through homing and initiated the spermatogenesis process. Perfusion bioreactor dynamic culturing system fosters spermatogenesis induction to generate haploid cells, in which long term (56 days) culturing host testicular tissue segments of the IVT group permitted spermatogenesis completion, giving rise to morphologically intact and mature spermatozoa.
Key words: Spermatogonial stem cells, Mouse, Transplantation, Azoospermia, Perfusion bioreactor.
 
O-19
Protective effect of crocin on electromagnetic field-induced testicular damage and heat shock protein A2 expression in male BALB/c mice
 
Vafaei Sh¹, Motejaded F¹, Ebrahimzadeh-bideskan A^{1, 2}.

1. Department of Anatomy and Cell Biology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
2. Microanatomy Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.

Email: ebrahimzadehba@mums.ac.ir
 
Background: Exposure to electromagnetic fields (EMF) emitted from mobile phones may cause a deleterious effect on human health and may affect the male reproductive system. Crocin, a carotenoid isolated from Crocus Sativus L. (Saffron), is a phar macologically active component of saffron.
Objective: This study was conducted to investigate the protective effect of crocin on the male reproductive system of 60 day old mice after EMF exposure.
Materials and Methods: Twenty-four male BALB/c mice were randomly divided into 4 groups: 1) electromagnetic (EM) group (2100 MHZ); 2) Crocin (Cr) group (50 mg/kg); 3) Em+Cr group (2100 MHZ+50 mg/kg), and 4) Control group. Sperm parameters (count, and abnormal percent), testis weight index, testis volume, seminiferous tubule diameter, germinal epithelium thickness, luteinizing hormone, follicle-stimulating hormone and testosterone serum level, testicular Heat shock protein A2 (HspA2) immunoreactivity, and apoptosis were evaluated.
Results: HspA2 immunoreactivity, apoptosis in the germinal epithelium and abnormal sperm were increased in EM group compared with the control group (p < 0.05). Sperm count, luteinizing hormone, and testosterone serum level were decreased in the EM group compared with the control group (p < 0.05). These parameters were improved in the EM+Cr group compared with EM group significantly (p < 0.05).
Conclusion: Our findings revealed that EMF exposure leads to harmful impressions on the male reproductive system, while crocin can attenuate EMF-induced destructive effects.
Key words: Apoptosis, Electromagnetic field, Crocin, Heat shock protein, Testis.

The original full text of this abstract has been published in Iranian Journal of Basic Medical Sciences 2020; 23(1): 102. https://doi.org/ 10.22038/IJBMS.2019.38896.9229.
How to cite to this article: Vafaei S, Motejaded F, Ebrahimzadeh-Bideskan A. Protective effect of crocin on electromagnetic field-induced testicular damage and heat shock protein A2 expression in male BALB/c mice. Iranian Journal of Basic Medical Sciences 2020; 23(1): 102.

O-20
Annexin-V MACS sperm selection method could be effective on separation of sperm with high expression of PLCZ1 gene and development of high blastocyst rate in male factor patients with high DNA fragment
 
Salehi Novin M, Zandieh Z, Bakhtiari M, Aflatoonian R.
Department of Anatomy, Iran University of Medical Sciences, Tehran, Iran.
Email: maryam.salehi74@gmail.com
 
Background: Sperm selection according to morphology and motility in assisted reproduction technologies (ART), is not sufficient for selecting the best sperm especially in patients who have male factor problems. Apoptotic sperm and non-apoptotic ones are distinguished from each other by negative selection in Annexin-V magnetic-activated cell sorting (MACS) technique. So, compaction rates in embryo quality are enhanced in this method. PLCZ1, which is one of the oocyte activating factors, starts oscillations of Ca²⁺ in oocyte and it has a significant impact on the fertilization and implantation process.
Objective: In this study, hypostasis of sperm selection relying on motility and morphology in ART techniques, not be sufficient for selecting the most qualified sperm especially in male factor patients are shown. Apoptotic sperm cells and non-apoptotic ones are distinguished from each other in the Annexin-V MACS-DGC technique. Therefore, this method enhanced the quality of embryo compaction rate.
Materials and Methods: Semen samples of 30 infertile couples who have male factor problem with DNA fragmentation index (DFI) above 30% were selected for this study. The samples of each patient were divided into two groups, control and experimental. The control group was washed with the routine density gradient centrifugation (DGC) method and the experimental group was selected by magnetic-activated cell sorting combined isolate density gradient centrifugation (MACS-DGC). Similarly, eggs in each female of patients were divided into 2 mentioned groups. Control was injected by DGC, on the other hand, the experimental group was injected by MACS-DGC. On both before and after processing sperm parameters were evaluated. DFI was reported based on the halo sperm method both before and after processing. Embryo quality, blastocyst formation rate, and fertilization rate were estimated, after ICSI. Expression of PLCZ1 was evaluated by real-time PCR. Comparison between results of two groups was determined by SPSS software.
Results: The research reported that sperm morphology and motility after the MACS-DGC method (1.7%, 45%) were significantly higher in comparison with the DGC method (1.1%, 40%) and before washing (0.9%, 35%). The percent of DFI in the MACS-DGC group (36%) was significantly reduced in comparison to DGC (45%) and primitive group (55%). The number of oocytes injected was 93 and 111 in DGC and MACS-DGC group, respectively. The fertilization rate in both groups was approximately equal (73.11 in DGC versus 72.07% in MACS-DGC). The rate of day 3 embryos with good grade was significantly higher than in the MACS-DGC group (72.5%) in comparison to the DGC group (51.47%) (p < 0.05). The blastocyst rate in the MACS-DGC group (69.69%) was significantly higher than the DGC group (48%). PLCZ1 gene expression in MACS-DGC was significantly increased compared to the DGC group (p = 0.046).
Conclusion: Results show that sperm selection based on the MACS-DGC method can enhance morphology, motility, and decrease sperm DFI. No significant difference was observed in fertilization rate, but the percent of the high-quality embryo on days 3 and 5 was significantly higher by this method. According to the mechanism of the MACS-DGC method, it can be suggested as a good choice for patients with high DFI.
Key words: MACS, PLCZ1, High DFI, Male factor.
 
O-21
Viability of isolated preantral follicles using decellularized ovarian tissue after grafting under the kidney capsule
 
Nikniaz H¹, Jameie S², Zandieh Z¹, Nouri M³, Aflatoonian R⁴, Gholipourmalekabadi M⁵.

1. Department of Anatomical Sciences, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
2. Neuroscience Research Center (NRC), Iran University of Medical Sciences, Tehran, Iran.
3. Department of Reproductive Biology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
4. Department of Endocrinology and Female Infertility at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
5. Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran.

Email: Jameie.sb@iums.ac.ir
 
Background: nowadays scientist use decellularized tissue as a novel technique in regenerative medicine. Recently the application of decellularized tissue in the field of human fertility preservation has been studied. Due to the high resemblance of decellularized ovarian tissue to the main organ, it can be used as a scaffold for follicle growth and development.
Objective: Mice ovarian follicles can be viable and seed into the acellular scaffold after 7 days of grafting.
Materials and Methods: In this study fragmented bovine ovarian cortex (2 × 2 mm) were decellularized by Sodium dodecyl sulfate, Triton X100 and Ammonium. 120 primordial follicles of NMRI mice were isolated and put into the decellularized scaffold   (n = 30/ 4 scaffold) then transplanted under the kidney capsule for 7 days. H&E staining was used to determine follicle morphology after transplantation. Follicular proliferation was measured by Ki-67 antibody. Apoptosis (TUNEL) and vessel formation (CD31) were analyzed.
Results: According to the H&E staining results, after 7 days of grafting, 38/120 follicles were viable (31.6%) and seeded into the scaffold. Ki67-positive OCs was found in 15.2% of cells of the grafted scaffold. While TUNEL-positive cells were in 13.7% of granulosa cells. According to H&E staining results and CD34-staining, vessels were found inside the scaffold after 7 days of grafting.
Conclusion: This study shows that follicles can survive and seed into the acellular matrix after 7 days of grafting.
Key words: Fertility preservation, Ovary, Scaffold.
 
O-22
Application of auto-crosslinked hyaluronic acid hydrogel loaded with the bone marrow mesenchymal stem cell-extracellular vesicles to prevent the formation of intrauterine adhesions in a rat model
 
Mansouri N¹, Hajari MA², Sadeghi Abandansari H^{2, 3}, Niknejadi N², Heidari Khoei H¹, Baharvand H^{4, 5}, Montazeri L².

1. Department of Biology and Anatomical Sciences, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
2. Department of Cell Engineering, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
3. Department of Cancer Medicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Babol, Iran.
4. Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
5. Department of Developmental Biology, University of Science and Culture, Tehran, Iran.

Email: montazeri@Royaninstitute.org
 
Background: Severe trauma of uterus may cause damage in the basal layer of endometrium leading to intrauterine adhesion (IUA) or Asherman’s syndrome (AS), and eventually infertility. Nowadays, conventional approaches including surgical adhesiolysis and following hormone therapy are used to treat AS in clinics. However, the high recurrence after these procedures reveals the importance and superiority of IUA prevention instead of treatment. As a preventive agent, auto-crosslinked hyaluronic acid (HA) hydrogels can provide an anti-adhesive barrier to decrease the incidence of IUA. In addition, MSC-derived extracellular vesicles (EVs) have been recently introduced as an effective and novel therapeutic agent to reduce inflammation and fibrosis. This study has been designed to investigate whether combining the auto-crosslinked HA hydrogel with mesenchymal stem cell-extracellular vesicles (MSC-EVs) could improve the efficiency of HA in endometrial regeneration in the rat model of AS.
Objective: To evaluate whether the combination of HA hydrogel and MSC-EVs could facilitates functional regeneration of injured uterus in experimental rats.
Materials and Methods: Forty eight-week-old female Wistar rats weighting 200–250 g were randomly assigned into 5 groups (n = 8/each): I) Intact group: without any intervention, II) AS Model group: model was established by three surgical steps of mechanical injury (incision, curettage, and suture), III) Sham surgery group: Subjected to the abdominal surgery, incisions and suturing, but not the curettage procedure, IV) HA + MSC-EVs: A mixture of 400 µl HA hydrogel + 160 µg/kg/dose MSC-EVs (around 200 µl hydrogel containing 20 µg EV per each horn) was injected into the uterine horn immediately after making the AS model and V) HA: Receiving an intrauterine injection of only 400 µl HA hydrogel immediately after making the AS model. Two weeks after the transplantation, four rats from each group sacrificed and uterine samples were harvested to be evaluated histologically by H&E and Masson’s trichrome staining. The remaining four animals in each group were coupled with fertile males (female: male = 2:1) 1 week after modeling for 3 months. The number of deliveries and the cumulative number of pups were assessed at the end of this time to survey reproductive function.
Results: Histology examination revealed significantly thicker endometrium, increased gland numbers and fewer fibrotic areas in the HA + MSC-EVs and HA transplantation groups compared with the model group. The cumulative number of pups and number of deliveries also showed a significant increase in the HA group compare to the model group. But our results displayed no significant differences between the HA + MSC-EVs and HA groups in terms of morphometric parameters and mating test outcomes.
Conclusion: MSC-EVs cannot amplify preventive properties of HA on the rat model of AS.
Key words: Intrauterine adhesions, Asherman’s syndrome, Hyaluronic acid hydrogel, Extracellular vesicle.
 
O-23
In vitro cytotoxicity of zinc oxide nanoparticles in mouse ovarian germ cells
 
Saber M¹, Hayaei Tehrani RS¹, Mokhtari S², Hoorzad P¹, Esfandiari F¹.

1. Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
2. Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
3. Department of Physics, Shahid Beheshti University, Tehran, Iran.
4. Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
5. Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.

Email: fereshtehesfandiari@royaninstitute.org
 
Background: Recently, metal oxide nanoparticles such as zinc oxide nanoparticles (ZnO-NPs) have received considerable attention and humans are exposed to them in everyday life. The increasing use of ZnO-NPs may lead to human health issues. However, little is known about their effects on female reproductive systems, particularly on female germ cells. Germ cells differentiation is a complex biological process that is sensitive to environmental insults and any negative effect on germ cells development may inhibit fertility.
Objective: The purpose of this study was to assess the effects of ZnO-NPs on mouse ovarian germ cells (OGCs) as an in vitro model for the assessment of nanotoxicity in the OGCs. To the best of our knowledge, no study has been conducted to determine the effects of ZNO-NPs on female OGCs. Our study provides a sensitive in vitro model to assess the toxic effects of ZNO-NPs and other NPs in the female OGCs. This study aimed to determine the impact of ZnO-NPs on mouse OGCs in an in vitro system.
Materials and Methods: Briefly, after isolation and culture of OGCs, the effects of ZnO-NPs on these cells were evaluated using light microscopy, cell proliferation assessment, reactive oxygen species (ROS) level determination, standard cytotoxicity assessment (cell viability assessed by PI staining) and gene expression analysis.
Results: Our results demonstrated that ZnO-NPs have cytotoxic effects in a concentration- and time-dependent manner in mouse OGCs. Exposure of cells to ZnO-NPs concentration-dependently enhanced ROS generation. Furthermore, molecular analysis of ZnO-NPs-treated cells showed a significant increase in expression of premeiotic germ cells markers but a decrease in meiotic and post-meiotic markers compared to un-treated cells.
Conclusion: Our data provides a preliminary insight into possible adverse effects of ZnO-NPs on mouse OGCs differentiation even at low concentrations.
Key words: Nanoparticle, Zinc oxide, Ovarian germ cell, Infertility.

The original full text of this abstract has been published Toxicology in Vitro 2021 Feb 1; 70: 105032. https://doi.org/10.1016/j.tiv.2020.105032.
How to cite to this article: Saber M, Hayaei-Tehrani RS, Mokhtari S, Hoorzad P, Esfandiari F. In vitro cytotoxicity of zinc oxide nanoparticles in mouse ovarian germ cells. Toxicology in Vitro 2021; 70: 105032.

 
O-24
Oxidative stress-dependent toxicity of dextran-coated superparamagnetic iron oxide nanoparticles on mouse embryo produced by in vitro fertilization
 
Alaee S¹, Bakhtari A¹, Nazari S¹, Kargar-Abarghouei E², Mirzaei E³.

1. Department of Reproductive Biology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran.
2. Department of Anatomy, Faculty of Medicine, Hormozgan University of Medical Sciences, Bandar Abbas, Iran.
3. Department of Medical Nanotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran.

Email: sanaz620@gmail.com
Background: Superparamagnetic iron oxide nanoparticles (SPIONs) are capable to penetrate the placenta. Also, small nanoparticles can cross the blood-testis barrier and aggregate in the testes. Thus, SPIONs might have adverse impacts on reproduction systems.
Objective: The influence of adding dextran-coated SPIONs (D-SPIONs) into the fertilization medium was investigated in a dose-dependent manner on gene expression of oxidative stress enzymes in the resultant blastocysts in a mouse model.
Materials and Methods: Mature oocytes were collected from superovulated female BALB/c mice and randomly divided into three groups (0, 50, and 250 µg/ml of D-SPIONs). These concentrations were mixed into fertilization medium as control, low, and high dose groups, respectively. The toxic effects of D-SPIONs on murine in vitro fertilization (IVF) were investigated by developmental competence and alterations in gene expression of antioxidant enzymes were assessed for glutathione peroxidase 1, superoxide dismutase 1, and catalase in the blastocysts derived from IVF. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test (SPSS 20) and presented as mean ± standard deviation.
Results: Mature oocytes were collected from superovulated female BALB/c mice and randomly divided into three groups (0, 50, and 250 µg/ml of D-SPIONs). These concentrations were mixed into fertilization medium as control, low, and high dose groups, respectively. The toxic effects of D-SPIONs on murine IVF were investigated by developmental competence and alterations in gene expression of antioxidant enzymes were assessed for glutathione peroxidase 1, superoxide dismutase 1, and catalase in the blastocysts derived from IVF. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test (SPSS 20) and presented as mean ± standard deviation.
Conclusion: Our results may suggest that increasing these antioxidant enzyme genes, as reactive oxygen species scavengers, meaningfully promoted overtime to protect the resultant blastocysts from oxidative damage. Despite considerable usage of D-SPIONs in numerous fields of science and technology, this study presented extensive worries about their toxicity towards IVF. Therefore, it is important to perform further studies to detect the potential risks of this nanoparticle in various areas of nanotechnology.
Key words: Blastocyst, Catalase, Developmental competence, Oxidatiove stress.
 
O-25
Evaluation of the effect of granulocyte-macrophage colony stimulating factor on sperm quality in oligoasthenoteratospermia men
 
Tanhaye Kalate Sabz F¹, Ashrafi M^{1, 2}, Amjadi F¹, Aflatoonian R³, Zandieh Z¹, Hosseini E⁴.

1. Department of Anatomical Science, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
2. Shahid Akbar Abadi Clinical Research Development Unit, Iran University of Medical Sciences, Tehran, Iran.
3. Department of Endocrinology and Female Infertility,Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
4. Department of Obstetrics and Gynecology, IVF Clinic, Mousavi Hospital, Zanjan University of Medical Sciences, Zanjan, Iran.

Email: Amjadi.bh@gmail.com
 
Background: Oligoasthenoteratospermia (OAT) is characterized by abnormalities in sperm count, motility and morphology. Poor sperm quality can adversely affect the results of assisted reproductive technique. Development of sperm media is necessary to improve the sperm parameters of these patients. Granulocyte-macrophage colony stimulating factor (GM-CSF) is a natural growth factor produced by the reproductive organs, previous studies show that this growth factor in the semen of infertile men is lower than that of fertile men. However, there is no study to assess the effect of GM‐CSF on sperm quality.
Objective: The aim of this study is to evaluate the effect of GM-CSF as a sperm medium supplement on sperm quality in OAT patients.
Materials and Methods: In the present study, semen specimens were collected from 20 OAT patients who have male infertility factors, according to WHO criteria. After the swim-up washing procedure, each of the samples was divided into two groups; experiment, and control. In the experimental group, samples were incubated with a medium containing 2 ng/ml GM-CSF for one hour, yet, in the control group, the sperms were incubated without GM-CSF for the same time. The sperm motility was examined with phase-contrast microscopy, Eosin-nigrosin staining method was used to assess sperm viability, and DNA fragmentation were evaluated by TUNEL test. The expression of sperm glucose transporters (GLUT 1, 3) was determined using Immunofluorescent staining, the phospho‐Akt/total Akt ratio was assessed by the Western blotting method. Data were analyzed by SPSS software and P-value < 0.05 was considered statistically significant.
Results: As compared to the control group, supplementation with GM-CSF improved sperm progressive motility, enhanced GLUT 1 and 3, and phospho‐Akt/total Akt expression (p < 0.05). In GM-CSF treated groups, DNA fragmentation was lower than control ones (p < 0.05). There was no significant difference between the viability of the control and experimental groups.
Conclusion: Our results showed that GM-CSF can improve sperm quality by influencing motility and energy metabolism in spermatozoa which can be affected by increasing the phosphorylation of AKT for the first time. This growth factor could be an appropriate supplement in sperm media for OAT patients.
Key words: GM-CSF, Oligoasthenoteratospermia, Sperm quality.
O-26
Alternation of interleukins expression from fallopian tube epithelial cells after co-incubation with spermatozoa
 
Mohammadi R¹, Mousavi SO¹, Sabbaghian M², Zandieh Z³, Maadani T¹, Aflatoonian R¹.

1. Department of Endocrinology and Female Infertility, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
2. Department of Andrology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
3. Shahid Akbarabadi Clinical Research Development Unit (ShACRDU), Iran University of Medical Science, Tehran, Iran.

Email: r.aflatoonian@gmail.com
 
Background: Fallopian tubes are important places in which spermatozoa are saved until fertilization with oocytes occurs. The immune system in the reproductive tract has an important role in overcoming pathogens while preparing a safe environment for allogenic spermatozoa. Interleukins are among the most important variables of the immune system which provide effective response in normal and pathological conditions. Therefore, many scientists are interested in better understanding of the role of interleukins in reproductive immunology.
Objective: The aim of this investigation was to find out more details about the role of interleukins in the interaction between spermatozoa and fallopian tube epithelial cells. Therefore, the expression of different interleukins from the fallopian tube cell line (OE-E6/E7) which had co-incubated with spermatozoa was investigated by PCR array.
Materials and Methods: We collected sperm samples from 10 healthy men. All samples were checked to ensure they have normal features according to WHO guidelines. Simultaneously, we cultured epithelial cell line in 6-well plates and incubated until 70% of each well was covered by the epithelial cells. Cells without spermatozoa were analyzed as the control group. The cells and the spermatozoa were co-cultured for 24 hr. Then, the cells were washed and followed by RNA extraction and cDNA synthesis. PCR array was performed to evaluate the transcriptomic changes of different interleukins. To confirm our results, the concentrations of IL-10 and IL-1β were also analyzed by ELISA.
Results: The results of our investigation indicated that the expression of some interleukins in the vicinity of sperm significantly changes. It has been shown that anti-inflammatory interleukins including IL-9 (p = 0.02) and IL-10 (p ≤ 0.01) from fallopian tube epithelial cells were significantly upregulated in the presence of spermatozoa. However, the expression of pro inflammatory interleukins such as IL-16, IL-17, IL-1A, and IL-1B was significantly (p < 0.05) lower in the case group than the control group. Moreover, the concentration of IL-10 in the case group was higher than the control. Although, the concentration of IL-1B in the case group was lower than the controls.
Conclusion: This study indicates that spermatozoa modulate the expression of interleukins from OE-E6/E7. Moreover, altered genes expression might have increasing survival chance of spermatozoa in fallopian tubes’ microenvironment.
Key words: Fallopian tube, Spermatozoa, Interleukins, PCR array.
 
O-27
Effects of knockout serum replacement on the quality of frozen-thawed human spermatozoa
 
Taher Mofrad SMJ¹, Rezaei Topraggaleh T², Ziarati N¹, Ashrafzade A³, Seifi S¹, Shahverdi A¹.

1. Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
2. Department of Anatomical Sciences, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran.
3. Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

Email: shahverdi@royaninstitute.org
 
Background: Cryopreservation of human spermatozoa is an important technique in the treatment of male infertility. This procedure is regularly used for preserving the fertility potential of young male patients with cancer before chemo- or radiotherapy. It is well known that the freeze-thaw practice leads to the functional and structural damages to human spermatozoa, mainly due to the formation of ice crystals, oxidative stress, and osmotic imbalance. In order to decrease sperm cryo-damages and improve the quality of post-thawed spermatozoa, factors, such as the composition of the freezing medium, methods of freezing (i.e., slow, rapid, and vitrification), and the way of packaging the samples (cryotube or straw) have been largely investigated. Knockout serum replacement has been recently used as an effective serum substitute for culture of several mammalian cells and cryopreservation of some cell types. It is a rich source of amino acids, antioxidants, vitamins, and some trace elements and would be preferable due to the components’ reliability, no varies between batches and the absence of probably microbial agents.
Objective: In this study, we used KSR as a serum substitute in the freezing medium for sperm cryopreservation. For this purpose, we designed a simple handmade freezing medium that consisted of sucrose and different concentration of HSA or KSR for the rapid freezing of human sperm cells and compared them with commercial freezing medium.
Materials and Methods: Twenty semen samples were taken from normozoospermic men who referred to the Royan Institute. After the swim-up process and the evaluation of fresh sample, supernatants were divided into five groups. The four aliquots were diluted with a sucrose solution containing different percent (5%, 10%) of HSA and KSR. The other aliquot was diluted with a freezing medium containing (Sperm-Freeze) as a control group (CON). The samples were hold in liquid nitrogen vapor for 15 min and then plunged into the liquid nitrogen and cryopreserved for 1 month. After thawing, sperm parameters including motility characteristics, viability, acrosome integrity and DNA intactness were assessed.
Results: All of sperm parameters were significantly decreased in cryopreserved samples compared to the fresh group. There were no significant differences in the motion characteristics of spermatozoa between the control and experimental groups. However, the highest sperm viability, acrosome integrity and DNA intactness was achieved by the addition of 10% KSR to the freezing medium, which was statistically significant when compared with other experimental groups.
Conclusion: In conclusion, our results demonstrated that the addition of 10% KSR to the sucrose-based freezing solution improves the quality of post-thawed human spermatozoa, including motility, viability, acrosome integrity, and DNA integrity and may have potential to develop chemically defined freezing medium.
Key words: Human sperm, Cryopreservation, Knockout serum replacement, Human serum albumin.
 
O-28
Reconstruction of the mouse uterine tissue using polycaprolacton/ gelatin/ polydimethylsiloxane hybrib scaffolds: In vitro and in vivo study
 
Dehghan M^{1, 2}, Nikukar H^{2, 3}, Khajeh Mehrizi M¹.

1. Textile Engineering Department, Faculty of Engineering, Yazd University, Yazd, Iran.
2. Medical Nanotechnology and Tissue Engineering Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
3. Department of Advanced Medical Sciences and Technologies, School of Paramedicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: mahdiyeh.dehghan60@gmail.com
 
Background: Serious endometrial damage in women of fertility age is often associated with the formation of uterine scars and a lack of functional endometrium prone to infertility or miscarriage. Uterine tissue engineering using biological materials and stem cells may replace the need for surrogacy and may prevent the necessary immune suppression therapy. However tissue engineering structures were also used as laboratory models to study the mechanisms of endometrial invasion.
Objective: Reconstruction of the mouse uterine tissue using polycaprolacton/ gelatin/ polydimethylsiloxane hybrib scaffolds (in vitro and in vivo study).
Materials and Methods: In the present study, according to the structure of mouse uterine tissue, a tubular nanofiber scaffold was designed and fabricated. Mouse cells were cultured on target scaffold. 3- (4،5-dimethylthiazoyl-2-yl) 2، 5-diphenyltetrazolium bromide (MTT) test was performed to evaluate cell viability on the scaffold. Hematoxylin and eosin staining examined cell growth and proliferation on the scaffold. Mouse embryos were cultured on the target scaffold and examined with immunofluorescents staining. Finally, a tubular scaffold replaced one of the branches of the rat uterus and was evaluated 30 days after surgery by hematoxylin and eosin staining and immunohistochemistry for tissue formation.
Results: The tubular scaffold designed in this study showed that due to the location of the cells between the scaffold layers, cell infiltration between the nanofibers was good due to the small porosity of nanofibers. It also had a higher performance than similar tubular scaffolds. In the present study, the mouse embryo was hatched on the scaffold and attached to the scaffold. This indicates that the embryo was compatible with the scaffold. Also, after tubular scaffold transplantation instead of the mouse uterine horn, many cells close to the injured site migrate inward and the tubular tissue of the mouse uterus was formed on the scaffold.
Conclusion: In the present study, we developed a scaffold with the standards needed for whole uterine tissue engineering. It also could be useful for multiple tissue engineering applications. In these scaffolds, cell proliferation and migration occured well while enhancing angiogenesis to regenerate new uterine horns. Electrospun polycaprolacton/ gelatin/ polydimethylsiloxane fibrous scaffolds were developed to use as promising uterine tissue engineering.
Key words: Mouse uterus, Tubular scaffold, Embryo, Nanofiber.
 
 
O-29
Designing a new delivery system containing quercetin and edible oils to treat male infertility induced by nonalcoholic fatty liver in rats
 
Mousavi SN^{1, 2}, Hosseini E^{1, 3}, Seyed Dorraji MS⁴, Sheikh Mohammadi Sh⁴, Pourmansouri Z⁵, Rasoulifard MH⁴, Doosti M⁴, Chiti H¹.

1. Zanjan Metabolic Diseases Research Center, Zanjan University of Medical Sciences, Zanjan, Iran.
2. Department of Nutrition, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran.
3. Department of Obstetrics and Gynecology, IVF Clinic, Mousavi Hospital, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran.
4. Applied Chemistry Research Laboratory, Department of Chemistry, Faculty of Science, University of Zanjan, Zanjan, Iran.
5. Department of Pharmacology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran.

Email: dorraji@znu.ac.ir
 
Background: Recent molecular and physiological studies have shown that adverse effects of nonalcoholic fatty liver diseases (NAFLD) extend far beyond the liver. NAFLD can impair male reproductive function by increasing reactive oxidative stress levels, reducing the expression of antioxidant genes and inducing damage in testes immune privilege. Antioxidant therapy and its effectiveness depend on whether the exogenous antioxidant will be readily absorbed to reach high enough that are required to decrease the pathological damages. Quercetin (Quer), as an antioxidant, is able to ameliorate oxidative stress but has low bioavailability in the body. Therefore designing new drug delivery systems are needed to reach the best effects.
Objective: We aimed to prepare a new delivery system containing edible oils for quercetin entrapment to slow release.
Materials and Methods: Bigels were prepared using cottonseed oil/cannabis oil/alginate/ferula gum. Sprague-Dawley rats were housed for 2 wk, then NAFLD was induced by 58% of dietary calorie as lard and 42 g/L fructose for 16 wk. The experimental protocol was approved by the ethical committee of Zanjan University of Medical Sciences, Zanjan, Iran. After confirming the NAFLD induction, animals were divided into five groups: Control, control NAFLD, received 2 mg/kg Quer loaded on bigels, free bigels, and free Quer for 45 days as daily gavage. Semen parameters (count, motility, and morphology), viability (Eosin-nigrosine staining) and serum testosterone levels were analyzed. In addition, histological sections of testicular tissues were investigated by Hematoxylin-Eosin staining method. In situ detection of apoptosis was performed using terminal deoxynucleotidyl-transferase dUTP nick end labeling (TUNEL) assay.
Results: The sperm count, sperm motility, normal morphology and testosterone level were significantly lower in the NAFLD group than those the controls. Moreover, higher head and tail abnormality percentages were seen in the sperm of these groups. Bigel-Quer significantly improved the serum testosterone level, sperm count, motility, and morphology compared with the NAFLD group. Spermatogenic cells in all stages of differentiation (spermatogonia, primary spermatocytes, early spermatids, late spermatids) are observed and preserved normally in the testicular tubules and lumen filled with mature sperms in the control group. Interestingly, atrophic changes in the testicular tubule architecture with swelling in spermatogonia cells, detachment from tubule membrane, reduced number of mature sperm, and reduced lumen thickness were seen in the NAFLD. In the Quer, bigel and bigel-Quer-treated groups, swelling and vacuolation rate of germ cells decreased. The testicular morphology, and tubule structure were significantly normalized, especially in the bigel-Quer-treated group. Serum testosterone levels significantly increased and reached the healthy control group in the bigel-Quer group. TUNEL-positive cells in testes increased significantly after NAFLD induction. Quantitative analysis showed a significant decrease in testicular TUNEL-positive cells following bigel-Quer treatment, but not in other groups.
Conclusion: The bigel showed synergistic effects with Quer for treating infertility in rats with NAFLD. Stability and bio-availability of Quer are important aspects that should be considered to justify its supplementation. Empowering antioxidant shield of NAFLD patients by Quer supplementation can improve various damage effects and clinical status of diseases.
Key words: Quercetin, Non-alcoholic fatty liver, Semen parameters, Bigel.

The original full text of this abstract has been published in International Journal of Biological Macromolecules 2021; 177: 157-165. https://doi.org/10.1016/j.ijbiomac.2021.02.121.
How to cite to this article: Mousavi SN, Hosseini E, Dorraji MS, Sheikh Mohammadi S, Pourmansouri Z, Rasoulifard MH, Doosti M, Chiti H. Synthesis of a green bigel using cottonseed oil/cannabis oil/alginate/ferula gum for quercetin release: Synergistic effects for treating infertility in rats. International Journal of Biological Macromolecules 2021; 177: 157-165.

 O-30
Debates on COVID-19 presence in semen: A systematic review
 
Moshrefi M^{1, 2}, Ghasemi-Esmailabad S^{2, 3}, Mangoli E^{2, 3}, Khalili MA^{2, 3}.

1. Medical Nanotechnology and Tissue Engineering Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
3. Department of Reproductive Biology, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: khalili59@hotmail.com
 
Background: Coronavirus disease 2019 (COVID-19) is a challenge not only for survival and being alive, but probably for ability to have a child. This concern is more exacerbated for men, because sex differences susceptibility has been reported for men and men are more prone to COVID-19. Also, orchitis is reported in autopsy of men, which may be due to the highly expression of ACE2 in testicular cells.
Objective: Some studies have reported the real time polymerase chain reaction (RT-PCR) detection of virus in semen of men, affected by COVID-19; while, others did not confirm these data. So, the aim of this study was a systematic review of published data regarding the detection of SARS-CoV-2 virus in semen.
Materials and Methods: The literature search was performed in PubMed, Scopus, and Google scholar based on the following key words: [“severe acute respiratory syndrome–coronavirus 2” OR “COVID-19”, OR “2019-nCoV”, OR “SARS-CoV-2”, OR “severe acute respiratory syndrome coronavirus 2” OR  “SARS CoV2” OR “SARS CoV 2”] AND [“semen” OR “sperm” OR “testis” OR “testicles” OR “seminal” OR “testes” OR “male reproduction” OR “orchitis” OR “testicular” OR “male fertility” OR “male infertility” OR “epididymis” OR “prostate” OR “testosterone” OR “DHT” OR “dihydrotestosterone” OR “azoospermia”. All published papers were screened from December 2019-december 2020. The literature search was conducted by manual screening of the titles and abstract. The full text of studies that detected the virus in semen by RT-PCR and RT-qPCR were reviewed.
Results: 10 papers published from April-December 2020 were suitable. one paper reported that 6.66% of cases (6 of 38) tested positive for virus in semen. Among them, 2 recovered and 4 patients (15 ≥ years) in acute phase were positive. Nine other studies reported that the semen samples were negative for detection of virus RNA in semen which were as follows. Song, Wang colleague tested semen and testicular biopsy of 12 young recovered patients (11 patients with mild symptoms, 1 asymptomatic and 1 patient in acute phase) and testis biopsy achieved from 1 dead patient. Xiao colleague collected semen samples from 34 Chinese men (18-57 yr) with generally mild symptoms and 6 patients (19%) developed scrotal discomfort. Pallotti colleague  checked one 31-yr-old man, 8 days after positive pharyngeal swap. Ning, Li colleague  also analyzed 17 patients with aged 23-46 yr. They showed symptoms or signs related to male reproductive system. Zhao colleague evaluated 23 patients in recovery phase. Nora, Philippos colleague  tested 18 patients, 8-54 days after absence of symptoms and 2 samples from patients with active infection. Temiz, Dincer colleague  tested 55 patients, 18 to 60-yr old. Li, Xiao colleague  tested 23 patients, aged above 18 yr. Ruan, Hu colleague analyzed 74 men, aged 20-50 yr. All these 9 studies reported negative results in different age categories and different COVID-19 phases.
Conclusion: The detection of CIVID-19 in semen was noted and the possibility of male factor infertility and sexual transmission should not be neglected.
Key words: COVID-19, SARS-CoV-2, Testis, Sperm, Semen.
 
O-31
Impact of sperm parameters on mRNA level of AnnexinA2, Sp17, SerpinA5, Prdx2, oxidative stress, and sperm DNA fragmentation
 
Afsari M¹, Talebi AR¹, Fesahat F².

1. Andrology Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Reproductive Immunology Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: proftalebi@gmail.com
 
Background: Today, it has been shown that having the normal sperm parameters cannot only represent fertility status of male partners of infertile couples. The potential role of several molecular and cellular factors associated with fertilization and embryo failure is not clearly identified.
Objective: The aim of this study was to investigate the association between sperm DNA fragmentation, oxidative stress as well as some sperm functional genes and the sperm parameters among both men of infertile couples with a history of recurrent early pregnancy loss and fertile men.
Materials and Methods: The mRNA levels of the AnnexinA2, Sperm protein 17 (Sp17), SerpinA5, and Peroxiredoxin-2 (Prdx2) genes were comparatively evaluated between sperm samples of infertile men with abnormal parameters (n = 25), male partners of infertile couples with normal parameters (n = 25), and the fertile men with normal sperm parameters (n = 25) as experimental group I, II and control, respectively by using quantitative real time polymerase chain reaction. The sperm DNA fragmentation (SDF) was assessed using Chromomycin A3 (CMA3), acridine orange (AO), annexin V (ANXV) staining and Propidium iodide (PI). Sperm maturity was evaluated by acrosom reaction test. To determining the stress oxidative, malondialdehyde (MDA) and total antioxidant capacity (TAC) levels were measured in seminal plasma.
Results: The gene expression profile of SP17 showed a significant down-regulation between experimental I as well as experimental II and control group (p < 0.005 and p < 0.0007, respectively). In contrast, SerpinA5 mRNA level was significantly down-regulated in experimental groups I (p < 0.05). Both experimental groups showed an increase in PRDX2 mRNA level. However, there was a significant association between experimental group II and controls based on PRDX2 gene expression. Also, there was no significant difference between three groups in accordance of AnnexinA2 gene expression levels. The results demonstrated significant higher rates of CMA3+ and AO+ sperm cells in both experimental group I and II compared to the controls. The most numbers of necrotic sperm cells were detected in experimental group I based on PI staining. However, we found no significant change in early apoptotic rates (ANXV+) of sperm specimen between all study groups. There was a significant decrease in acrosome-reacted spermatozoa in experimental group I in comparison with controls. Furthermore, a significant positive correlation was seen between seminal MDA and TAC concentration.
Conclusion: The data indicates that Sp17 not only has potential functions in the fertilization process, but also in the developing embryo at stages of implantation and pregnancy maintenance. SerpinA5 gene expression is strongly associated with abnormal sperm morphology. SDF plays a role as a major cause of male infertility independent of the sperm parameters.
Key words: Sperm parameter, Gene expression, DNA fragmentation, Reactive oxygen species.
 
O-32
Morphological evaluation of oocytes with image processing methods in patients undergoing intracytoplasmic sperm injection
 
Eslamdoost AR¹, Mehrafza M², Aghajani S².

1. Department of Engineering, Lahijan Branch, Islamic Azad University, Lahijan, Iran.
2. Mehr Fertility Research Center, Guilan University of Medical Sciences, Rasht, Iran.

Email: alireza.eslamdoost.ec@gmail.com
 
Background: Morphological assessment of oocyte quality is one of the most essential and sensitive steps in infertility treatment and deciding on treatment type. Oocytes abnormalities can be seen and detected under a microscope by embryologists. Diagnosis of the type and severity of abnormalities in in vitro fertilization centers is done by embryologists. Diagnosis is based on the appearance of the egg and according to scientific standards. Factors such as fatigue, inexperience and taste can cause differences in the outcome. Using image processing and morphological detection techniques, the egg and its cytoplasm can be identified and its features extracted. Finally, with the help of the decision tree, the normal egg can be distinguished from abnormal.
Objective: This study aimed to evaluate human oocyte abnormalities by image processing. Our goal was to develop a diagnostic tool that can analyze microscope images of human oocytes and derive a detection of the oocyte cytoplasm and zona-pellucida that is functional for quality evaluation in assisted insemination.
Materials and Methods: The approach of the present study includes four main phases: 1) segmentation, 2) feature extraction, 3) learning model (decision tree), and 4) model assessment. In the segmentation phase we use two algorithms, first, the oocyte was identified, and then the required features are extracted with the help of the second algorithm using the Hough transform. In the second phase, the extracted features are used to diagnose oocytes according to 11 different, including cytoplasmic and zona-pellucida abnormalities. This approach is made by wavelet transforming and Fourier-conversion. To this aim, we evaluate some statistics in the Haar wavelet transform domain. In the third phase, the normal oocyte was distinguished using decision tree model learning from the abnormal. Finally, a program was written using the Python programming language that has the ability to distinguish normal from abnormal oocyte.
Results: In this study, using innovative oocyte and cytoplasm algorithms, it was identified with great accuracy. 700 photos were received from Mehr Infertility Center Rasht. In all cases, the designed algorithms succeeded in distinguishing normal from abnormal oocyte. Software was developed using the Python programming language to distinguish normal from abnormal oocyte.
Conclusion: This study reported experimental results on a collection of microscope images of oocytes Indicated the proposed approach's effectiveness. It seems that, measuring the quality of oocytes with image processing helps classify oocytes into normal and abnormal without human intervention.
Key words: Biomedical image processing, Decision tree, Human oocyte, Intracytoplasmic sperm injection.
 
O-33
Does dual trigger with human chorionic gonadotropin and gonadotropin releasing hormone agonist improve the outcome of IVF in poor responder women?
 
Lubis HP, Halim B.
Halim Fertility Center IVF Clinic, Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia.
Email: hilmaputrilubis@gmail.com
Background: Poor responder has lower number of follicle and mature oocytes after ovarian stimulation. There are various protocols that have been performed to improve In vitro fertilization (IVF) outcome in poor responders. Dual trigger is one of the triggering protocol to increase the number of retrieved oocytes, the number of mature oocytes, fertilized embryos, implantation and pregnancy rates in normal and high responder women. Several studies have investigated the efficacy of dual triggers in poor responders and the results are still controversial.
Objective: To investigate the efficacy of dual trigger consisted of human chorionic gonadotropin (hCG) plus gonadotropin-releasing hormone agonist (GnRH-a) for final oocyte maturation in increasing number of oocytes retrieved, number of mature oocyte and cleavage embryo in poor responder women.
Materials and Methods: A retrospective analytic study was performed in 260 cycles fulfilling the POSEIDON group 3 and group 4 criteria from January 2018 until October 2019 in Halim Fertility Center IVF Clinic. All poor ovarian responder women underwent modified natural cycle protocol for IVF cycles. Final maturation of oocytes was divided into two groups: group I (114 cycles) received 250 µg of recombinant hCG alone as the single trigger and group II (146 cycles) triggering was done with coadministration of 250 µg of recombinant hCG plus 1 mg GnRH-a simultaneous as dual-trigger. Baseline characteristics and cycle parameters, as well as IVF outcomes of two groups were compared.
Results: In this study, there was no significant difference in the number of retrieved oocytes between the two groups but the number of mature oocytes (MII) was higher in the dual trigger group than single trigger but there was no significant differences between them (p > 0.05). The number of fertilized oocytes (2PN) and the number of cleavage embryo were higher in the dual trigger than single trigger groups but there were no significant differences between the two groups (p > 0.05).
Conclusion: Dual trigger for final oocyte maturation might improve the outcome of IVF cycles in poor responder women.
Key words: Dual trigger, Poor responder, In vitro fertilization.
 
O-34
The experience and needs of puerperal women who have had a child following assisted reproductive technologies (ART): A qualitative study
 
Warmelink JC.
Department of Midwifery Science, Amsterdam Public Health Research Institute, University of Amsterdam and University of Groningen, Department of General Practice and Elderly Medicine, Section Midwifery Science, Academy Midwifery Amsterdam and Groningen, Amsterdam, Groningen, The Netherlands.
Email: catja.warmelink@inholland.nl
Background: There is a steadily increasing number of newborns born following assisted reproductive technologies (ART), in Europe. Women who became pregnant ART in the Netherlands are under the care of a primary midwife. It is known that during pregnancy, these formal infertile women might have specific experiences such as anxiety and insecurity and paradoxical needs in maternity care. Little is known about how they experience the first few weeks after birth (puerperium).
Objective: The aim of this research is to investigate how women who have had a child after ART experience puerperium and what care needs these women have.
Materials and Methods: From 2017 till 2020, we interviewed sixteen women were interviewed who had a child after fertility treatment by in vitro fertilization or intracytoplasmic sperm injection. This explorative, qualitative study was based on the constructivist paradigm, using a comparison/grounded theory design.
Results: The three themes that emerged from the analysis were 1) the puerperal woman, 2) the caregiver and 3) parenting. The main need of the he puerperal women was to be able to talk about their experiences “when the baby arrived, I just couldn't believe it was my child”. From the care provider, they needed understanding “She only had to say one sentence ‘so I know it's different for you” coordinated information and continuity of care. The processes that underlie this are the transition to parenthood, insecurities “Oh I think it's all scary", the unreality “I never learned how to take care of a child because I did not believe that a child would come” and gratitude in having a child.
Conclusion: Fertility treatment and the additional uncertainties are mentioned as reasons whether or not to prepare for the puerperium and to have little expectations regarding puerperium. It is important for care providers to be aware of the experiences of the women, to make space for emotions, show understanding and give tailored information and care. In further research, we would like to explore the views of the partners and couples with different ethnic and cultural backgrounds.
Key words: Intracytoplasmic sperm injection, Puerperium, ART.
 
O-35
Immunology and immunotherapy in RIF and recurrent miscarriage
 
Emami F.
Research and Clinical Center for Infertility, Yazd Reproductive Sciecnes Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: miss.dremami@gmail.com
 
The most RSA is mainly due to immune factors and Mainly affects 30~40 years old women. Three main players of immune system that affecting implantation are 1) TH1–TH2 balance 2) natural killer cells, and 3) autoantibodies. In pregnancy we have shift toward TH2 dominance and Th2 cells act as an antagonist against embryo cytotoxicity of Th1 cells. There is no screening tests for Th1 and Th2 and rising progesterone levels can stimulate Th2 and inhibit Th1 secretions. Association between autoantibodies and miscarriage is clear but association between autoantibodies ART failure is inconclusive. In RIF, ACLA and anti-β2glycoprotein1 antibodies are not detect but LAC is more often detected and some evidence suggest that it is reasonable to test these antibodies in RIF. Implantation is interaction between maternal killer immunoglobulin-like receptors (KIRs) (expressed by (uNKs)) and fetal human leukocyte antigen (HLA) (expressed by extravillous trophoblasts). There is different haplotypes for KIR and HLA and if there is haplotype KIR AA and HLA-C2C2, they may lead to RIF, recurrent miscarriage, preeclampsia, and (IUGR). Excessive inhibition of uNK cell may lead to pre-eclampsia and low birth weight. And strong activation of uNK cells can lead to macrosomia. uNK measurement is possible directly by uterine biopsy but it is inaccurate and indirectly by assessment of peripheral blood NK cells (CD56 (majority of these cells)-CD16-CD38). There is dramatic increase in the mid-secretory phase starting 6-7 days after the LH surge but routine testing in RIF is not advised. There is different chimeric fetal cells during pregnancy and H-Y antigene is one of them and is male-specific minor antigens and H-Y antibody produce against it in patients with secondary RPL who had a firstborn male. HLA class II alleles and HLA-G polymorphisms in conjunction with H-Y Antibodies may lead to RPL and RIF (in male and female fetuses) and homosexuality in male fetuses (due to inhibitory effect on the masculinization of the brain). Due to different immunological problems there is different Immunotherapies like Intravenous human immunoglobulin, steroids, anti-TNF drugs, intra lipid, immunosuppressant drugs and Immunization with lymphocytes that is the most studied immunologic treatment for RM and it consiste of PBMCs that were isolated by centrifugation of patients’ husband`s blood and administered intradermally. And increase blocking antibody (kind of IgG) that Inhibits lymphocyte reaction and deters the immune system’s attack on embryos. And also Increase concentration of TGF-β1 and Restoring balance in the Th1/Th2 and Treg cells. It should use fresh, intradermally, befor, and during pregnancy with low dose (less than 1 × 10⁸ lymphocytesc) and paternal source is better than other sources.
 
O-36
The serum levels of insulin-like growth factor-1 as a prognostic and diagnostic tool in IVF
 
Jalaliani S.
Research and Clinical Center for Infertility, Yazd Reproductive Sciecnes Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: Samane.jalaliani@gmail.com
 
Insulin-like growth factor 1 (IGF-1), is a small single-chain polypeptide, secreted by liver in response to GH. The IGF-1 is expressed in most tissues but has a specific role in amplification of gonadotropin hormonal action during follicular growth and development.we want to discuss about some possible benefit of checking IGF-1 serum level in ART.
Measuring GH levels in the serum may not reflect true GH status as the hormone is released in a pulsatile manner, mainly during the night. We can check IGF-1 serum level in order to determine which women may benefit from GH as an adjuvant therapy. It would also be useful to know whether the baseline serum IGF-1 level has any predictive value in women with normal ovarian reserve. High levels of IGF-1 in day 2 in these patients predict a poorer response than expected based on traditional ovarian reserve markers so, this marker could be used to guide the starting dose and protocol selected for these patients. The most important uses of checking IGF-1 serum level is in poor responders group. The poor responder group demonstrated more than two fold increase in the mean serum level of IGF-1 in cycle day 2 compared with normal responders, and a three fold increase compared with the high responder group . IGF-1>72 ng/ml in day in the poor responder group had 70% sensitivity and 78% specificity for a negative outcome. Cycle day 2 IGF-1 serum levels are predictive for a negative outcome to COH in the poor responders group. We can check IGF-1 serum level for improving IVF outcome and this parameter could be used to: 1) reflect true GH status and AGHD diagnosis to determine which women may benefit from GH as an adjuvant therapy. 2) to predict a poorer response than expected based on traditional ovarian reserve markers. 3) to guide the starting dose and protocol selected for patients. 4) to predict negative outcome to COH in the poor responders group. 5) to determine which women benefit from LE pretreatment.
 
O-37
Use of menstural blood drived-mesenchymal stem cells in infertility field
 
 
Pejman A.
Research and Clinical Center for Infertility, Yazd Reproductive Sciecnes Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Email: dr.atpejman@yahoo.com
 
Mesenchymal stem cells (MSC) are highly important in regenerative medicine because of their inherent regenerative properties. They can be harvested from several adult tissues, such as the bone marrow, umbilical cord, peripheral adipose tissue, placenta, menstrual blood, fluid, and amniotic fluid. They are an excellent source of growth factors/ cytokines. Menstural blood-mesenchymal stem cells (MB-MSCs) can be isolated from menstrual blood. These cells have high proliferative, self-renewal, and multiple differentiation potentials. MB-MSCs expressed surface markers CD9, CD29, CD41a, CD44, CD59, CD73, CD90, and CD105; human telomerase reverse transcriptase, etc. It has been demonstrated that MB-MSCs could differentiate into ovarian tissue-like cell and differentiation of MB-MSCs into germ cells. MB-MSCs were easy to access compared with bonemarrow-MSCs and umbilical-MSCs. Using of HMB-MSC in field of infertility such as POF, endometriosis, and Asherman syndrome has been investigated. Appliance of MSC to treat female infertility such as a Asherman syndrome, endometriosis, premature ovarian failure and poor ovarian responder studied in some article and the researchers believed that MB-MSC might improve infertility in the disease mentioned above by gene expression.
 
O-38
The effect of autologous platelet-rich plasma on in vitro maturation of immature human oocytes
 
Faizi F¹, Allahveisi A².

1. Department of Animal Sciences, Faculty of Biological Sciences, Shahid Beheshti University of Sciences, Tehran, Iran.
2. Department of Anatomy, Infertility Center, Besat Hospital, Kurdistan University of Medical Sciences, Sanandaj, Iran.

Email: farid.faizi69@gmail.com
 
Background: Elements in the culture medium can have an increasing effect on the growth and development of follicles and oocytes in the laboratory. In this study, the effect of autologous platelet enriched plasma on the maturation of immature human oocytes in vitro was investigated.
Objective: Blood contains many platelets that can be used enriched. Platelet-rich plasma (PRP) has been used for more than a decade now in a variety of fields, such as surgical wound healing or unhealed fractures. The use of autologous PRP has the advantage that it does not cause allergic reactions in the patient and can be easily used.
Materials and Methods: The follicles were cultured in culture medium for 12 days and the medium contained 5 and 10% of platelet extract .The culture medium was changed every other day and finally, the percentage of survival and growth of oocytes was examined under a microscope at the end of culture. After 12 days of culture, oocytes showed significant growth in environments containing platelet extract and were able to reach the size of mature oocytes in the control environment, but at the end of 12 days of culture, the highest survival belonged to the experimental medium containing 5% platelet and the number of oocytes in other groups showed a significant decrease compared to this experimental group.
Results: As a result of this study, it was shown that PRP improves the culture medium of immature oocytes and is effective in its growth and survival rate.
Conclusion: At the end of the culture, the best survival was related to 5% platelet-rich plasma, but using different doses of PRP and other tests with more immature oocytes are recommended. It seems that platelet can be used as a supplement or a suitable alternative to regular serums.
Key words: Immature oocytes, In vitro maturation, Plated-rich Plasma.
4^th Congress of Reproductive Genetics
 
O-39
A novel biallelic missense variant in cyclin B3 is associated with failure of oocyte meiosis II and recurrent fetus triploidy
 
Fatemi NS^{1, 2}, Salehi N², Pignata L^{3, 4}, Palumbo P⁵, Vittoria Cubellis M⁶, Ramazanali F⁷, Ray P^{8, 9}, Varkiani M², Reyhani-Sabet F², Biglari A¹, Sparago A³, Acurzio B^{3, 4}, Palumbo O⁵, Carella M⁵, Riccio A^{3, 4}, Totonchi M^{2, 10}.

1. Department of Genetics and Molecular Medicine, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran.
2. Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
3. Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Università Degli Studi Della Campania Luigi Vanvitelli, Caserta, Italy.
4. Institute of Genetics and Biophysics, Adriano Buzzati-Traverso, Consiglio Nazionale delle Ricerche, Naples, Italy.
5. IRCCS-Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG), Italy.
6. Department of Biology, Università Degli Studi di Napoli, Federico II, Napoli, Italy.
7. Department of Endocrinology and Female Infertility, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
8. Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, INSERM 1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France.
9. Unité Médicale de génétique de l'infertilité et de diagnostic pré-implantatoire (GI-DPI), Centre Hospitalier Universitaire Grenoble Alpes, Grenoble, France.
10. Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.

Email: totonchimehdi@gmail.com
 
Background: Recurrent pregnancy loss (RPL) is an important infertility-related complication affecting up to 5% of clinical gestations. The both parental and embryonal factors are associated with RPL. Triploidy is one of the common chromosomal abnormalities affecting pregnancy and accounts for an important portion of first-trimester abortions. Triploidy has been reported in some cases of RPL but its underlying molecular mechanism remains unknown.
Objective: The aim of this study was to determine the genetic causes of RPL associated with fetus triploidy in an Iranian family.
Materials and Methods: We examined the status of genomic imprinting, short tandem repeat (STR) markers and performed the whole exome sequencing in a family including two sisters with RPL history. Additionally, we assessed oocyte maturation in vivo and in vitro and effect of the candidate protein variant in silico.
Results: Triploidy of maternal origin was confirmed in the aborted fetuses by STR markers genotyping. All the maternally inherited pericentromeric STR alleles were homozygous in the fetuses and oocytes maturation was deficient. A new deleterious missense variant (c.T4050A, p.V1251D) of the cyclin B3 gene (CCNB3) was identified by whole exome sequencing. The homozygous mutation affecting a residue conserved in placental mammals and located in a region that can interact with the cyclin-dependent kinases co-segregated in homozygosity with RPL.
Conclusion: Here, we report a family in which a novel damaging variant in cyclin B3 is associated with the failure of meiosis II in oocyte and recurrent fetus triploidy, implicating a rationale for CCNB3 testing in RPL patients.
Key words: Recurrent pregnancy loss, Triploidy, Whole exome sequencing, CCNB3.

The original full text of this abstract has been published in Journal of Medical Genetics 2020 Sep 16; 0:1-6. https://doi.org/ 10.1136/jmedgenet-2020-106909.
How to cite to this article: Fatemi NS, Salehi N, Pignata L, Palumbo P, Vittoria Cubellis M, Ramazanali F, Ray P, Varkiani M, Reyhani-Sabet F, Biglari A, Sparago A, et al. Biallelic variant in cyclin B3 is associated with failure of maternal meiosis II and recurrent digynic triploidy. Journal of Medical Genetics 2020; 0: 1-6.

O-40
Challenges in herbal personalized medicine
 
Mosadegh AM¹, Shams Ardakani M².

1. Traditional Pharmacy, School of Persian Medicine, Tehran University of Medical Sciences, Tehran.
2. School of Persian Medicine, Tehran University of Medical Sciences, Tehran, Iran.

Email: ali.moh.mosadegh@gmail.com
 
Traditional medicines have been used for years. They are the oldest and most diverse forms of healing that form the foundation of medical systems in many regions of the world. Every continent has its version of traditional medicine. Although modern medicine is the foundation of treatment nowadays, herbal and traditional medicine can help this system in various ways farther than it is estimated. WHO has been studying and working on traditional and herbal medicine for years and defines traditional medicine as “the sum total knowledge, skills and practices based on the theories, beliefs and experiences indigenous to different cultures, whether explicable or not, that are used to maintain health, as well as to prevent, diagnose, improve or treat physical and mental illness.” and is trying to help the world benefit from its different potentials. The positive benefits of T&M medicine are that they may not be as costly as modern therapies and medications, they are accessible for local communities, making them a vital part of well-being and the belief systems in these parts of the world. The practitioners use plants and herbal elements to treat a wide variety of ailments and diseases. Now it should be noted that the key element in medication in traditional and herbal medicine is that it is a personal based therapy. Because of the lack of genetics knowledge in the older ages the factor interfered to dissociate patients for receiving the proper therapy they need was their phenotypical properties from skin color to height, weight, body mass, and etc. Herbal and traditional medicine does not have a specific pathway to cure an ailment because of the multicomponent structure of the drugs and therapies that are usually built up of a few herbal plants. Therefore comparing it to precision medicine is unsustainable. Precision drugs are medicines that trigger a reaction in the body that is easily measurable and identical. Modern practitioners although using traditional and herbal medicine in their personal life usually don’t have the confidence and knowledge needed to interact with the medical situations they confront. In Iranian traditional medicine, ‘’Mezaj” is a key concept in defining human health and disease. In this view, just as the fingerprints of no two people are the same, the “Mezaj” and composition of no two people are the same, and also in many diseases, certain changes occur in the “Mezaj” of the individual. It is believed that by dividing patients based on the type of disease and considering the individual “Mezaj” and the “Mezaj” of the disease and combining this issue with the specific “Mezaj” of drugs it can be more successful in predicting the effectiveness of the drug or the possibility of side effects. In other words, it is possible to shorten the path to pharmacogenetic goals based on the “Mezaj” phenotype.
Key words: Herbal and traditional medicine, Pharmacogenomics, Mezaj.
 
O-41
Investigating the expressions of miRNA-125b and TP53 in endometriosis: Does it underlie cancer-like features of endometriosis?
 
Hajimaqsoudi E¹, Darbeheshti F^{2, 3}, Kalantar SM⁴, Javaheri A⁵, Mirabutalebi SHR ⁶, Sheikhha MH⁴.

1. Department of Genetics, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences,Yazd, Iran.
2. Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences,Tehran, Iran.
3. Breast Cancer Association (BrCA), Universal Scientific Education and Research Network (USERN), Tehran, Iran.
4. Abortion Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi Universityof Medical Sciences, Yazd, Iran.
5. Department of Obstetrics and Gynecology, Faculty of Medicine, Shahid Sadoughi Hospital, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
6. Student Research Committee, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: sheikhha@yahoo.com
 
Background: Endometriosis is generally considered as a benign condition, but there is a possibility for it to become cancerous. miR-125b was upregulated in both endometriotic tissues and serum samples of women with endometriosis but its potential targets in endometriosis are still not fully understood.
Objective: The role of miR-125b in the regulation of TP53 expression in endometriosis was tested with a bioinformatics approach. In addition, the expression of miR-125b and TP53 in both eutopic endometrium (Eu-p) and ectopic endometrium (Ec-p) in endometrium tissues of patients with endometriosis was compared to these in the normal endometrium tissues of controls (Normal).
Materials and Methods: In this case-control study, the eutopic and ectopic samples were collected from 20 patients who underwent laparoscopic surgery and the normal endometrium tissues were collected from 20 controls with no evidence of endometriosis. For bioinformatics approach a protein-protein interaction network was constructed based on co-expressed potential targets of miR-125b. Quantitative PCR technique was used for measurement of miR125b and TP53 expression.
Results: Our results showed that miR-125b was significantly overexpressed in Ec-p. In addition, there was a significant TP53 underexpression in both Ec-p and Eu-p samples compared with normal tissues.
Conclusion: There was a negative correlation between miR-125b and TP53. In addition we observed a noticeable decreased expression of TP53 in both Ec-p and Eu-p samples. These findings may be interpreted as the roles of miR-125b/TP53 axis in the pathogenesis of endometriosis. With the help of bioinformatics analyses we conclude that there is a possible role of miR-125b in cancer-like features of endometriosis.
Key words: Endometriosis, TP53, miR-125b, Ectopic endometrium.

The original full text of this abstract has been published in Int J Reprod BioMed 2020; 18 (10): 825-836. https://doi.org/ 10.18502/ijrm.v13i10.7767.
How to cite to this article: Hajimaghsoudi E, Darbeheshti F, Kalantar SM, Javaheri A, Mirabutalebi S H, Sheikhha MH. Investigating the expressions of miRNA-125b and TP53 in endometriosis. Does it underlie cancer-like features of endometriosis? A case-control study. Int J Reprod BioMed 2020; 18 (10) :825-836.

 
O-42
Evaluation of the miR-144 and its candidate target gene expression in cumulus cells and its impact on in vitro maturation of oocyte in patients with polycystic ovary syndrome (PCOS)
 
Shafienia H¹, Montazeri F², Heydari L³, Khalili MA³, Sheikhha MH⁴, Mazloomzadeh S⁵, Biglari A¹.

1. Department of Genetics and Molecular Medicine, School of Medicine, Zanjan University of Medical Sciences (ZUMS), Zanjan, Iran.
2. Abortion Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences and Health Services, Yazd, Iran.
3. Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences and Health Services, Yazd, Iran.
4. Biotechnology Research Center, International Campus, Shahid Sadoughi University of Medical Sciences and Health Services, Yazd, Iran.
5. Department of Epidemiology and Statistics, School of Medicine, Zanjan University of Medical Sciences (ZUMS), Zanjan, Iran.

Email: Biglari63@hotmail.com
Background: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of reproductive age. One of the problems in IVF cycles in PCOS women is predisposing to develop ovarian hyperstimulation syndrome. Therefore, in vitro maturation (IVM) of the oocytes has grown as an alternative treatment. Transcriptomic signatures of cumulus cells (CC) have the potential to serve as valuable non-invasive biomarkers for oocyte competence. Recent studies suggest miRNA involvement in regulating follicular growth, differentiation and development. miR-144 is one of the miRNAs that has been shown to involve in oocyte maturation.
Objective: In this study, the expression level of miR-144 and cyclooxygenase-2 (COX-2) as its candidate target gene was examined in women with PCOS, then its impact on IVM outcome of oocyte was evaluated.
Materials and Methods: A total of 30 cumulus-oocyte complexes with oocyte at GV stage were retrieved from 20 women with PCOS during IVF cycles and cultured in IVM medium for 24 hr at 37°C. After IVM, maturity of oocytes was assessed through morphological criteria and the samples were divided into two groups: matured and unmatured oocytes. The expression level of miR-144 and COX-2 in CCs of each group were detected by qRT-PCR and the relation between the expression level of them and IVM of oocytes was evaluated.
Results: In the 30 retrieved GV oocytes, 18 oocytes (60%) were matured after IVM and placed in matured group, whereas 12 oocytes (40%) could not mature and placed in U group. We found that the expression level of miR-144 was lower (P-value: 0.0008) and the COX-2 mRNA level was higher (P-value: 0.005) in CCs of matured group than in CCs of unmatured group. So, the selected miRNA was related to oocyte nuclear maturation in PCOS women.
Conclusion: We determined that the expression profile of miR-144 and COX-2 were different in CCs isolated from oocytes that could mature after IVM compared with those that could not in PCOS women. Since oocyte competence has an important role in formation of normal zygote and blastocyst, the expression level of this miRNA can be used for predicting oocyte quality before IVM process.
Key words: Polycystic ovary syndrome, In vitro maturation, miR-144, COX-2, Cumulus cells.
 
O-43
Evaluating senescence of amniotic fluid mesenchymal stem cell in different passages by Q-PCR analysis of FoxM1 gene
 
Zare E¹, Javid A², Hoseini SM³, Montazeri F⁴.

1. Biology Department, Faculty of Sciences, Science and Art University, Yazd, Iran.
2. Department of Mulecular Genetics Faculty of Basic Science and Advanced Technologies in Biology, University of Science and Art, Yazd, Iran.
3. Biotechnology Research Center, International Campus, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
4. Abortion Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: F_montazeri@outlook.com
 
Background: Stem cells are undifferentiated cells that have the ability to transform and differentiate into different cell types. These cells have two important characteristics that differentiate them from others. They have the ability to reproduce unlimited and remain in the undifferentiated state. The most important mature types of these cells are mesenchymal cells, which become more susceptible to accumulation of cell damage with passing time and increase longevity. These damages can help to improving recovery, senescence and finally death of cells. Various factors cause the intrinsic and harmful process of senescence such as internal factors as genetics, the expression of some genes as P53, Nuclear factor NF-kappa-B and Forkhead Box M1 (FoxM1). free radicals and external factors such as environmental changes that affect the function of the cell. FoxM1 is a member of the Forkhead transcription family, which has been actively involved in regulating organism growth, differentiation and cell proliferation and is important for the expression of cell cycle-dependent genes in the G2 phase.
Objective: The main goal of this study was to evaluate the expression level of FoxM1 gene as a marker of senescence in mesenchymal stem cells isolated from human amniotic fluid at different passages.
Materials and Methods: Totally 37 amniotic fluid samples were obtained from pregnant mothers referred to the PND Department of Yazd Reproductive Sciences Institute. After culturing successive passages and examining the cells morphologically and characterizing them by flow cytometry, their aging status was evaluated in several passages by using beta-galactosidase (X-gal Cinna Gene) staining. After RNA extraction by Tripure kit and cDNA synthesis by Thermo Fisher kit, 10 samples in passages 4 and 7 were evaluated for FoxM1 gene expression change as a marker of aging and GAPDH gene as internal reference using (quantitative PCR) technique. The data were analyzed using GraphPad Prism and SPSS version16 software.
Results: Microscopic examination and staining of beta-galactosidase showed that mesenchymal stem cells isolated from amniotic fluid enter the aging stage in different passages. Comparing the results of FoxM1 gene expression in different passages (2, 4, and 7), showed a statistical meaningful increase of expression in old cells compared to young cells (F = 10.43; P < 0.001). Despite the increased expression of FoxM1 gene in the passage 4 compared to the young cells in passage 2, indicated that there was no significant difference between two groups (t = 1.134, p<0.05). Comparison of FoxM1 gene expression in aging cells of passage 7 compared with young cells of passage 2, showed that the increase was statistically significant (t = 3.758; p < 0.003).
Conclusion: FoxM1 gene expression in cellular aging has an effective role in preventing cellular aging, and control of aging-related traits includes reducing cell doubling time, regenerative power, and differentiation potential.
Key words: Mesenchymal stem cells, Amniotic fluid, Cellular senescence, FoxM1 gene expression, Q-PCR.
 
O-44
Comparison of polymorphism 139 C> A (rs737008) of protamine 1 gene in infertile men with diagnosis of oligospermia and asthenospermia referred to Gerash Infertility Treatment Center from 2016 and 2017
 
Mohsenzadeh M¹, Dehghani Ashkezari M², Pirouzi A³, Saadat S².

1. Gerash Al-Zahra Fertility Center, Gerash University of Medical Sciences, Gerash, Iran.
2. Medical Biotechnology Research Center, Ashkezar Branch, Islamic Azad University, Ashkezar, Yazd, Iran.
3. Cellular and Molecular Gerash Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.

Email: mdashkezary@yahoo.com
 
Background: The male infertility accounts for about half of infertility in couples. The idiotypic asthenospermia and oligospermia, which mostly occur as a result of genetic mutations, are among the main causes of male infertility.
Objective: Until now, the relationship between different SNPs in the protamine1 (PRM1( gene and male infertility has been reported. In this study, we evaluated the possible correlation between 139 C> A (rs737008) SNP in the PRM1 gene and asthenospermia/oligospermia in patients who referred to the Gerash Infertility Center.
Materials and Methods: Three groups were considered in this study including healthy fertile males, asthenospermia patients, and the patients suffering from oligospermia. After DNA extraction from their blood samples, the PCR was carried out to amplify a 558 bp PRM1 gene fragment. Then, the RFLP technique was performed to identify the SNP in the PCR products.
Results: Our results showed that the frequency of the 139 C> A (rs737008) SNP in the population study was 41%. We found no significant differences between the SNP and asthenospermia/oligospermia in the current study. According to the demographic data, no significant differences were also found between smoking or alcohol consumption and male infertility in this study.
Conclusion: In this study, no significant relationship between male infertility and the frequency of the rs737008 polymorphism was observed. It seems that a wider investigation on the other SNPs within the protamin gene will help us to provide more reliable information in this context.
Key words: Polymorphism, Protamin, Asthenospermia, Oligospermia, Male infertility.
O-45
Exploring the dysregulated mRNAs–miRNAs–lncRNAs interactions associated to idiopathic non-obstructive azoospermia
 
Sabetian S¹, Zarei M², Namavar Jahromi B^{1, 3}, Morowvat MH², Tabei SMB⁴, Cava C⁵.

1. Infertility Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
2. Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
3. Department of Obstetrics and Gynecology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
4. Department of Medical Genetics, Shiraz University of Medical Sciences, Shiraz, Iran.
5. Institute of Molecular Bioimaging and Physiology (IBFM), National Research Council (CNR), Milan, Italy.

Email: soudabehsabet@gmail.com
 
Background: Non-obstructive azoospermia (NOA) is the most clinical problem in case of infertility. About 70% of NOA patients are idiopathic with uncharacterized molecular mechanisms.
Objective: This study aimed to analyze the possible pathogenic miRNA-target gene interaction and lncRNA-miRNA association involved in NOA.
Materials and Methods: In the current study, differentially expressed (DE) nRNAs, miRNAs and lncRNAs were determined using the microarray dataset and statistical software R. miRNAs–mRNA and miRNA-lncRNA interactions were identified and the base-pair binding between the seed region of miRNAs and complementary nucleotides in 30 UTR of      mRNAs were analyzed. The influence of the validated single nucleotide polymorphisms was described by calculating the minimum free energy (MFE) of the interaction.
Results: A total of 74 mRNAs, 14 miRNAs, and 10 lncRNAs were identified to have significant differential expression in testicular tissue between patients and the fertile group. Four of the DE-mRNAs and all of the reported DE-miRNAs were upregulated. In addition, all of the represented DE-lncRNAs were showed to be downregulated. miR-509-5p and miR-27b-3p were found to interact with target gene polo-like kinase 1 (PLK1) and Cysteine-rich secretory protein2 (CRISP2), respectively. Rs550967205 (A > G) positioned at 30 UTR CRISP2 and rs544604911 (T > C) located at 30 UTR PLK1, with lowest MFE in miRNA-mRNA interaction, were assumed to have possible pathogenic roles linked to spermatogenesis arrest.
Conclusion: The results of the study provide new clues to understand the regulatory roles of miRNAs and lncRNAs in the pathogenesis and diagnosis of idiopathic azoospermia.
Key words: Azoospermia, mRNA, miRNA, lncRNA, Gene expression.

The original full text of this abstract has been published in Journal of Biomolecular Structure and Dynamics 2021: 16: 1-9. https://doi.org/10.1080/07391102.2021.1875879.
How to cite to this article: Sabetian S, Zarei M,  Namavar Jahromi B, Morowvat MH, Tabei SM, Cava C. Exploring the dysregulated mRNAs–miRNAs–lncRNAs interactions associated to idiopathic non-obstructive azoospermia. Journal of Biomolecular Structure and Dynamics 2021: 16: 1-9.

O-46
Testicular expression of TDRD1, TDRD5, TDRD9, and TDRD12 in azoospermia
 
Babakhanzadeh E^{1, 2}, Khodadadian A¹, Rostami S³, Alipourfard I^{4, 5}, Aghaei M¹, Nazari M¹, Hosseinnia M⁶, Vahidi Mehrjardi MY^{1, 2}, Jamshidi Y⁷, Ghasemi N^{1, 8}.

1. Department of Medical Genetics, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Medical Genetics Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
3. Department of Cellular and Molecular Biology, Faculty of Science, Azarbaijan Shahid Madani University, Tabriz, Iran.
4. Center of Pharmaceutical Sciences, Faculty of Life Sciences, University of Vienna, Vienna, Austria.
5. School of Pharmacy, Faculty of Sciences, University of Rome Tor Vergata, Rome, Italy.
6. Department of Biology, Faculty of Science, University of Guilan, Rasht, Iran.
7. Genetics Centre, Molecular and Clinical Sciences Institute, St George’s University of London, London, UK.
8. Abortion Research Center, Yazd Reproductive Sicences Institue, Shahid sadoughi University of Medical Sciences, Yazd, Iran.

Email: nghasemi479@gmail.com
 
Background: Tudor domain-containing proteins (TDRDs) play a critical role in piRNA biogenesis and germ cell development.
Objective: piRNAs, small regulatory RNAs, act by silencing of transposons during germline development and it has recently been shown in animal model studies that defects in TDRD genes can lead to sterility in males.
Materials and Methods: Here we evaluate gene and protein expression levels of four key TDRDs (TDRD1, TDRD5, TDRD9 and TDRD12) in testicular biopsy samples obtained from men with obstructive azoospermia (OA, n = 29), as controls, and various types of non-obstructive azoospermia containing hypospermatogenesis (HP, 28), maturation arrest (MA, n = 30), and Sertoli cell-only syndrome (SCOS, n = 32) as cases. One-way ANOVA test followed by Dunnett’s multiple comparison post-test was used to determine inter-group differences in TDRD gene expression among cases and controls. 
Results: The results showed very low expression of TDRD genes in SCOS specimens. Also, the expression of TDRD1 and TDRD9 genes were lower in MA samples compared to OA samples. The expression of TDRD5 significantly reduced in SCOS, MA and HP specimens than the OA specimens. Indeed, TDRD12 exhibited a very low expression in HP specimens in comparison to OA specimens. All these results were confirmed by Western blot technique.
Conclusion: TDRDs could be very important in male infertility, which should be express in certain stages of spermatogenesis.
Key words: Spermatogenesis, Non-Obstructive Azoospermia, piRNAs, TDRD genes.

The original full text of this abstract has been published in BMC Medical Genetics 2020; 21(1): 1-7. https://doi.org/10.1186/s12881-020-0970-0.
How to cite to this article: Babakhanzadeh E, Khodadadian A, Rostami S, Alipourfard I, Aghaei M, Nazari M, Hosseinnia M, Vahidi Mehrjardi MY, Jamshidi Y, Ghasemi N. Testicular expression of TDRD1, TDRD5, TDRD9 and TDRD12 in azoospermia. BMC Medical Genetics 2020; 21(1): 1-7.

 
O-47
Correlation between long non-coding RNA MALAT1 and HOTAIR expression with sperm parameters and MDA level in infertile men
 
Jaberi Asl A¹, Dashti GhR¹, Sharifi M².

1. Department of Anatomical Sciences, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
2. Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

Email: grd_dashti@yahoo.com
 
Background: Infertility is a common complete disorder, which can be caused by oxidative stress. Accumulating evidence suggest that long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and HOX transcript antisense RNA (HOTAIR) is involved in the regulation of the oxidative stress responses.
Objective: We aimed to investigate the possible expression status of MALAT1 and HOTAIR in the sperm and its correlation between sperm parameters and malondialdehyde (MDA) levels.
Materials and Methods: Specimens were obtained randomly from 25 fertile men and 25 infertile men, aged between 25-55 yr old. Sperm parameters were evaluated by computer-aided sperm analysis. Sperm chromatin quality were assessed by acridine orange staining method. Seminal MDA levels were determined by thiobarbituric acid reaction method. The expression of MALAT1 and HOTAIR was detected by RT-PCR.
Results: A decreased level of MALAT1 and HOTAIR expression was observed to be associated with the infertile patients (p < 0.001). The relative expression level of MALAT1 and HOTAIR were positively correlated with motility and morphology (p < 0.001). Meanwhile we found the expression levels of genes were negatively correlated with sperm chromatin damage and MDA levels (p < 0.001).
Conclusion: The decreased expression of MALAT1 and HOTAIR resulted in high level of MDA, DNA denaturation and abnormal semen parameters. These findings exhibited the important implications of lncRNAs serving as a potential therapeutic indicator to assess male infertility in assisted reproductive procedures.
Key words: LncRNA, MALAT1, HOTAIR, Sperm, Infertile.
 
O-48
Y chromosome microdeletion in azoospermia fctor region in globozoospermic man
 
Hojati S¹, Miresmaeili SM¹, Montazeri F², Amiri S¹, Hoseini SM³, Kalantar SM², Fesahat F⁴.

1. Biology Department, Faculty of Sciences, Science and Art University, Yazd, Iran.
2. Abortion Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
3. Biotechnology Research Center, International Campus, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
4. Reproductive Immunology Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: smkyzd@gmail.com
 
Background: Thin long tail deletion of Y chromosome is the most common molecular genetic cause of infertility. It is considered to be severe in men which occurs in the three region of the azoospermic factor; AZFa, AZFb and AZFc. These region contain multiple genes involved in spermatogenesis.
Objective: The aim of this study was to investigate the Y chromosome deletion pattern among infertile men with globozoospermic referring to Yazd Infertility Treatment Center.
Materials and Methods: 19 infertile men referred to Yazd Reproductive Science Institute with globozoospermia (from 2014 to 2016) were studied considering microdeletions in Y chromosome. Using multiplex PCR and six different STS (Sequence-Tagged Site) markers microdeletions of Y chromosome in AZFa, AZFb and AZFc regions was analysed.
Results: In our samples, the deletion of AZF regions of the Y chromosomes was not abserved in any blood sample of globozoospermic man.
Conclusion: In 19 samples, no defect was observed in the AZF regions of the Y chromosomes was not the cause of globozoospermia.
Key words: Male infertility, Globozospermia, Y chromosome deletion, Azoospermic factor, multiplex PCR.
 
O-49
Evaluation of the expression level of miR-1271 and its association with the GRB2 gene expression in tissue samples of patients with endometriosis
 
Yarahmadi G¹, Vahidi Mehrjardi MY², Kalantar SM³.

1. Department of Genetics, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Medical Genetics Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
3. Research and Clinical Center for Infertility, Yazd Reproduction Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: mmvahidi@gmail.com
 
Background: Endometriosis, a relatively prevalent gynecologic disorder, affecting 6 to 10 percent of women in reproductive ages around the globe. Primary recognition can help to decrease its progression and morbidity. Many studies demonstrated that microRNA has a vital role in the pathogenesis of endometriosis. miR-1271 and its direct target gene, GRB2, expression have been studied in gynecologic cancers and found to be involved in cell proliferation, migration, and metastasis, while their role in endometriosis has not been studied.
Objective: In this study, we measured miR-1271 and GRB2 genes expression in the endometrial tissues of patients (eutopic and ectopic tissues) compared to the control samples.
Materials and Methods: In our study, the endometriosis tissue samples of 15 patients with endometriosis and 15 women without endometriosis were collected. We used quantitative polymerase chain reaction to check the level of miR-1271 and GRB2 genes expression in these samples.
Results: We observed a significant decrease in miR-1271 expression level in both ectopic and eutopic samples of patients with endometriosis compared with control samples, while there was a noticeable increase in the expression level of its target gene, GRB2, in tissues of endometriosis patients compared with normal control samples.
Conclusion: We discovered an inverse relationship between the reduction of miR-1271 expression level and increase in the expression level of GRB2. Therefore, increased GRB2 expression in endometriosis tissues can be due to decreased expression of this microRNA. Our findings suggested that miR-1271 maybe play the role as a biomarker in the diagnosis of patients with endometriosis.
Key words: Biomarker, Endometriosis, miR-1271, GRB2.
 
O-50
Evaluation of the expression level of miR-337-3p and its association with the RAP1A gene expression in tissue samples of patients with endometriosis
 
Dehghanian M¹, Vahidi Mehrjardi MY^{1, 2}, Kalantar SM³, Dehghani MR².

1. Department of Medical Genetics, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Medical Genetics Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
3. Research and Clinical Center for Infertility, Yazd Reproduction Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: mmvahidi@gmail.com
Background: Endometriosis, a common and multifactorial disease in women, has different symptoms such as pelvic pain and infertility. Recent studies demonstrated that genetic factors have an important role in its pathogenesis so that dysregulation of many genes and microRNAs have been reported in this disease. Based on previous studies, we know that decreased expression level of miR-337-3p in ovarian and cervical cancers can lead to increase its target genes like RAP1A, which plays role in the pathogenesis of these diseases. miR-337-3p expression also downregulated in serum samples of endometriosis patients. However, the role of miR-337-3p and its direct target gene RAP1A in endometriosis tissues have not been investigated.
Objective: The goal of this study was to compare the expression level of miR-337-3p and its direct target gene, RAP1A, in endometriosis tissues and control samples to find their relationship with pathogenesis of endometriosis.
Materials and Methods: We measured miR-337-3p and RAP1A expression levels by quantitative polymerase chain reaction (qRT-PCR) in 15 ectopic and eutopic tissue samples from patients with endometriosis and 15 normal endometrium tissue samples from women without endometriosis.
Results: The results showed a significant increase in the expression level of RAP1A gene in the endometriosis tissue samples (both of ectopic and eutopic tissues), while miR-337-3p expression level decreased significantly in these tissue samples compared with the normal endometrium samples.
Conclusion: In this study, we found an opposite relationship between miR-337-3p and RAP1A gene expression in endometriosis so that decrease in miR-337-3p expression can lead to increase in RAP1A gene expression in endometriosis tissues. Changes in the expression of these genes in our study can also interpret as the role of them in the pathogenesis and progression of endometriosis.
Key words: Endometriosis, microRNA, miR-337-3p, RAP1A.
 
O-51
GM3-synthase (hST3Gal V) gene expression in endometriotic tissues
 
Emami Joo N^{1, 2}, Chitsazian F², Mashayekhy M³, Shahhoseini M^{2, 4}.

1. Department of Basic Sciences and New Biological Technologies, Science and Culture University, Tehran, Iran.
2. Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
3. Department of Endocrinology and Female Infertility, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
4. Reproductive Epidemiology Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

Email: m.shahhoseini@royaninstitute.org
Background: Endometriosis is a gynecological disease, affects 10%-15% of women in their reproductive ages under the influence of hormonal, genetic, epigenetic, and environmental factors. According to Sampson's theory, endometrial cells are implanted and proliferated outside the uterine cavity, attacking the pelvic structures and causing chronic inflammation. Hence endometriosis can be considered benign cancer. Changes in the cell surface glycosylation is a common phenotype observed during cell differentiation, tissue development cancers, and oncogenesis, a key feature associated with the potentiality of cancer cells for metastasis and invasion. Studies are indicative of changes in the expression of the human ST3 beta-galactoside alpha-2,3-sialyltransferase 5 (hST3Gal V) gene, which encodes the GM3 synthase enzyme (the producer of the GM3 ganglioside).
Objective: In this study, we examined changes of hST3gal V gene expression in ectopic and eutopic endometrial tissues of women with endometriosis compared with the control group.
Materials and Methods: Samples were collected from 20 women with endometriosis (10 eutopic and 10 ectopic samples) and also 10 normal endometrium samples were enrolled as the control group. Ectopic biopsies were obtained with the use of the laparoscopic procedure, eutopic and control biopsies were obtained with the use of pipelle. RNA extraction and cDNA synthesis were performed for all samples and then gene expression levels were measured by real time-PCR, using designed primers for hST3Gal V and also GAPDH as the housekeeping gene. Data analysis performed using One-way ANOVA as the statical method. Values were expressed as mean ± SEM and the results were considered significant at the level of p < 0.05.
Results: Results showed that the hST3Gal V gene expression was reduced in eutopic samples than control group (p = 0.538) gene and hST3Gal V gene expression in ectopic samples was reduced than both eutopic and control groups (p = 0.696 and p = 0.153, respectively).
Conclusion: Results shows a decrease in the gene expression profile of hST3Gal V in endometriotic samples. Since GM3 ganglioside is a substrate for the extension and branching of other gangliosides, it seems that the lower expression of the hST3Gal V gene can be involved in the etiology of the disease. This study is limited by the number of samples in each group. Further studies with larger samples numbers can provide more accurate results in this regard.
Key words: Endometriosis, hST3Gal V, Gm3 Synthase, Ganglioside, Gene expression.
 
O-52
Endometrial scratching affects gene expression of NLRP3 in patients with unexplained repeated implantation failure: A randomized control trial
 
Aghajanpour S¹, Hosseini E², Amirchaghmaghi E³, Zandieh Z⁴, Amjadi F⁴, Yahyaei A¹, Ashrafi M¹, Aflatoonian R¹.

1. Department of Endocrinology and Female Infertility, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
2. Department of Obstetrics and Gynecology, IVF Clinic, Mousavi Hospital, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran.
3. Department of Regenerative Biomedicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
4. Department of Anatomical Sciences, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.

Email: r.aflatoonian@gmail.com
 
Background: Alternative strategies have been used to augment success rate of implantation in IVF/ICSI cycles in unexplained repeated implantation failure patients. Endometrial scratching is one of these procedures. It seems scratching can affect NLRP3 gene expression which has an important role on receptivity of endometrium. NLRP3 is an intracellular sensor that detects a broad range of harmful sterile or infectious stimuli, resulting in the formation and activation of the NLRP3 inflammasome
Objective: In the present study, we investigated whether gene expression of NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) is affected by endometrial injury during proliferative phase of menstrual cycle before embryo transfer.
Materials and Methods: Twenty women with unexplained repeated implantation failure who failed to conceive during three or more IVF/ICSI cycles and embryo transfer were selected. The patients randomly classified into two study groups (N = 10 in each group). In the intervention group (not in the control group), endometrial scratching was done on day 9-13 in the proliferative phase of the preceding menstrual cycle. Then, endometrial biopsies of the intervention and control groups were performed in the luteal phase (on 19-21 day). The RNA of all samples was extracted and cDNA synthesis was performed. The expression of NLRP3 was quantified by quantitative real-time PCR.
Results:NLRP3 gene expression from all samples was investigated. Relative expression of NLRP3 was lower in the intervention samples compared to the controls.
Conclusion: The inflammasome components are suggested as a novel family of endometrial biomarkers. This result is in consistent with other studies that showed dysregulated inflammasome activation has involved in the disruption of maternal–fetal immune-tolerance and in pregnancy complications.
Key words: Endometrial scratching, NLRP3, Unexplained repeated implantation failure.
 
O-53
The influence of single blastomere biopsy on human embryo expansion and pregnancy result
 
Aghajani Sh¹, Mehrafza M², Hosseini A², Salehzadeh A¹, Ghasemian F³.

1. Department of Biology, Rasht Branch, Islamic Azad University, Rasht, Iran.
2. Mehr Fertility Research Center, Guilan University of Medical Sciences, Rasht, Iran.
3. Department of Biology, Faculty of Science, University of Guilan, Rasht, Iran.

Email: shahrzadaghajani1987@gmail.com
 
Background: The use of preimplantation genetic testing (PGT) in fresh and frozen intracytoplasmic sperm injection cycles and the possible damage is still unclear. Studies on aspects of this method, such as the prevalence of expansion on day 5 and pregnancy rate, are limited.
Objective: This study aimed to assess the rate of embryo expansion on day 5 in PGT patients and particular developmental components (expansion stage, inner cell mass, and trophectoderm) of euploid blastocysts influence on pregnancy outcomes.
Materials and Methods: A total of 433 embryos from 115 patients from intracytoplasmic sperm injection with or without PGT using fluorescence in situ hybridization method for X, Y, 13,18, and 21 chromosomes in fresh or freeze cycles between february 2018 and June 2020 was evaluated. The zona pellucida of fresh embryo transfer patients as a control group was untreated. In the PGT group, 6-8 cell embryos on the day 3 with grade A were hatched by laser, and extract one blastomere for PGT. Following evaluation, embryo transfer was done on day 5. Statistical analyzes were performed using SPSS 23 and p < 0.05 was considered statistically significant.
Results: In embryos that screened with X, Y, 13,18, and 21 probes in the fresh and freeze PGT cycles, more euploid embryos reached blastocyst with expansion 3, 4, and 5 (p < 0.001). Single blastomere biopsy (SBB) in PGT groups increases blastocyst expansion grade, and pregnancy outcomes compare with blastocyst embryos without blastomere biopsy and PGT (p < 0.01). Embryos with an expansion grade A compared with C had a higher pregnancy rate (p < 0.01). Blastocysts with a trophectoderm and inner cell mass grade of A or B compared with C had a higher likelihood of pregnancy rate (p < 0.01).
Conclusion: Among euploid embryos, expansion grade is the best predictor of sustained implantation; however, a composite score of embryo morphology on day 5 may provide additional guidance. Therefore, this investigation shows that the laser zona hatching may positively affect embryo expansion grade and pregnancy rates.
Key words: Preimplantation genetic testing, Zona pellucida, Fluorescence in situ hybridization.
 
O-54
Investigation of immunosuppressive- immunomodulatory markers in amniotic fluid-derived mesenchymal stem cells from women who experienced recurrent pregnancy loss
 
Hossini SM¹, Kalantar SM², Aflatoonian B³, Bahrami A¹, Montazeri F⁴, Moghaddam Matin M¹.

1. Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran.
2. Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
3. Stem Cell Biology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
4. Abortion Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: f_montazeri@Outlook.com
 
Background: The amniotic fluid contains a heterogeneous population of different cells that are produced prior to the gastrulation process. Therefore, it is expected that mesenchymal stem cells derived from the amniotic fluid will have high plasticity between mature and pluripotent stem cells. Due to unique features of these cells such as high cloning potential, high self-renewal capacity along with chromosomal stability and low immunogenicity as well as anti-inflammatory and immune-modulating properties, it has attracted more and more attention from researchers.
Objective: The aim of this study was to investigate the immunosuppressive genes in mesenchymal stem cells isolated from amniotic fluid of women with a history of recurrent pregnancy loss (RPL) and the effect of gamma interferon as an immunological stimulus on the expression of these genes.
Materials and Methods: The study group included pregnant women with a history of unexplained RPL. The control group consisted of pregnant women with at least one healthy child, no history of miscarriage, and normal hormonal and immunologic profiles. In this study, mesenchymal stem cells (MSCs) isolated from amniotic fluid from RPL and non-RPL women. On the other hand, each cell line was examined under 5 different treatment groups, control and 4 groups with 20 and 100 IU IFN-γ per ml of medium over two periods of 24 h and 72 h. Finally, the relative mRNA expression level of immune-suppressive/modulator gene including two indole amine-2 and 3-dioxygenase 1 and 2 in AF-MSCs in both groups were evaluated and compared using Q-PCR.
Results: The average expression of candidate gene IDO1 and IDO2 showed a significant increase in the RPL group rather than non-RPL, specially under treatment with 100 IU IFN-γ and after 24 h. Interestingly, expression of both genes IDO1 and IDO2 decrease after 72 h in RPL and non-RPL groups (p = 0.05).
Conclusion: Immunosuppression by MSCs, which is currently recognized as a powerful tool in preventing acute rejection, graft therapy, and regenerative medicine, is not an inherent potential but is induced by environmental factors. Various studies have identified that some potential causes of unexplained RPL are due to immunological factors. The results of this study, especially for indole amine-2 and 3-dioxygenase genes, do not rule out such a possibility. Despite the unknown role of AF-MSCs in the abortion mechanism, the results of this study suggest that there is a significant difference between the mRNA level of understudied genes between AF-MSCs in the RPL and non-RPL group. Due to the absence of such a similar study, it cannot be fully interpreted, however, these cells appear to represent genetic compartments of couples with a history of RPL that may defective in immunological factors. However, planning for further investigation of these uncertain immunological mechanisms appears to be valuable in the future.
Key words: Recurrent pregnancy loss, Immunosuppressive gene, Mesenchymal stem cells, Amniotic fluid, Quantitative gene expression.
 
O-55
Evaluating cell free DNA in spent embryo culture media in cleavage and blastocyst stage
 
Jahanara M¹, Montazeri F², Sharifiyazdi H³, Kalantar SM², Hoseini SM⁴, Moshrefi M^{5, 6}.

1. Department of Biotechnology, School of Veterinary Medicine, Shiraz University, Fars, Iran.
2. Abortion Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical, Yazd, Iran.
3. Department of Clinical Science, School of Veterinary Medicine, Shiraz University, Fars, Iran.
4. Biotechnology Research Center, International Campus, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
5. Medical Nanotechnology and Tissue Engineering Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
6. Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Yazd, Iran.

Email: malihejahanara@gmail.com
 
Background: Chromosomal abnormalities are one of the most important causes of failure in in vitro fertilization. Preimplantation genetic testing can be a way to prevent the transfer of aneuploid embryos. It entails the use of invasive techniques to obtain embryonic DNA, with major technical limitations and ethical issues today. Therefore, the use of new non-invasive methods is a suitable solution to this problem. One of the non-invasive methods is to use the embryo spent culture medium. The origin of cell free DNA in embryo spent culture medium is trophectoderm cells and the internal cell mass.
Objective: Cell-free genomic DNA in the embryonic culture medium can be a non-invasive method for genetic assessment.
Materials and Methods: This study reviewed 25 spent embryo culture mediums. The spent culture medium used between day 3 and day 5 of embryonic development. Patients were undergoing intracytoplasmic sperm injection, and each embryo was in one drop of culture medium. We had two control samples: the culture medium contaminated with purified DNA from human blood and the culture medium without embryonic development. All samples were evaluated with nanodrop for dsDNA and ssDNA concentration. Among the collected medium, ten samples (group 1) concentrated by heating, then evaluating SRY and FMR1 genes with real-time polymerase chain reaction (RT-PCR) (group 1). Six samples were three days, and four samples were five days. The rest of the samples were classified into three groups. The cell-free DNA from the medium was purified with the blood DNA extraction kit. in group 2 with Genet bio kit, group 3 with YTzol pure DNA kit (yekta Tajhiz), and group 4 with High Pure Viral Nucleic Acid extraction kit (Roche). They evaluated by RT-PCR. Nine samples were three days, and six samples were five days. 
Results: Although cell-free DNA was confirmed in the samples using nanodrop (with a range of 160 to 225 ng per microliter), the cycle of threshold did not observe in the RT-PCR product of group 1. The Purified samples were amplified in group 2,3 and 4 for SRY and FMR1 genes with RT-PCR and observed only acceptable cycle of threshold in the fourth group.
Conclusion: The high protein and solutes in the culture medium and the low amount and quality of DNA are restrictive. For better results, it is necessary to purify the genomic DNA and amplify it with precise kits. Our research is underway to improve DNA collection, amplification, and testing to isolate genomic DNA.
Key words: Cell free DNA, Spent embryo culture media, Preimplantation genetic testing.
 
O-56
In vitro implantation of euploid and aneuploid embryos
 
Amiri S^{1, 2}, Amjadi F^{1, 2}, Aflatoonian R³, Ashrafi M², Akbari Sene A², Mehdizadeh M¹, Zandieh Z^{1, 2}.

1. Anatomy Department, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
2. Akbarabadi IVF Clinic, Akbarabadi Hospital, Iran University of Medical Sciences, Tehran, Iran.
3. Department of Endocrinology and Female Infertility at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

Email: s.amiri7@yahoo.com
 
Background: Although embryo selection for transfer is usually based on morphology, 70% of embryos with high morphological quality have chromosomal abnormalities. The results of implantation and pregnancy rate assessments following preimplantation genetic screening (PGS) are controversial. There is still no in vitro study to compare the implantation of human euploid and aneuploid embryos.
Objective: This study was designed to compare the ability of aneuploid embryos to attach to endometrial cells with euploid embryos by simulating the human endometrium using a three-dimensional scaffold.
Materials and Methods: After informed consent, 10 endometrial biopsies were taken from fertile women. Endometrial cells were isolated and expanded in 2D cultures to achieve enough cells. The fibrin-agarose scaffold was made and stromal cells were cultured into the scaffold, after 24 hr, the epithelial cells were seeded on the scaffold. Cell culture continued for 5 days to reach the appropriate confluence. Then, cell proliferation was assessed by MTT assay. The simulated endometrial construct was confirmed by H&E and immunohistochemistry (IHC). The embryos were also examined by performing PGS following conventional comparative genomic hybridization array. 10 euploid and 10 aneuploid blastocysts were selected for co-culturing. Partial hatching of blastocysts was performed using a laser system. Blastocysts were co-cultured with the 3D structure of human endometrial cells for 72 hr. The blastocyst's attachment to the endometrial-like structure was examined under a phase-contrast microscope and scanning electron microscopy.
Results: The MTT OD of scaffolds increased during 5 days of cell culture (p < 0.05). The histological evaluation of the co-culture systems was done under light microscopy by H&E staining. On the top of the 3D culture system, epithelial cells shaped a constricted cell monolayer. Stromal cells combined with the fibrin-agarose scaffold got lengthened and expanded, displaying that the 3D culture systems supplied a suitable environment for the growth of endometrial cells. In the 3D culture, the origins and locations of epithelial and stromal cells were defined by cytokeratin and vimentin immunostaining, respectively. IHC for cytokeratin was only positive for epithelial cells in the surface epithelium. IHC for the vimentin was positive for the stromal cells located in the 3D matrix. These results showed that fibrin-agarose scaffold could simulate the human endometrial structure. Using scanning electron microscopy and phase-contrast microscopy, it was found that only euploid embryos were able to attach to the endometrial construct while aneuploid embryos weren't.
Conclusion: Our findings determined that PGS allows us to transfer top-quality embryos with higher implantation potential. It improves implantation and pregnancy rate during assisted reproductive technologies cycles, especially in patients with recurrent implantation failure.
Key words: Three-dimensional culture, Implantation, Human endometrial cells, Aneuploid and euploid embryos, CGH array.
O-57
Non-invasive preimplantation genetic diagnosis (PGD) for X-linked disease by sex determination through cell-free DNA
 
Mosavi A¹, Amjadi F², Barati M³, Aflatoonian R⁴, Amiri S¹, Mehdizadeh M¹, Mirsanei J¹.

1. Department of Anatomy, Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran.
2. Shahid Akbarabadi Clinical Research Development Unit , Iran University of Medical Sciences, Tehran, Iran.
3. Department of Medical Biotechnology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran.
4. Department of Endocrinology and Female Infertility, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

Email: amjadi.bh@gmail.com
 
Background: Preimplantation genetic diagnosis (PGD) is a useful clinical tool to identify embryos with or at risk of specific genetic malady before embryo implantation. Current procedures for embryo chromosomal screening require an invasive biopsy of the embryo. Blastomere biopsy has a potential lesion to the embryos may result in developmental defects or abortion. Thus, a non- invasive PGD is needed.This study hypothesized that embryonic DNA is present in the spent culture medium. We focused on X-linked disorders, these single-gene diseases due to the presence of defective genes on the X chromosome are dominant in males.
Objective: Therefore, the objective of this study was to discriminate between female (XX) and male (XY) embryos by detecting Y chromosome- specific genes in cell-free DNA and comparing to PGD results. It opens a new window for the development of a non-invasive PGD method.
Materials and Methods: Embryo´s spent media from day 3 and day 5 embryos development were collected. The modified phenol-chloroform solution was used for DNA extraction from spent media. DNA from spent media was evaluated using SRY, TSPY, and AMELOGENIN as targets using the qPCR method. IBM SPSS and Medcals were used for statistical analyses, to compare sex determination of embryos using spent medium with PGD results.
Results: Yield and purity of the extracted DNA as well as repeatability of the method were performed well using the modified phenol-chloroform solution. The amount of DNA at day 5 embryo culture medium was significantly higher than day 3. Results of sex determination using spent medium by Q-PCR were consistent with the results of PGD and 12^th wk sonography. This invasive PGD method using a spent culture medium gave a sensitivity of 66.7%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 67.6 (N = 56, Nxx = 23, Nxy = 33).
Conclusion: This investigation provides a potentially effective procedure that can help to avoid the invasive preimplantation genetic diagnosis, especially about X-linked diseases. Results of sex determination using spent medium by Q-PCR were consistent with the results of PGD. Improvements in DNA collection, amplification, and testing may allow for PGD without biopsy in the Future.
Key words: PGD, X-Link diseases, cfDNA.

 Poster Presentations
 
P-1
Effects of maternal voluntary wheel running during pregnancy on the neonatal rat ovary
 
Baradaran R¹, Yousefi B².

1. Department of Anatomy and Cell Biology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
2. Department of Anatomical Sciences, School of Medicine, Semnan University of Medical Sciences, Semnan, Iran.

Email: baradaranr941@mums.ac.ir
 
Background: Regular maternal exercise in pregnancy enhance the physiological, metabolic, and psychological health of mother and fetus. Probing the effects of maternal exercise in gestation, on the developmental programming of pups all over intrauterine and postnatal life, is known as a novel and favorable research field.
Objective: The purpose of the present study was to evaluate the effects of maternal voluntary wheel running during mid or late gestation on rat neonatal estrogen and progesterone plasma concentration; ovarian development and its angiogenesis; and development of the primary oocyte, primordial follicle, and their apoptosis.
Materials and Methods: 21 female Wistar rats were accidentally distributed into experimental groups (doing exercises during the 2^nd and 3^rd wks of pregnancy, n = 14) and control (n = 7). In the exercise groups, each rat had access to a running wheel (diameter = 34.5 cm, width = 9.5 cm) that was embedded in their cage and during the 2^nd and 3^rd wk of pregnancy, it rotated freely during the resistance of 100 g. In this regard, it is notable that each wheel was linked to a counter that recorded its rotations. Pregnant rats in the control group were put in the cages without any access to a running wheel. After birth, the neonate's blood was obtained and the estrogen and progesterone concentration were evaluated. Thereafter, the ovaries were removed and used for histological investigations and apoptic assessment.
Results: A significant increase was found in estrogen and progesterone concentration in neonates of experimental groups (p = 0.001). The experimental groups had an increased ovarian diameter (2^ndW: p = 0.044 and 3^rdW: p = 0.005) and angiogenesis (2^ndW: p = 0.003 and 3^rdW: p = 0.001). In addition, significant enhances were seen in the number (2^ndW: p = 0.017 and 3^rdW: p = 0.035) and diameter (2^ndW: p = 0.046 and 3^rdW: p = 0.004) of primordial follicles as well as in the diameter of primary oocytes (2^ndW: p = 0.037 and 3^rdW: p = 0.019) of the experimental groups compared to the control group. Moreover, maternal voluntary wheel running reduced the number (2^ndW: p = 0.001 and 3^rdW: p = 0.001) of apoptotic primordial follicle in the experimental groups compared to the control group.
Conclusion: It was shown that maternal voluntary wheel running of the pregnant rats during mid or late gestation increase estrogen and progesterone plasma concentrations, and ovarian size and its angiogenesis in neonates. Furthermore, this type of exercise increases the primordial follicle/primary oocyte numbers and diameters as well as oocyte nuclei, while inversely decreases the numbers of apoptotic primordial follicles.
Key words: Apoptosis, Exercise, Neonatal, Ovary, Rat.
 
P-2
The effect of cysteine and glutamine on human sperm functional parameters during vitrification
 
Koohestanidehaghi Y¹ , Torkamanpari M², Shirmohamadi Z³, Lorian K⁴, Vatankhah M⁵.

1. Department of Anatomical Sciences, Faculty of Medicine, Guilan University of Medical Sciences, Rasht, Iran.
2. Department of Biology, Payam e Nour University, Tehran, Iran.
3. Department of Biostatistics, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
4. Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
5. Department of Physiology, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran.

Email: mahsaneh.vatankhah@gmail.com
 
Background: Assuming the adverse effects of reactive oxygen species (ROS) on sperm function, this study was conducted to assess the effects of cysteine and glutamine as effective antioxidants on human sperm parameters under vitrification.
Objective: The present study aimed to investigate the protective effect of cysteine and glutamine on motility parameters, plasma membrane potential, mitochondrial membrane potential, DNA damage and human sperm intracellular ROS during vitrification.
Materials and Methods: Twenty normozoospermic samples were used. The samples were subjected to a vitrification process and cysteine (5 and 10 mM) and glutamine (10 and 15 mM). The sperm motility parameters, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI), DNA damage and intracellular ROS damage were assessed for each sample.
Results: Statistical analyses showed that motility, mitochondrial membrane potential and DNA damage decreased in the vitrified groups with cysteine 5, 10 mM and glutamine 10, 15 mM separately. Also intracellular ROS increased significantly compared to the fresh group (p < 0.05). No significant differences were observed for PMI compared with the fresh group (p > 0.05). Supplementation of cysteine and glutamine in both concentrations separately decreased intracellular ROS and DNA damage of spermatozoa with significant increase in PMI, MMP and progressive motility compared to vitrified control group (p < 0.05).
Conclusion: The results showed no significant effect of a specific concentration in cysteine and glutamine on sperm parameters compared to other concentrations. Both amino acids have the potential to improve the harmful effects of freezing on sperm parameters.
Keywords: Cysteine, Glutamine, Human, Sperm parameters, Vitrification.

The original full text of this abstract has been published in Andrologia 2021;53:e13870. https://doi.org/10.1111/and.13870.
How to cite to this article: Koohestanidehaghi Y, Torkamanpari M, Shirmohamadi Z, Lorian K, Vatankhah M. The effect of cysteine and glutamine on human sperm functional parameters during vitrification. Andrologia 2021; 53: e13870.

P-3
Bone morphogenetic protein 15 induces differentiation of mesenchymal stem cells derived from human follicular fluid to oocyte-like cell
 
Taheri Moghadam M^{1, 2, 3}, Saki Gh², Nikbakht R³, Eftekhari Moghadam AR^{1, 2}.

1. Cellular and Molecular ResearchCenter, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
2. Department of Anatomical Science, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
3. Fertility, Infertility and Perinatology Center,Imam Khomeini Hospital, Ahvaz JundishapurUniversity of Medical Sciences, Ahvaz, Iran

Email: mahintaherimoghadam@yahoo.com
 
Background: Follicular fluid (FF) is essential for developing ovarian follicles. Besides the oocytes, FF has abundant undifferentiated somatic cells containing stem cell properties, which are discarded in daily medical procedures. Earlier studies have shown that FF cells could differentiate into primordial germ cells via forming embryoid bodies, which produced oocyte‐like cells (OLC).
Objective: This study aimed at isolating mesenchymal stem cells (MSC) from FF and evaluating the impacts of bone morphogenetic protein 15 (BMP15) on the differentiation of these cells into OLCs.
Materials and Methods: Human FF‐derived cells were collected from 78 women in the assisted fertilization program and cultured in human recombinant BMP15 medium for 21 days. Real‐time polymerase chain reaction and immunocytochemistry staining characterized MSCs and OLCs.
Results: MSCs expressed germline stem cell (GSC) markers, such as OCT4 and Nanog. In the control group, after 15 days, OLCs were formed and expressed zona pellucida markers (ZP2 and ZP3), and reached 20-30 μm in diameter. Ten days after induction with BMP15, round cells developed, and the size of OLCs reached 115 μm. A decrease ranged from 0.04 to 4.5 in the expression of pluripotency and oocytespecific markers observed in the cells cultured in a BMP15‐supplemented medium.
Conclusion: FF‐derived MSCs have an innate potency to differentiate into OLCs, and BMP15 is effective in promoting the differentiation of these cells, which may give an in vitro model to examine germ cell development.
Key words: OLC, Follicular fluid, Bone morphogenetic protein 15, Mesenchymal stem cell.

The original full text of this abstract has been published in Cell Biology International 2021; 45(1): 127-139. https://doi.org/10.1002/cbin.11475.
How to cite to this article: Taheri M, Saki G, Nikbakht R, Eftekhari AR. Bone morphogenetic protein 15 induces differentiation of mesenchymal stem cells derived from human follicular fluid to oocyte‐like cell. Cell Biology International 2021; 45(1): 127-139.

 
P-4
Exploration of couple's experiences of long-term marital satisfaction: A qualitative study
 
Samadi P¹, Alipour Z², Kohan Sh³, Salehi K³, Hashemi M³.

1. Department of Midwifery and Reproductive Health, School of Nursing and Midwifery, Tehran University of Medical Sciences, Tehran, Iran.
2. Department of Midwifery and Reproductive Health, School of Nursing and Midwifery, Qom University of Medical Sciences, Qom, Iran.
3. Nursing and Midwifery Care Research Center, School Of Nursing and Midwifery, Isfahan University of Medical Sciences, Isfahan, Iran.

Email: kanom_alipour@yahoo.com
 
Background: Marital satisfaction is a multidimensional phenomenon, which refers to the quality of marital relationship, or the general view of marriage status and reflection of happiness and marital performance. Repetition of certain positive behaviors can make a big difference in the success of continued married life, and that awareness of such behaviors seems to be critical to recognizing certain warnings.
Objective: This study with qualitative approach conducted to promoting long term marital satisfaction by exploring couple`s experiences.
Materials and Methods: This study was conducted using descriptive phenomenology method. The participants were 12 person (six couples) with a history of 20-30 yr of married life expectancy and a marital satisfaction score of above 65. The data were collected by purposeful sampling and semi-structured interviews, analyzed using Colaizzi method. By categorizing the codes, subcategories, and main categories were extracted.
Results: An analysis of the experiences of the participants resulted in emergence of eight subcategories, and three main categories: “strong foundation for living together”, “mutual commitment to protecting marital cohesion”, and “striving to improve sexual relations”.
Conclusion: A long-term marriage associated with a variety of variables, including a strong foundation for living together, a mutual commitment to protect marital cohesion, and an effort to improve sexuality. And the results showed that the type of relationship will change during the years after marriage in a way that takes on certain meanings and concepts and can be interpreted in physiological, cultural and other specific contexts.
Key words: Marital satisfaction, Long married life, Phenomenology.
P-5
Potential therapeutic effect of bee pollen and metformin combination on testosterone and estradiol levels, apoptotic markers and total antioxidant capacity in a rat model of polycystic ovary syndrome
 
Naseri L¹, Khazaei MR², Khazaei M².

1. Student Research Committee, Kermanshah University of Medical Sciences, Kermanshah, Iran
2. Fertility and Infertility Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran.

Email: mkhazaei1345@yahoo.com
 
Background: Polycystic ovary syndrome (PCOS) is associated with metabolic disorders as well as infertility. Many traditional remedies have been reported to show estrogenic and antioxidant potential. Bee pollen (BP) is a natural compound, reported as one such remedy.
Objective: The present study aimed to investigate the effects of BP extract (BP) and metformin (MET) on estradiol (E2) and testosterone (T) levels, apoptotic markers, and total antioxidant capacity (TAC) in a rat model of PCOS.
Materials and Methods: In this experimental study, 54 female Wistar (n = 6/group) rats received 2 mg of estradiol valerate (EV) intramuscularly and 6 additional rats were considered the control without EV injection. The rats were treated with BP (50, 100, and 200 mg/kg), MET (300 mg/kg) and BP+MET (50 BP+300 MET, 100 BP+300 MET, and 200 BP+300 MET mg/kg). Serum levels of E2 and T were assessed by the ELISA method. TAC of serum was also determined. The expressions of Bcl2, Bax, Caspase3 (Cas-3), and Sirt-1 genes were evaluated by real-time polymerase chain reaction (PCR). Data were statistically analyzed using one-way ANOVA.
Results: In the untreated PCOS group E2 and T levels (p < 0.01), and Bcl2 (p = 0.007) expression were increased, but TAC (p = 0.002) and expression of Bax (p = 0.001), Cas-3 and Sirt-1 (p < 0.01) were decreased significantly. The levels of E2 and T, as well as the expressions of Bcl2, were decreased in all treated groups compared to the untreated PCOS group (p < 0.01). On the other hand, TAC and expression of Bax, Cas-3and Sirt-1 were increased in the BP- and MET-treated groups (p < 0.05).
Conclusion: BP and MET synergistically improved serum E2, T, and TAC levels, and expression of apoptotic genes.
Key words: Metformin, Apoptosis, Bee pollen, Estradiol, Polycystic ovarian syndrome.

The original full text of this abstract has been published in Int J Fertil Steril 2021; 15(2): 101-107. https://doi:10.22074/IJFS.2020.134604.
How to cite to this article: Naseri L, Khazaei MR, Khazaei M. Potential therapeutic effect of bee pollen and metformin combination on testosterone and estradiol levels, apoptotic markers and total antioxidant capacity in a rat model of polycystic ovary syndrome. Int J Fertil Steril2021; 15(2): 101-107.

P-6
Prevention of uterine fibrosis in rabbit model by intrauterine stem cell conditioned media injection immediately after endometrial curettage
 
Bazoobandi S¹, Tanideh N^{1, 2}, Rahmanifar F³, Zare S¹, Koohi-Hosseinabadi O^{4, 5}, Razeghian-Jahromi I⁶, Dianatpour M¹, Ahmadi M¹, Khoradmehr A⁷, Nabipour I⁷, Khodabandeh Z¹, Tamadon A⁷.

1. Stem Cells Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
2. Department of Pharmacology, Medical School, Shiraz University of Medical Sciences, Shiraz, Iran.
3. Department of Basic Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran.
4. Laparoscopy Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
5. Central Research Laboratory, Shiraz University of Medical Sciences, Shiraz, Iran.
6. Cardiovascular Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
7. The Persian Gulf Marine Biotechnology Research Center, The Persian Gulf Biomedical Sciences Research Institute, Bushehr University of Medical Sciences, Bushehr, Iran.

Email: amintamaddon@gmail.com
 
Background: Uterine fibrosis or Asherman's syndrome is a uterine acquired disorder with symptoms of implantation disturbances, menstrual irregularities and abortion.
Objective: The main goal of this study was evaluation of the effects of conditioned media of bone marrow-derived mesenchymal stromal/stem cells (BM-MSCs) in prevention of uterine fibrosis immediately after uterine curettage in rabbit.
Materials and Methods: This study included 12 female rabbits (24 uterine horns in total) were randomly divided into four groups of intact negative control, curettage positive control, stem cell therapy, and stem cell conditioned media injection in the way that two corresponding uteri from a rabbit were assigned in different groups.
Results: The BM-MSC-conditioned media treated uterus showed regenerated endometrial layer compared with lower diameter of endometrium in the control group. It showed that BM-MSC-conditioned media play a positive role in the regeneration of the uterine wall. Area of fibrotic tissue in the treated groups was lower than the control groups.
Conclusion: Injected stem cell conditioned media had preventing effect on occurrence of uterine fibrosis. Therefore, BM-MSC-conditioned media can be suggested to be injected immediately after endometrial curettage.
Key words: Mesenchymal stromal/stem cell, Conditioned media, Uterine fibrosis.

The original full text of this abstract has been published in Stem Cells International 2020, Article ID 8849537, 10 pages. https://doi.org/10.1155/2020/8849537.
How to cite to this article: Bazoobandi S, Tanideh N, Rahmanifar F, Zare S, Koohi-Hosseinabadi O, Jahromi IR, Dianatpour M, Khodabandeh Z, Ahmadi M, Khoradmehr A, Nabipour I. Preventive effects of intrauterine injection of bone marrow-derived mesenchymal stromal cell-conditioned media on uterine fibrosis immediately after endometrial curettage in Rabbit. Stem Cells International 2020, Article ID 8849537.

P-7
Evaluation of the effect of folic acid and nicotinic acid on malondialdehyde levels of semen in Oligospermia men after cryopreservation
 
Sadeghi Z, Dashti G.
Department of Anatomy, Isfahan University of Medical Sciences, Isfahan, Iran.
Email: dashti@med.mui.ac.ir
 
Background: Reactive oxygen species and free radicals are one of the most important detrimental factors on sperm quality, especially during the freezing process. One of the important factor is reactive oxygen species which increases the lipid proxidation of cell membranes.
Objective: In this study, an attempt was made to measure the concentration of malondialdehyde levels in semen in oligospermia men before and after cryopreservation process and to evaluate the effect of folic acid and nicotinic acid on the malondialdehyde concentration after freezing.
Materials and Methods: For this purpose, semen fluid sample was collected from 25 oligospermia men in the age range of 25 to 45 yr. Each sample was divided into 5 groups: fresh group, freeze group without antioxidants (control), freeze group with nicotinic acid (10 mM), freeze group with folic acid (50 nM) and freeze group with a combination of nicotinic acid (10 mm) + folic acid (50 nM). The concentration of malondialdehyde was measured in nmol/ml in each group.
Results: Our study showed that the concentration of malondialdehyde in the semen increased after freezing compared to before freezing (p > 0.001). Also, the concentration of malondialdehyde in the group of folic acid + nicotinic acid was lower compared to other groups after freezing (p > 0.001).
Conclusion: The combination of folic acid and nicotinic acid antioxidants with sperm freezing medium reduced the level of Malondialdehyde and lipid peroxidation of sperm membrane during the freezing process and thereby maintains the fertility potential in oligospermia men.
Key words: Cryopreservation, Sperm, MDA, Nicotinic acid, Folic acid.
 
P-8
Effects of nicotinic acid and Folic acid on sperm motility, Viability and DNA integrity in oligospermia men during cryopreservation
 
Sadeghi Z, Honarvar A, Dashti G.
Department of Anatomy, Isfahan University of Medical Sciences, Isfahan, Iran.
Email: dashti@med.mui.ac.ir
 
Background: Sperm freezing is an important technique for treating infertility and maintaining sperm, but because of the increased production of reactive oxygen species (ROS), threats such as chromatin damage and sperm DNA, threaten sperm motility and consequently reduce fertility. Antioxidants are important compounds to minimize the deleterious effects of freezing sperm and maintaining fertility potential.
Objective: The aim of this study was to investigate the effect of nicotinic acid and folic acid antioxidants on the sperm motility, survival and deoxyribonucleic acid of oligospermia men during cryopreservation.
Materials and Methods: For this purpose, 25 semen samples of Oligospermia men who referred to the Fertility and Infertility Center of Shahid Beheshti Hospital in Isfahan province were randomly taken into sterile containers and after fluidization, sperm parameters including morphology, motility, and concentration, life and quality of chromatin and their DNA  were Measure according to WHO criteria. Then, each sample was divided into 4 parts with freeze-dried medium for freezing: antioxidant-free (control group), 50 nM folic acid, 10 mM nicotinic acid, 50 nM folic acid + 10 mM nicotinic acid. After freezing, the samples were thawed and reexamined for sperm parameters.
Results: Our study showed that the process of freezing sperm by damaging the spermatozoa and cell membrane, resulted in decreased motility and competence of sperm to fertilize with oocytes and decrease the fertility potential of males (p < 0.001).
Conclusion: The use of folic acid and nicotinic acid antioxidants during sperm freezing reduces the harmful effects of free radicals created during the freezing process and helps preserve the fertility potential of Oligospermia men (p < 0.001).
Key words: Folic Acid, Nicotinic Acid, Deoxyribonucleic Acid, Oligospermia, Cryopreservation.
 
P-9
Effects of monosodium glutamate on apoptosis of germ cells in testicular tissue of adult rat: An experimental study
 
Rahimi Anbarkeh F¹, Baradaran R¹, Ghandy N¹, Jalali M¹, Nikravesh M¹, Soukhtanloo M².

1. Department of Anatomy and Cell Biology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
2. Department of Clinical Biochemistry, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.

Email: jalalim@mums.ac.ir
 
Background: Monosodium glutamate (MSG) is used as a flavoring and food seasoning. Some studies have reported the oxidative effects of using this substance on various tissues.
Objective: This study has investigated the effects of MSG and the protective effect of vitamin C (Vit C) on apoptosis of testicular germ cells and biochemical factors.
Materials and Methods: In this experimental study, 24 adult male Wistar rats were randomly divided into four groups: control (received distilled water), Vit C group (150 mg/kg), experimental group 1 (MSG 3 gr/kg), experimental group 2 (MSG 3 gr/kg + Vit C 150 mg/kg). The rats were gavaged for 30 days and then were sacrificed, the right testis was isolated for biochemical examinations for the glutathione, malondialdehyde, and left testis used in histological experiments. Tunnel staining was used to determine the number of apoptotic cells.
Results: The results showed that apoptotic cells in the MSG group had a significant increase compared to the control group (p = 0.001), but the number of these cells in the MSG co-administered with Vit C and Vit C groups was significantly lower than the MSG group. Germinal epithelial thickness also decreased in the MSG group compared to the control group.
Conclusion: MSG can lead to increase apoptotic changes in the germinal epithelial of the testicle, and Vit C as an antioxidant can modify the pathological and biochemical changes induced by MSG.
Key words: Apoptosis, Monosodium glutamate, Rat, Testis, Vitamin.

The original full text of this abstract has been published in Int. J Reprod BioMed 2019; 17(4): 261. https://doi.org/10.18502/ijrm.v17i4.4551.
How to cite to this article: Rahimi Anbarkeh F, Baradaran R, Ghandy N, Jalali M, Nikravesh MR, Soukhtanloo M. Effects of monosodium glutamate on apoptosis of germ cells in testicular tissue of adult rat: An experimental study. Int J Reprod BioMed 2019; 17: 261-270.

 
P-10
Cryoprotective effect of pentoxifylline on spermatogonial stem cell during transplantation into azoospermic torsion mouse model
 
Malekzadeh M, Rastegar T.
Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Email: trastegar2002@gmail.com
 
Background: Preserving the spermatogonial stem cells (SSCs) in a long periods of time during the treatment of male infertility using stem cell banking systems and transplantation is an important issue.
Objective: This study was conducted to develop an optimal cryopreservation protocol for SSCs using 10 mM pentoxifylline (PTX) as an antioxidant in basal freezing medium.
Materials and Methods: Testicular torsion - a mouse model for long-term infertility- was used to transplant fresh SSCs (n = 6), fresh SSCs treated PTX (n = 6), cryopreserved SSCs with basal freezing medium (n = 6) and cryopreserved SSCs treated PTX (n = 6). 8 wk after transplantation, samples were assessed for proliferation (through evaluation of MVH and ID4 markers) and differentiation (via evaluation of c‐Kit and SCP3, Tnp1, Tnp2, and Prm1 markers).
Results: Morphological and flow cytometry results showed that the SSCs were the population of cells able to form colonies and to express ID4, α6‐integrin and β1‐integrin markers, respectively. We found positive influence from PTX on proliferative and differentiative markers in SSCs transplanted to azoospermic mice.
Conclusion: In the recipient testis, donor SSCs formed normal spermatogenic colonies and sperm. These data indicate that adding the PTX is an effective way to efficiently cryopreserve germ cells enriched for SSCs in cryopreservation, and this procedure could become an efficient method to restore fertility in a clinical setup, but more studies are needed to ensure its safety in the long term.
Key words: Male infertility, Testicular torsion, Spermatogonial stem cells, Transplantation, Pentoxifyllin.
 
P-11
The effect of low-dose aspirin on the pregnancy rate in frozen-thawed embryo transfer cycles: A randomized clinical trial
 
Davar R¹, Pourmasumi S^{2, 3}, Mohammadi B¹, Mortazavi Lahijani M⁴.

1. Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Non-Communicable Diseases Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran.
3. Clinical Research Development Unit (CRDU), Moradi Hospital, Rafsanjan University of Medical Sciences, Rafsanjan, Iran.
4. Department of Obstetrics and Gynecology, Rafsanjan University of Medical Sciences, Rafsanjan, Iran.

Email: m_mortazavi58@yahoo.com
 
Background: The results of previous studies on the effect of low-dose aspirin in frozenthawed embryo transfer (FET) cycles are limited and controversial.
Objective: To evaluate the effect of low-dose aspirin on the clinical pregnancy in the FET cycles.
Materials and Methods: This study was performed as a randomized clinical trial from May 2018 to February 2019; 128 women who were candidates for the FET were randomly assigned to two groups receiving either 80 mg oral aspirin (n = 64) or no treatment. The primary outcome was clinical pregnancy rate and secondary outcome measures were the implantation rate, miscarriage rate, and endometrial thickness.
Results: The endometrial thickness was lower in patients who received aspirin in comparison to the control group. There were statistically significant differences between the two groups (p = 0.018). Chemical and clinical pregnancy rates and abortion rate was similar in the two groups and there was no statistically significant difference.
Conclusion: The administration of aspirin in FET cycles had no positive effect on the implantation and the chemical and clinical pregnancy rates, which is in accordance with current Cochrane review that does not recommend aspirin administration as a routine in assisted reproductive technology cycles.
Key words: Aspirin, Embryo transfer, Pregnancy rates.

The original full text of this abstract has been published in Int J Reprod BioMed 2020; 18: 693-700. https://doi.org/10.18502/ijrm.v13i9.7664.
How to cite to this article: Davar R, Pourmasumi S, Mohammadi B, Mortazavi Lahijani M. The effect of low-dose aspirin on the pregnancy rate in frozenthawed embryo transfer cycles: A randomized clinical trial. Int J Reprod BioMed 2020; 18: 693-700.

P-12
The ameliorating effect of hydroalcoholic extract of date palm (Phoenix Dactilifera L.) fruit on formaldehyde reproductive toxicity of male NMRI mice
 
Zare M¹, Haghpanah T², Asadi-Shekari M³, Eftekhar-Vaghefi Sh².

1. Health Policy Research Center, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
2. Department of Anatomy, Afzalipour Faculty of Medicine, Kerman University of Medical Sciences, Kerman, Iran.
3. Neuroscience Research Center, Institute of Neuropharmachology, Kerman, Iran.

Email: thaghpanah1984@gmail.com, sheftekharv@yahoo.com
 
Background: Formaldehyde (FA) is one of the most widely used materials in industrial and sciences. Prolonged contact with FA might have harmful effects on fertility due to increasing the reactive oxygen species level. On one hand, date palm (Phoenix Dactilifera L.) fruit extract (DPFE) contains a high concentration of natural antioxidants that could scavenge free radicals.
Objective: The aim was to investigate the prophylactic effects of DPFE, with strong antioxidant properties, on FA-induced testicular toxicity in male mice.
Materials and Methods: Thirty-two adult NMRI male mice were randomly divided into four groups: control group (CTL; distilled water, orally, 35 days), FA group (FA; 0.25 mg/kg intraperitoneally (i.p.), 20 days), treatment group (DT+FA; DPFE, 4 mg/kg, for 35 days followed by FA administration, 0.25 mg/kg, i.p., 20 days), and date fruit extract group (DT; DPFE, 4 mg/kg, orally, 35 days). After this period, the blood collected and left epididymis and testis tissues were isolated to evaluate the sperm parameters and histological examination, respectively.
Results: The FA administration increased the sperm morphological anomalies and reduced the sperm count, viability and motility and also testosterone versus the control group (p < 0.001). In addition, histological studies of the testes showed that FA causes changes in the testis seminiferous tubules such as destruction of germinal epithelium and vacuolization of the tubules. The DPFE consumption before FA administration could partially ameliorate the reduced testosterone, sperm and testicular parameters due to FA.
Conclusion: The date palm fruit extract use might have discount effects on FA-induced testicular toxicity.
Key words: Formaldehyde, Date fruit, Testis, Sperm, Testosterone.

The original full text of this abstract has been published in Int J Reprod BioMed 2020; 18: 275-286. https://doi.org/10.18502/ijrm.v13i4.6890.
How to cite to this article: Zare M, Haghpanah T, Asadi Shekari M, Eftekhar-Vaghefi SH. The prophylactic effect of date palm (Phoenix dactylifera L.) fruit extract on testicular toxicity induced by formaldehyde: An experimental study. Int J Reprod BioMed 2020; 18: 275-286.

P-13
The role of HLA-G in recurrent pregnancy loss: A case-control study
 
Adib Rad H¹, Basirat Z¹, Mostafazadeh A², Faramarzi M¹, Bijani A³, Aghajanpour-Mir S².

1. Infertility and Reproductive Health Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, Iran.
2. Cellular and Molecular Biology Research Center, Health Research Institute, Department of Immunology and Microbiology., Babol University of Medical Sciences, Babol, Iran.
3. Social Determinants of Health Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, Iran.

Email: basiratzahra@yahoo.com
 
Background: Human leukocyte antigen (HLA)-G is the main molecule for maternal acceptance of the semi-allogenic fetus by adjusting the maternal immune system in the pregnancy.
Objective: The aim of this study was to determine the role of sHLA-G in recurrent pregnancy loss (RPL) in North of Iran.
Materials and Methods: This case-control study was done on two different groups including 40 women with recurrent miscarriage, and 40 non-pregnant healthy women. Soluble HLA-G (sHLA-G) levels were measured using a BioVendor sHLA-G ELISA kit.
Results: Findings show that women with recurrent abortion had significantly higher sHLA-G concentrations than fertile women (mean ± SD: 220.62 ± 223.48 u/ml and 87.77 ± 91.65 u/ml, respectively p < 0.0001, Mann-Whitney test).
Conclusion: There is many argumentation about the role of HLA-G in the pregnancy and RPL. Therefore document in this context remains obscure. So it can be concluded that sHLA-G may not act in the implantation of the embryo, but its role in the preservation of maternal tolerance to fetus, because serum sHLA-G level increased in the after abortion and postpartum in both women who had recurrent spontaneous abortion and normal vaginal delivery.
Key words: HLA-G, Recurrent pregnancy loss, Early pregnancy, Reproduction.

The original full text of this abstract has been published in Ann Trop Med Public Health 2018; 13.
How to cite to this article: Adib Rad H, Basirat Z, Mostafazadeh A, Faramarzi M, Bijani A, Aghajanpour-Mir SM. The role of HLA-G in recurrent pregnancy loss: A case-control study. Ann Trop Med Public Health 2018; 13: SX738.

 
P-14
Protective effect of the co-administration of testosterone and sodium hydrosulfide on testicular H2S levels and serum testosterone in experimental model of varicocele
 
Shafie A¹, Seifi B¹, Ashabi G¹, Kadkhodaee M¹, Hajiaqai M¹, Lorian K².

1. Department of Physiology, Faculty of Medicine, Tehran University of Medical Science, Tehran, Iran.
2. Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: b-seifi@tums.ac.ir
 
Background: Androgen secretion is reduced in varicocele. Hydrogen sulfide (H2S), is known as an antioxidant and antiapoptotic molecule.
Objective: This study aimed to assess the effects of co-administration of testosterone and NaHS on sperm count, H2S levels in testicular tissues and serum testosterone in varicocele-induced male rats.
Materials and Methods: Adult male rats were randomly assigned to 5 groups: sham, varicocele, varicocele+testosterone, varicocele+NaHS, varicocele+tetstosterone+NaHS. In the varicocele groups, the left renal vein was partially ligated. In treatment groups, five wk after the induction of varicocele, testosterone (200 µg/kg, subeffective dose) was given subcutaneously for four wk and NaHS (15 µmol/L in drinking water, subeffective dose) were given for four wk. The Left testis tissue samples resected for evaluation H2S levels. The left epididymis tissue also resected for sperm count. blood samples were taken from the inferior vena cava.
Results: Varicocele caused significant reduction in sperm count, testicular H2S levels and serum testosterone compared with the sham group. Administration of testosterone+NaHS significantly increased these parameters compared with varicocele group. But there were no significant changes in these parameters in varicocele+NaHS and varicocele+testosterone group compared with the varicocele group. However, there was a significant enhancement in serum testosterone levels in varicocele+testosterone group compared with the varicocele group but this enhancement was lesser than varicocele+tetstosterone+NaHS group that may due to synergistic effect of NaHS and testosterone.
Conclusion: This study suggested that long term testosterone and NaHS co-administration could improve testicular H2S levels and serum testosterone in varicocele male rats. Therefore, testosterone+NaHS appears to be a useful treatment against varicocele.
Key words: Varicocele, Testosterone, Hydrogen sulfide, Testicular H2S levels, Serum testosterone.
 
P-15
The effectiveness of mindfulness-based cognitive therapy on sexual function in reproductive age
 
Bokaie M¹, Mohammad alian F², Farzinrad B³, Dehghani A³.

1. Research Center for Nursing and Midwifery Care, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Collage of Nursing and Midwifery, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
3. Shahid Sadoughi University of Medical Sciences,Yazd, Iran.

Email: fsmdan1393@gmail.com
 
Background: Sexuality is an important part of the human life.
Objective: The aim of this study was to investigate the effectiveness of group counseling mindfulness-based cognitive therapy on sexual function of reproductive age women.
Materials and Methods: This study is a parallel randomized controlled trial with pre-test, post-test and follow-up. Fifty reproductive age women in randomly allocated in two intervention and control group. For intervention group (25 persons) 8 sessions of mindfulness intervention (90 minutes weekly) was done and control group received routine clinic services. FSFI questionnaire were complete by two groups before, after and one month after intervention. Data analysis was performed using SPSS 24 and p < 0.05.
Results: In intervention group main score of FSFI were reported 22.43 ± 5.66, 26.43 ± 4.96 and 26.26 ± 4.57 before, after and one month after intervention respectively and in control group were 24.00 ± 5.66, 18.50 ± 5.46 and 18.83 ± 5.35 before, after and one month after intervention respectively. The results of the study show that group counseling mindfulness-based cognitive therapy has a meaningful effect on sexual function of on women of reproductive age.
Conclusion: Mindfulness counseling significantly improve sexual function of reproductive age women.
Key words: Sexual function, Mindfulness, Reproductive age women, FSFI.
 
P-16
Antioxidant effects of royal jelly on lead induced sperm DNA damage and sperm abnormality in male mice
 
Kakebaraei S¹, Jalili C².

1. Student Research Committee, Kermanshah University of Medical Sciences, Kermanshah, Iran.
2. Department of Anatomy, School of Medicine, Kermanshah Medical University, Kermanshah, Iran.

Email: seyranbaraie@yahoo.com
 
Background: Royal jelly, a secretion vintage of the salivary glands of worker bees, is an extremely impressive antioxidant and possesses eminent free radical scavenging trait. It has been shown to have anti-tumor, antibiotic, anti-inflammatory, immunomodulatory and as well as antioxidant properties. Royal jelly has remarkable positive effects on reproductive system and fertility. On the other hand, lead, which is widely used in industry, can be detected in foods, drinking water, ambient air, dust, and various cosmetics products. Its toxicity causes excess levels of reactive oxygen species (ROS) and cellular oxidative stress. More importantly, our knowledge of the effects of lead on sperm DNA integrity is significantly limited.
Objective: We investigated the effect of Royal jelly against reproductive toxicity caused by lead exposure in mice.
Materials and Methods: Male mice received lead acetate (50 mg/kg) and plus Royal jelly (25, 50 or 100 mg/kg) via oral gavage for 28 days. Adult male mice were divided into five groups (n = 5). Caudal epididymal sperm characteristics, lipid peroxidation and in vitro fertilizing capacity were evaluated after 4 weeks.
Results: Lead acetate-intoxicated mice exhibited testicular tissue injury and decreased serum levels of SOD, TAC, testosterone and increased serum level of MDA and nitric oxide. The count, viability, motility and normal morphology of the sperms were decreased in lead-induced group. Royal jelly prevented testicular injury, increased serum levels of SOD, TAC and improved the semen quality and decreased serum level of MDA and nitric oxide. However, Royal Jelly can reduce the regulation of Bax and Caspase-3 pro-inflammatory factors in lead-treated mice by reducing oxidative stress.
Conclusion: Our findings showed that Royal jelly with its antioxidant effects reduces inflammation and cell death in testis and sperm DNA structure following exposure to lead. However, further studies are needed to illuminate other mechanisms of Royal jelly's effect on testis function.
Key words: Royal jelly, Lead acetate, Sperm, Caspase3, Bax.
 
P-17
Correlation between serum TGF-ß levels and recurrent implantation failure during implantation window in women undergoing IVF treatment
 
Saifi B¹, Nasiri R², Parvanehhosseini M¹.

1. Department of Anatomy, Islamic Azad University, Mashhad Brach, Mashhad, Iran.
2. Department of Obstetrics and Gynecology, Islamic Azad University, Mashhad Brach, Mashhad, Iran.

Email: bita_saifi@yahoo.com
 
Background: Frequent implantation failure is a common problem among women who underwent in vitro fertilization (IVF) procedure. Therefore, it is necessary to know the factors affecting recurrent implantation failure following IVF treatment.
Objective: The aim of present study was to investigate the relationship between serum TGF-ß levels and recurrent implantation failure during the implantation window in women undergoing IVF.
Materials and Methods: This study was performed on 39 patients including 20 women with recurrent implantation failure (case group) and 19 women with successful pregnancies in the first IVF cycle (control group). Serum TGF-ß levels were measured using enzyme-linked immunosorbent assay (ELISA) method. Demographic information including age, body mass index (BMI) and number of implantation failure were recorded.
Results: The mean serum levels of TGF-ß in the individuals with recurrent implantation failure was significantly lower than the control group (663.48 pg/ml vs. 1028.49 pg/ml). Moreover, the serum TGF-ß levels were significantly different among case and control groups based on the age and BMI grouping. However, there was no relationship between serum TGF-ß levels in the case group and age, BMI and number of implantation failures.
Conclusion: Serum TGF-ß levels may play a crucial role in the physiopathology of recurrent implantation failure. Measurement of this factor in the patients with recurrent implantation failure is recommended which may reduce the incidence of recurrent implantation failure following IVF treatment. However, further randomized clinical studies are required to clarify the definite correlation.
Key words: Recurrent implantation failure, TGF-ß, In vitro fertilization.
 
P-18
L-carnitine reduces inflammation and oxidative stress in mouse ovarian tissue following autotransplantation
 
Sanamiri K¹, Soleimani Mehranjani M¹, Shahhoseini M², Shariatzadeh S¹.

1. Department of Biology, Faculty of Science, Arak University, Arak, Iran.
2. Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

Email: m-soleimani@araku.ac.ir
 
Background: Transplantation of ovarian tissue is a fertility restoration technique in patients undergoing chemotherapy and radiotherapy. A major issue associated with ovarian transplantation is ischemia/reperfusion injury that leads to depletion and apoptosis of follicles. L-carnitine has antioxidant and anti-inflammation properties and can therefore be used to reduce ischemic damages.
Objective: The aim of this study was to investigate the effect of L-carnitine injection on transplanted mouse ovarian tissue.
Materials and Methods: The Naval Medical Research Institute (NMRI) mice at the age of 4-5 weeks, were divided randomly into groups of: Control, autograft and autograft + L-carnitine (200 mg/kg daily intrapritoneal injections). Seven days post ovary autografting, serum levels of Malondialdehyde (MDA), total antioxidant capacity, tumor necrosis factor alpha (TNF-α), interleukin (IL)-6 and IL-10 were measured. Data were analyzed using one-way analysis of variance (ANOVA) and Tukey test, and the means were considered significantly different at p < 0.05.
Results: A significant increase was found in the serum level of IL-6, TNF-α and MDA in the autograft group compared to the control counterpart whereas the mentioned parameters reduced significantly in the autograft+L-carnitine group. The Total antioxidant capacity and the serum level of IL-10 also revealed a significant decrease in the autograft group when compared to the control while they significantly increased in the autograft+L-carnitine group.
Conclusion: L-carnitine could reduce oxidative stress and inflammation following mouse ovarian tissue transplantation.
Key words: Ovary transplantation, L-Carnitine, Ischemia-reperfusion, Inflammation.
P-19
Assessment of sperm parameters in type 1 and 2 diabetes mellitus male mice C57
 
Pouriayevali F^{1, 2}, Tavalaee M¹, Nasr-Esfahani MH¹.

1. Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
2. Faculty of Veterinary Medicine, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran.

Email: farnaz.pouriayevali@gmail.com
 
Background: Diabetes mellitus could have multiple effects on various organs of the body. One of the organs that are sensitive to these effects is testis and spermatogenesis process.
Objective: Aim of this study was to compare the effects diabetes type 1 (DM1) and type 2 (DM2) on sperm parameters.
Materials and Methods: Forty male mice C57 (8 weeks; 22 gr) were divided into 4 groups (n = 10/each). Mice were fed with standard-chow diet except DM2 group that was fed with a 60%-kcal high-fat diet for 8 weeks. Furthermore, sham group received a single dose of sodium citrate buffer (0.005 mg/kg) as soluble of streptozotocin (STZ), DM1 group was induced by multiple low-dose injections of STZ (45 mg/kg/day for 5 consecutive days), and DM2 group after four weeks was given a single dose of STZ (110 mg/kg). After eight weeks, the mice were sacrificed and sperm was extorted from the cauda epididymis for tests on sperm parameters.
Results: This study showed that the effects of diabetes on sperm parameters were compared between groups. The mean percentage of sperm non progressive motility significantly was higher in DM2 group than control group (p = 0.05), however sperm total motility difference between groups wasn’t remarkable. Moreover, the mean percentage of sperm concentration was lower in DM1 group compared to other groups (p < 0.02).
Conclusion: Sperm parameters in type 1 and 2 diabetes mellitus male mice C57 could effect on reproductive system. This result showed that reduction of sperm concentration and progressive motility of sperm in DM1 and DM2 model mice were lower compared to control group.
Key words: Diabetes mellitus, Sperm parameters, Infertility.
 
P-20
Comparison the scores of couples' sexual knowledge and attitude before and after sexual education
 
Sadat Z¹, Ghofranipour F².

1. Faculty of Midwifery, Kashan University of Medical Sciences, Kashan, Iran.
2. Department of Health Education and Health Promotion, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.

Email: sadat_z2003@yahoo.com
Background: Sexual health education is an essential human need. This education enriches their sexual knowledge and information, and help them to understand their responsibilities, commitments and rights in order to be able to achieve an enjoyable life.
Objective: The purpose of this study was to assess the effect of educational package on couples' sexual knowledge and attitude.
Materials and Methods: An interventional study was performed on a sample of 160 couples referred to premarital counseling center in Kashan City, Iran. Couples were divided randomly into the intervention and control groups (n = 80/each couples). Intervention group received an educational program in ten sessions on weekends for three consecutive weeks and control group received routine program. Sexual knowledge and attitude scale was completed before, after intervention, and in 6 months follow up. The data were analyzed by independent Student's t test, Mann Whitney U, Kruska-wallis, Chi-square, and ANOVA in SPSS V16.
Results: Results showed no significant differences in demographic characteristics and sexual knowledge and attitudes between two groups before intervention. The results demonstrated that after intervention and six months follow up, the scores of sexual knowledge increased significantly in the intervention group compared to control group. Mean score of sexual knowledge in intervention and control groups after intervention was 21.72 ± 7.73 and 14.08 ± 7.16 (p < 0.001) and after six months follow up was 21.89 ± 7.01 and 15.52 ± 7.73 (p < 0.001), respectively. In addition, the results demonstrated after intervention and six months follow up scores of sexual attitudes increased significantly in the intervention group compared to controls. Mean score of sexual attitudes in intervention and control groups after intervention was 134.90 ± 12.84 and 122.62 ± 14.15 (p = 0.001) and after six months follow-up was 134.22 ± 13.43 and 124.78 ± 13.48 (p = 0.001), respectively.
Conclusion: Sexual educational programs can be effective on couple's sexual knowledge and attitude.
Key words: Couples, Sexual health, Educational program, Marriage, Attitude.
 
P-21
Correlation between sexual knowledge and couple relationship among cardiovascular patients
 
Taghizadeh Firoozjaei I¹. Taghadosi M², Sadat Z².

1. Medical Surgical Nursing Department, Faculty of Nursing and Midwifery, Kashan University of Medical Sciences, Kashan, Iran.
2. Trauma Nursing Research Center, Kashan University of Medical Sciences, Kashan, Iran.

Email: sadat_z2003@yahoo.com
 
Background: Lack of good couple relationship can lead to reduced health, lifespan, satisfaction with marital life, and disruption of growth and excellence of couples.
Objective: The study aimed to evaluate correlation between sexual knowledge and couple relationship quality among cardiovascular patients.
Materials and Methods: This correlational study was conducted on 200 cardiovascular patients with myocardial infarction or patients undergoing coronary artery bypass grafting during the past two to five months. They referred to the Department of Rehabilitation of Shahid Beheshti Hospital of Kashan, Iran in 2017. Patients who met the inclusion criteria filled the individual characteristics questionnaire, couple quality Relationship scale and sexual knowledge scale. Data analysis was performed in SPSS version 22 using t test, analysis of variance, as well as Pearson correlation coefficients. P-value less than 0.05 was considered statistically significant.
Results: In this study, the mean score of participants' age was 52.68 ± 6.88 and 62% of them were male. The score of sexual knowledge scale of the participants was 55.85 ± 11.61 of total score 100 and mean scores of quality relationship scale was 17.82 ± 8.66 of total score 33. According to the results, there was a significant and positive correlation between the score of sexual knowledge scale and the score of quality relationship scale (r = 0.530, p < 0.001).
Conclusion: Results showed that the sexual knowledge score and couple relationship score in participants was moderate, and promotion of the sexual knowledge in cardiovascular patients can improve their couple relationship.
Key words: Sexual knowledge, Couple relationship, Cardiovascular patients.

Part of the results presented in this abstract has been published inInt J Reprod BioMed 2021; 19: 261-270. https://doi.org/10.18502/ijrm.v19i3.8574.
How to cite to this article: Taghizade Firoozjaei I, Taghadosi M, Sadat Z. Determining the sexual quality of life and related factors in patients referred to the department of cardiac rehabilitation: A cross-sectional study. Int J Reprod BioMed 2021; 19: 261-270.

P-22
Validity and relibility of the persian version of the sexual quality of life-male questionnaire
 
Sadat Z¹, Ghofranipour F², Goshtasebi A³, Azin SA³.
1.Trauma Nursing Research Center, Kashan University of Medical Sciences, Kashan, Iran.
2.Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
3.Health Metrics Research Center, Iranian Institute for Health Sciences Research, ACECR, Tehran, Iran.
Email: sadat_z2003@yahoo.com
 
Background: A valid and reliable instrument that can evaluate the impact of sexual dysfunction on quality of life in men is needed.
Objective: The aim of this study was to evaluate the psychometric properties of sexual quality of life-male (SQOL-M) questionnaire in a sample of Iranian men.
Materials and Methods: In this cross-sectional study, using a standard "forward-backward" translation technique, the English language version of the SQOL-M questionnaire was translated into Persian. One hundred and forty eight men (21-57 yr old) that referred to a health center in Kashan city were enrolled in this study. Exploratory and confirmatory factor analysis, convergent and known groups validity by using the international index of erectile function (IIEF) and content validity were assessed. The reliability was evaluated by test re test reliability correlation coefficient (ICC).
Results: Exploratory and confirmatory factor analysis confirmed a one-factor solution with good item-total correlations. Convergent validity showed total score of the international index of erectile function and its subscales were correlated with scores of the the SQOL-M questionnaire. Evaluating known groups validly showed men without erectile dysfunction scored more than men with erectile dysfunction (p < 0.001). Content validly was performed by 10 specialists. Reliability evaluation was demonstrated excellent internal consistency and test-retest reliability (Cronbach’s alpha and intraclass correlation coefficient were 0.94 and 0.95 respectively).
Conclusion: The results of the study showed the Persian version of SQOL-M instrument has a good structural characteristic and is a valid and reliable tool for measuring the SQOL-M.
Key words: Sexual quality of life, Men, Reliability, Validity, Erectile dysfunction.

The original full text of this abstract has been published in Payesh 2017, 16(1). https://www.sid.ir/en/journal/viewpaper.aspx?id=519445.
How to cite to this article: Sadat Z, Ghofranipour F, Goshtasebi A, Azin S. Validity and relibility of the persian version of the sexual quality of life-male questionnaire. Payesh 2017, 16(1), 73-80.

 
P-23
Evaluation the effects of vitamin D supplementation of the extender on sperm quality after freeze-thaw process in normozoospermic and asthenozoospermic Holstein bulls
 
Taravat M¹, Asadpour R¹, Rahbar M¹, Khoshniat M¹, Hamidian GhR².

1. Department of Clinical Science, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran.
2. Department of Basic Science, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran.

Email: r_asadpour@tabrizu.ac.ir
 
Background: Asthenozoopermia is a usual male infertility factor, characterized by decreased sperm motility. There is evidence that vitamin D regulates widespread biological function.
Objective: This study aimed to evaluate the effects of vitamin D on sperm kinematics and apoptosis in normozoospermic and asthenozoospermic bulls’ semen after the freeze-thaw process.
Materials and Methods: The effect of vitamin D on sperm quality factors such as sperm kinematic, sperm plasma integrity, acrosomal membrane integrity, reactive oxygen species (ROS) and apoptosis statues following freezing and thawing process in asthenozoospermic bulls were examined. For this purpose, 32 semen samples of four Holstein bulls (normozoospermic, progressive motility > 70%) and 32 semen samples of four bulls (asthenozoospermic progressive motility < 40%) were collected. Then, the poll semen samples of each group (normozoospermic and asthenozoospermic) were diluted into four equal aliquots of extender containing different vitamin D concentrations (0, 5, 10, and 50 ng/mL) and aspirated into 0.5 mL straw. Semen straws were frozen in liquid nitrogen. After thawing, sperm kinematics parameters, viability, plasma membrane integrity, acrosome integrity, apoptosis statues, and ROS production levels were evaluated.
Results: The percentage of sperm motility and viability were significantly higher in 50 ng/mL of vitamin D in both groups (p < 0.05). Normozoospermic bull semen samples had significantly higher curvilinear and average path velocity levels in 50 ng/mL vitamin D groups compared to the control group (p < 0.05). However, no significant differences were observed in post-thaw sperm kinematics parameters in asthenozoopsrmic samples. No significant differences were identified in membrane integrity and acrosome integrity in both normozoospermic and asthenozoospermic samples. The percentage of early-apoptosis (p = 0.049) and late-apoptosis (p = 0.005) in the asthenozoospermic group were significantly higher than the normozoospermic group. Generally, in the asthenozoospermic group, the level of ROS production was significantly higher (p = 0.049) compared to the normozoospermic samples.
Conclusion: According to our results, it can be concluded that the vitamin D supplementation of the asthenozoospermic semen extender had no significant effect on the quality of semen after the freeze-thaw process.
Key words: Vitamin D, Apoptosis, Sperm kinematic, Asthenozoospermic, Normozoospermic.
 
P-24
The effect of alpha lipoic acid on human sperm cryopreservation
 
Shaygannia E¹, Ghandehari-Alavijeh R², Tavalaee M³, Nasr-Esfahani MH⁴.
Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
Email: mh.nasr-esfahani@royaninstitute.org
 
Background: Nowadays, infertility problems are dramatically elevating around the world. In this regard, assisted reproductive techniques are developing productively. The cryopreservation technique of sperm cells is one of the common daily processes in infertility centers. However, the freezing-thawing process induces the production of reactive oxygen species (ROS) which is strongly harmful to sperms and reduces their quality post thawing procedure. Accordingly, the addition of some antioxidants to the sperm freeze medium is one way to come over this problem.
Objective: In the current study, considering the therapeutic effect of alpha-lipoic acid (ALA) on improving sperm parameters in the literature, we decided to investigate its impact on preserving freeze-thaw sperms from ROS damages.
Materials and Methods: 20 normozoospermic samples were obtained from the Isfahan Fertility and Infertility Center. Different concentrations of ALA (0, 0.05, 0.1, 0.2, 0.4, 0.8, and 8 mM) were added to the sperm freeze medium to gain the best concentration. With the optimum concentration, its’ protective impact on sperm motility and DNA fragmentation was investigated.
Results: 0.2 mM of ALA showed the best effect on sperm motility. Consequently, assessment of sperm DNA damage was carried out before and after the thawing procedure with and without using the optimal concentration of ALA. Our result indicated a significant reduction in DNA damage at the presence of ALA (0.2 mM, p < 0.05).
Conclusion: ALA could have a cryoprotective effect on sperm motility and DNA damage through its’ antioxidant capacity and its ROS scavenging capacity.
Key words: Alpha lipoic acid, Freez-thaowing, DNA damage.
 
P-25
Anti-cancer properties of ethanol extracts of grey mangrove leaves on a breast cancer cell line
 
Afshar A¹, Zare M¹, Khoradmehr A¹, Mohebi G¹, Barmak A², Bargahi A¹, Daneshi A¹, Azari H¹, Nabipour I¹, Mahmudpour M³, Tamadon A¹.

1. The Persian Gulf Marine Biotechnology Research Center, The Persian Gulf Biomedical Sciences Research Institute, Bushehr University of Medical Sciences, Bushehr, Iran.
2. Food Lab, Bushehr University of Medical Sciences, Bushehr, Iran.
3. The Persian Gulf Tropical Medicine Research Center, The Persian Gulf Biomedical Sciences Research Institute, Bushehr University of Medical Sciences, Bushehr, Iran.

Email: amintamaddon@yahoo.com
 
Background: The breast cancer, as the most incident cancer in women, is rising up the concerns of women mortality worldwide. Herbal medicine showed to be effective in treatment of oncological disorders. Mangrove includes several kinds of phytochemical compounds including terpenoids, alkaloids, flavonoids, saponins, and glycosides which some of them shown to have anti-cancer effects.
Objective: In the current study, the anti-cancer effects of ethanolic (EtOH) extract of the grey mangrove (Avicennia marina) leaves of the Persian Gulf on a breast cancer cell line was evaluated.
Materials and Methods: The leaves of A. marina were collected from Asaluyeh mangrove forest, shores of the Persian Gulf, Iran. The EtOH extract of A. marina leaves were provided according to standard procedures. The phytochemically analysis including total phenolic and flavonoid contents were measured. In addition, the extracts were analyzed by gas chromatography-mass spectroscopy (GC-MS) and their compounds were analyzed. The extracts were used for in-vitro study. MTT analysis, population doubling time assay, and western blot analysis were performed on MCF-7 breast cancer cell line and Vero cell line. IBM SPSS Statistics 26 and GraphPad prism were used for statistical analysis. The mean differences between groups were analyzed by one-way ANOVA and post hoc Tukey test.
Results: The GC-MS analysis showed that there was 12 potent anti-cancer, anti-proliferation, and anti-oxidant compounds in EtOH extract. Four of these compounds including levoglucosan, 2,4-Di-Tert-Butylphenol, Pentadecanoic acid, and linoleic acid showed to have anti-cancer effects on MCF-7 cell line in previous studies. The MTT proliferation assay showed that the 120 and 160 µg/mL concentrations of ETOH extract had lower value than the control group (p < 0.05). Moreover, this result in Vero cell line was only seen in 160 µg/mL concentration (p < 0.05). In contrast with other concentrations, total cell counts of the 120 and 160 µg/mL concentrations was significantly lower than control group in all 7 days (p < 0.01). Furthermore, in Vero cell line, the 120 µg/mL concentration at day 3, 4, 5 and 7, and the160 µg/mL concentration at day 4 to 7 had lower value than the control group (p < 0.01). However, at the MCF-7 cell line, the cell viability rate of 160 µg/mL concentration was only different from control group at day 2 (p < 0.01). Moreover, at the Vero cell line, the 120 and 160 µg/mL concentrations had lower value than the control group at day 5 (p < 0.001 and p = 0.041, respectively). Additionally, the western blot analysis of the EtOH extract showed that the Bax, Cleaved-caspase-1, Cleaved-caspase-3 and Cleaved-caspase-7 proteins’ expression were significantly higher than the control group; represented that A. marina EtOH extract induced apoptosis in MCF-7 cell line.
Conclusion: The anti-cancer effects of EtOH extract of A. marina leaves at the concentrations of 120 and 160 µg/mL was shown through anti-proliferation and apoptosis of MCF-7 breast cancer cell line.
Key words: Mangrove, Avicennia, Anti-cancer, Apoptosis, Ethanol.
 
P-26
The comparison of depression and anxiety between fertile and infertile couples: A meta-analysis study
 
Fallahzadeh H¹, Zareei Mahmood Abadi H², Momayyezi M¹, Malaki Moghadam H³, Keyghobadi N³.

1. Departments of Biostatistics and Epidemiology, Center for Healthcare Data Modeling, School of Public Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Department of Psychology, Faculty of Humanities, Yazd University, Yazd, Iran.
3. Department of Statistics and Epidemiology, School of Public Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: mahdieh_momayyezi@yahoo.com
Background: Depression and anxiety are 2 of the most common reactions in infertile couples. Several studies have been conducted to examine the psychiatric disorders among infertile and fertile couples.
Objective: This meta-analysis was conducted to compare the depression and anxiety in fertile and infertile couples in various studies.
Materials and Methods: The authors searched articles published in multiple databases including World Health Organization, PubMed, Cochrane Library, Scopus, Science Direct, Medline EMBASE and Persian databases including SID and Iran Medx between 2005 and 2017. The main keywords used for searching the databases were: Depression, anxiety, infertility, and fertility. Statistical analyses were performed using comprehensive meta-analysis /2.0 software.
Results: The authors found 42 related articles after searching the databases. 11 articles entered the meta-analysis after considering the inclusion and exclusion criteria. Finally, eight articles were chosen for the comparison of depression and anxiety, two published articles for the comparison of depression, and one published article to compare anxiety in fertile and infertile couples. The results of the heterogeneity test showed a significant heterogeneity among all articles that were analyzed in this meta-analysis in the field of depression and anxiety. The results showed that depression (p = 0.0001; Hedges’g = 1.21; 95% CI 0.63-1.78) and anxiety (p = 0.00001; Hedges’g = 0.63; 95% CI 0.54-0.73) were higher in infertile couples than fertile couples and that the possibility of a publication bias does not exist in this study.
Conclusion: The analysis of articles used in this meta-analysis showed that depression and anxiety scores in infertile couples were higher than fertile couples.
Key words: Depression, Anxiety, Infertility, Meta-analysis.

The original full text of this abstract has been published in Int J Reprod BioMed 2019; 17: 153-162. https://doi.org/10.18502/ijrm.v17i3.4514.
How to cite to this article: Fallahzadeh H, Zareei Mahmood Abadi H, Momayyezi M, Malaki Moghadam H, Keyghobadi N. The compar-ison of depression and anxiety between fertile and infertile couples:A meta-analysis study. Int J Reprod BioMed 2019; 17: 153-162.

 
P-27
Waiting anxiety in infertile women referring to Yazd Infertility Center
 
Momayyezi M¹, Fallahzadeh H¹, Anbari Nogyni Z², Anoosheh VS³, Farzaneh F⁴.

1. Departments of Biostatistics and Epidemiology, Center for Healthcare Data Modeling, School of Public Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Department of Aging Health, School of Public Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
3. Department of Ergonomy, School of Public Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
4. Department of Statistics and Epidemiology, School of Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: mahdieh_momayyezi@yahoo.com
Background: Infertility has become a serious problem in societies, which may cause inevitable harm to the mental health in individuals. People suffering from their illness may experience increased anxiety if they wait for some time to receive services.
Objective: The present study was conducted to determine the status of waiting anxiety in infertile women.
Materials and Methods: This descriptive-analytic study was conducted on 200 infertile women who consulted the Infertility Center of Yazd for treatment in 2017. The method of sampling was conducted based on convenience sampling (availability sampling). Data were collected with waiting anxiety questionnaire. Statistical analysis was done by using SPSS software (version 16.0). The analysis included: (1) descriptive statistics [mean and standard deviation], (2) chi-square, Student's t test, ANOVA, and Pearson correlation coefficient.
Results: The total mean of waiting anxiety in infertile women was 20.69 ± 5.82. Based on the results, the mean of the dimensions were as follows: cognitive dimensions (5.31 ± 2.25), physiologic dimensions (5.24 ± 2.55), emotional dimensions (5.01 ± 2.13) and behavioral dimensions (14.32 ± 2.03). The results also showed that a significant relationship between the total mean of waiting anxiety and cognitive, physiology and behavioral dimensions with duration of infertility exists (p < 0.05). In addition, there was a significant relationship between the mean scores of behavioral dimensions with the duration of marriage (p = 0.04) and education (p = 0.015).
Conclusion: The results of this study showed that infertile women who consulted to the centers were in a moderate condition in terms of waiting anxiety. Therefore, designing and performing effective interventions to reduce the anxiety of infertile women is recommended.
Key words: Infertility, Women, Waiting anxiety.

The original full text of this abstract has been published in Zahedan J Res Med Sci; 21(3): e90877. https://doi.org/10.5812/zjrms.90877.
How to cite to this article: Momayyezi M, Fallahzadeh H, Anbari Nogyni Z, Anoosheh VS, Farzaneh F. Waiting aAnxiety in infertile women referring to Yazd Infertility Center. Zahedan J Res Med Sci Online ahead of Print; 21(3): e90877.

 
P-28
Dietary fat and minerals intake are related to semen quantity and quality in men referring to an Iranian Reproductive Sciences Institute: A cross sectional study
 
Haeri F¹, Shirani M¹, Nouri M², Dehghan Marvast L³, Ghiasvand R⁴.

1. Department of Community Nutrition, School of Nutrition and Food Science, Isfahan University of Medical Sciences, Isfahan, Iran.
2. Department of Community Nutrition, School of Nutrition and Food Science, Shiraz University of Medical Sciences, Shiraz, Iran.
3. Andrology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
4. Food Security Research Center, Isfahan University of Medical Sciences, Isfahan, Iran.

Email: rghiasvand@yahoo.com
 
Background: Some epidemiological studies have reported a relationship between infertility and lifestyle patterns including dietary habits.
Objective: Our objective was to identify the relation between sperm parameters and dietary fatty acid and mineral intake among Iranian infertile men.
Materials and Methods: This cross sectional was performed on 400 newly diagnosed infertile men in Yazd Reproductive Sciences Institute from July to December 2019. Men were recruited when their infertility was confirmed by the expert andrologist based on World Health Organization criteria. They delivered a semen sample and answered a 168 items semiquantitative food frequency questionnaire. All data were analyzed using SPSS V. 22 software. P-value less than 0.5 considered as significant.
Results: We found a positive association between poly-unsaturated fatty acid intake, total motility, and normal morphology (p = 0.03). Also, there was a significant negative association between the second quartile of sodium and calcium intake and sperm volume (ptrend: 0.04), compared with the first quartile.
Conclusion: We concluded that dietary of poly-unsaturated fatty acid intake, sodium and calcium intake are related to sperm morphology, volume and total motility in Iranian infertile men. However, more research is needed to confirm these relations and provide the evidence needed to exert these findings into clinical practice.
Key words: Sperm parameters, Male infertility, Fatty acid, Minerals.
 
P-29
Food groups intake and sperm variables in men referring to an Iranian Reproductive Sciences Institute: A cross sectional study
 
Haeri F¹, Shirani M¹, Shariatpanahi SP³, Dehghan Marvast L³, Ghiasvand R⁴.

1. Department of Community Nutrition, School of Nutrition and Food Science, Isfahan University of Medical Sciences, Isfahan, Iran.
2. Department of Epidemiology and Biostatistics, School of Health, Isfahan University of Medical Sciences, Isfahan, Iran.
3. Andrology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
4. Food Security Research Center, Isfahan University of Medical Sciences, Isfahan, Iran.

Email: rghiasvand@yahoo.com
 
Background: Infertility had an increasing trend between couples in the world. Several factors such as unhealthy dietary habits are associated with sperm abnormality.
Objective: This study was conducted to investigate the association between food groups intake and sperm variables in men referring to an Iranian Reproductive Sciences Institute.
Materials and Methods: 400 infertile Men 20-55 yr of age admitted to an Iranian Reproduction Research Institute, were selected for this cross-sectional study according to the World Health Organization Fifth Edition Laboratory Guidelines. Usual dietary intake was collected by using a 168 items semiquantitative food frequency questionnaire. The relationship between food groups and sperm factors was measured by a multiple linear regression model while other confounding variables were adjusted. All data were analyzed using SPSS V. 22 software. P-value less than 0.5 considered as significant.
Results: According to this study, after adjusting for potential confounders, there was a significant relationship between sperm count with refined grains and soft drink, a significant association between normal morphology with whole grains, low-fat dairy intake and fruit, semen volume is significantly related to red meat intake, low-fat dairy, fruit and tea intake and progressive motility had a significant association between progressive motility with whole grains, low-fat dairy, fruit, soft drink and coffee intake (p-trend < 0.05).
Conclusion: We concluded that there is a relationship between grains, dairy, fruits, meat, caffeine and tea dietary intake with sperm parameters, which are sometimes in line or in contradiction with the results of previous studies.
Key words: Diet, Male infertility, Food groups, Semen analysis.
 
P-30
Upregulation of elafin expression in the fallopian tube of ectopic tubal pregnancies compared to the normal tubes
 
Zakizadeh F¹, Mahmoudzadeh-Sagheb H^{1, 2}, Asemi-Rad A³, Ghasemi M^{4, 5}, Moudi B^{1, 2}, Sheibak N¹, Asadikalameh Z⁵, Heidari Z^{1, 2}.

1. Department of Histology, School of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran.
2. Infectious Diseases and Tropical Medicine Research Center, Zahedan University of Medical Sciences, Zahedan, Iran.
3. Department of Anatomy, School of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran.
4. Department of Gynecology and Obstetrics, Pregnancy Health Research Center, Zahedan University of Medical Sciences, Zahedan, Iran.
5. Department of Gynecology and Obstetrics, Zahedan University of Medical Sciences, Zahedan, Iran.

Email: zakizade.fateme72@gmail.com
 
Background: Ectopic pregnancy is one of the most important causes of maternal mortality and fallopian tubes are the location of 95% of ectopic pregnancies. Elafin is a natural antimicrobial molecule that plays an important role as an anti-inflammatory agent in mucosal surfaces and has been found in the female reproductive tract.
Objective: The aim of this study was to investigate elafin expression, in the fallopian tube mucosa of ectopic pregnancies compared to the normal tubes using immunohistochemistry techniques and quantitative reverse transcription (qRT-PCR).
Materials and Methods: In this case-control study, uterine tube samples were obtained from patients with an indication for surgical removal of the tubes. The case group (n = 20) consisted of patients who were undergoing salpingectomy due to an ectopic pregnancy, the control group (n = 20) included patients who had a salpingectomy and hysterectomy. Using qRT-PCR and immunohistochemistry, the expression of elafin was investigated in both study groups.
Results: Immunohistochemical expression of elafin in the epithelium and connective tissue was significantly increased in the implantation site of the patients in comparison with the control group (p < 0.001). The level of elafin mRNA increased in the mucous membrane of the fallopian tube from patients with the ectopic pregnancy compared to the normal mucosa (p < 0.001).
Conclusion: Increasing expression of elafin during an ectopic pregnancy may be a mechanism for enhancing innate immune response and be involved in related pathological conditions such as infection and ectopic implantation.
Key words: Elafin, Ectopic pregnancy, Immunohistochemistry, Reverse transcriptase quantitative PCR.

The original full text of this abstract has been published in Journal of Reproductive Immunology 2020. https://doi.org/10.1016/j.jri.2020.103136.
How to cite to this article: Zakizadeh F, Mahmoudzadeh-Sagheb H, Asemi-Rad A, Ghasemi M, Moudi B, Sheibak N, Asadikalameh Z, Heidari Z. Upregulation of elafin expression in the the fallopian tube of ectopic tubal pregnancies compared to the normal tubes. Journal of Reproductive Immunology 2020; 141: 103136.

 
P-31
Emotions towards potential genetic offspring among oocyte donors: A cross sectional study
 
Khosravi S¹, Kazemi A¹, Ahmadi S².

1. Isfahan University of Medical Sciences, Isfahan, Iran.
2. Isfahan Fertility and Infertility Center, Isfahan, Iran.

Email: kazemi@nm.mui.ac.ir
 
Background: The presence of maternal emotions towards the offspring resulting from assisted reproductive techniques (ART) has been previously reported in oocyte donors. However, there is limited information about the presence of these emotions in oocyte donors during the ART process and before pregnancy.
Objective: The aim of this study was to evaluate these emotions of women towards the potential genetic offspring and tocompare them with women treated with ART by using own oocytes.
Materials and Methods: A cross sectional study was conducted on 150 women who were divided into two groups of oocyte donors and those treated with ART and using autologous oocyte. At the time of oocyte retrieval and using a validated questionnaire, the emotions toward potential offspring (EPO) resulting from ART and its three dimensions (including imagination, sense of ownership, and importance of treatment outcome) were measured and compared in two groups.
Results: Out of 150 women, the mean score of EPO was 39.34 in oocyte donors and 49.52 in own oocyte women. Comparison of the EPO in the two groups showed that the emotions in all three dimensions were lower in oocyte donors than the other group (p < 0.0001). Moreover, in oocyte donors, the mean score of the scale of the importance of treatment outcome dimension was higher than the other two scales (p < 0.0001).
Conclusion: The results of the study showed that there is a significant emotion toward the potential offspring in oocyte donors. The presence of these emotions thus should be considered in formulating the ethical principles of ART by using oocyte donation.
Key words: Oocyte donation, Emotion, Offspring, Assisted reproductive techniques.
 
P-32
Reproductive health literacy and its relationship with some demographic factors in men referring to one infertility center in Mashhad, Iran
 
Saifi B¹, Mostafavian Z², Abtahi S³, Vakili N⁴.

1. Department of Anatomy, Mashhad Branch, Islamic Azad University, Mashhad, Iran.
2. Department of Community Medicine, Mashhad Branch, Islamic Azad University, Mashhad, Iran.
3. Department of Pediatric Cardiology, Mashhad Medical Sciences Branch, Islamic Azad University, Mashhad, Iran.
4. Faculty of Medicine, Islamic Azad University, Mashhad Branch, Mashhad, Iran.

Email: dr.mostafavian@mshdiau.ac.ir, soofy5@yahoo.com
 
Background: Infertility has been one of the most important problems of human societies throughout history, which has always caused many problems for people, depending on the perspective of societies. This disorder, which is defined as infertility after one year of unprotected sex, has directly and indirectly affected about 10% of the world's population. Men have rarely been the focus of research and their knowledge and attitude have been less studied. Researchers must consider the importance of each couple's awareness of infertility issue and also the fact that before any intervention in the field of health literacy and necessity to be aware of the current situation.
Objective: The present study aimed to investigate reproductive health literacy and attitudes toward infertility in men referring to one infertility center in Mashhad in 2019 and its relationship with variables such as age, education, occupation, income, duration of infertility and history of assissted reproductive therapy (ART).
Materials and Methods: In this cross-sectional study, men with infertility who referred to one infertility center in Mashhad in 2019 were entered by convenience sampling. Data collection tools included questionnaires of reproductive health literacy and attitudes toward fertility, as well as a checklist of demographic information. The reproductive health literacy questionnaire includes 20 questions and the attitude questionnaire includes 5 questions. Relationship between age, education, occupation, income, duration of infertility and ART history with reproductive health literacy and attitude was investigated in SPSS V. 20 with t test, ANOVA and linear regression with a significance level of less than 0.05. The present study has been approved by the ethics committee of the medical school of the Islamic Azad University of Mashhad with the code IR.IAU.MSHD.REC.1397.020.
Results: Mean and standard deviation of age and duration of infertility in 196 men included 32 ± 6 yr (21 to 60) and 3.11 ± 1.36 yr (1 to 7), respectively. Reproductive health literacy score (3.2 ± 0.3) didn’t show significant relationship with age (p = 0.336), education (p = 0.33), job (p = 0.493), income (p = 0.856), Infertility duration (p = 0.136) and history of ART (p = 0.057). All attitude questions were not related to education and history of ART. Attitude about surrogacy (p = 0.011) and the possibility of separation of each couple in case of infertility (p = 0.001) (p = 0.015) was different according to age.
Conclusion: Considering the equality of knowledge level scores of study participants who had different levels of job and university degree and the fact that the average score of most knowledge questions was around 3, it means that their answers to most questions were “I don’t know”. Therefore, it can be concluded that information about infertility in this group of men is low and the need for educational planning in this regard, especially in infertility treatment centers for specific groups and also through the public media for the general public is felt.
Key words: Health literacy, Attitude, Infertility, Men, Reproductive.
 
P-33
The effect of L-arginine on the menopausal estradiol
 
Lakzaei F¹, Karami M¹, Jalali Nadoushan MR².

1. Department of Biology, Faculty of Basic Sciences, Shahed University, Tehran, Iran.
2. Department of Pathology, Faculty of Medicine, Shahed University, Tehran, Iran.

Email: karami@shahed.ac.ir
 
Background: Menopause in women is associated with many complications such as hot flashes, osteoporosis, and infertility that most of them are related to the decrease of estrogen levels in this period. Treatment with high doses of estrogen is common but has side effects.
Objective: In this study, the effect of L-arginine administration on the level of this hormone in elderly rat was investigated.
Materials and Methods: Elderly Wistar rats were first studied with the help of Papanicolaou smear to identify the stage of female sexual cycle. If confirmed to have diestrus phase, the rats were randomly classified into the following groups: control (saline 1 mL/kg), intraperitoneally, and L-arginine dose groups (5, 25 and 50 mg/kg). They were injected saline or L-arginine over a period of at least three to nine days. At the end, the rats were anesthetized by intraperitoneally injection of ketamine 100 mg/kg and xylazine 20 mg/kg, and the blood samples were collected, and the estrogen levels were measured with enzyme-linked immunosorbent assay kit. The rats’ ovaries and uteri were also biometrically examined and fixed in the formalin. They were stained by hematoxylin and eosin and the number of cysts in the ovaries were counted. The data were analyzed by the Analysis of variance (ANOVA).
Results: L-Arginine at all doses (5-25 mg/kg) during all injection periods from three to nine days significantly increased the estradiol levels, but prominently reduced the ovarian cysts at the lowest doses (5 mg/kg).
Conclusion: Low doses of estrogen over short periods of time can relieve menopausal problems in animal, including estrogen levels and ovarian status, and this may be due to the modulatory role of estrogen in the animal's natural processes.
Key words: Menopause, L-Arginine, Estradiol level, Rat.
 
P-34
COVID-19 and male infertility
 
Mosleh H, Shabani R, Mehdizadeh M, Moradi F.
Department of Anatomical Sciences, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran.
Email: Shabani.r@iums.ac.ir
 
Background: Lately in 2019, a new type of coronavirus (Sars-CoV-2) was identified in China. SARS-CoV-2 may damage male reproductive tissues and cause infertility.
Objective: Despite the rare information in the literature on the relation of coronavirus diseases in the productiveness of humans and animals, it is important to be conscious about the effect of the coronavirus disease 2019 (COVID-19) pandemic on male fertility.
Materials and Methods: We searched on PubMed and Google Scholar databases in June 2020 to find papers and studies about COVID-19 and its effects on fertility. The search on PubMed found 98 papers and on Google Scholar found 224 papers. We exclude the papers which their titles or abstracts were not relevant. At last, we select the most related papers to use in this article.
Results: Reports recognized that COVID-19 is closely related to the cells that secrete the angiotensin-converting enzyme 2 (ACE2). ACE2 is one of the enzymes involved in the renin-angiotensin system and is widely secreted in several tissues, for example, testis tissue; and organs that have a high expression of ACE2 are volunteers for infection. Analyses showed that in testicular cells, such as spermatogonia, seminiferous duct cells, Sertoli, and Leydig cells, there is a high expression level of ACE2.
Conclusion: Male infertility is an important problem, so scientists are evaluating if the ACE2 in the Covid-19 pandemic can influence male fertility. To date, there is no evidence if the SARS-CoV-2 virus uses ACE2 receptors in the reproductive system and what, or any affect this can have on human infertility. Although, side effects of COVID-19 pandemic can influence on infertility too, like drugs that use for treatment, chemical disinfectants, and psychological disorders.
Key words: COVID-19, Sars-CoV-2, ACE2, Infertility.
 
P-35
The effect of three-dimensional nanocomposite scaffolds on spermatogonial stem cells differentiation
 
Mosleh H¹, Shabani R¹, Moeinzadeh A¹, Bagher Z², Mehdizadeh M¹.

1. Department of Anatomical Sciences, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran.
2. ENT and Head and Neck Research Center and Department, The Five Senses Institute, Hazrat Rasoul Akram Hospital, Iran University of Medical Sciences, Tehran, Iran.

Email: Shabani.r@iums.ac.ir
 
Background: Concern of azoospermia is common in male survivors of childhood cancer. Therefore, the culture of spermatogonial stem cells (SSC) for future treatment method is required, because these cells are important in spermatogenesis.
Objective: SSC isolation and differentiation are really important. 3D (Three-Dimensional) scaffolds play important role in cell culture, these scaffolds simulating a microenvironment similar to an extracellular matrix for differentiation of cells. The present study aimed to evaluate the efficiency of spermatogonial cells culture on a 3D microenvironment containing the Chitosan-Alginate (CA) scaffolds that contain graphene oxide (GO) nanocomposite for investigated the differentiation improving.
Materials and Methods: We isolated spermatogonial cells from neonatal 3‐ to 6‐day‐old NMRI mice. Then we prepared the scaffolds (Based on our last studies, which we added GO concentrations of 5, 15, 30, 45, 75 µg/ml to the CA, the seeded cells have shown strong attachment on CA/GO 30 µg and had the best biocompatibility compared with other concentrations. So, CA/GO 30 µg become our selected scaffold for this study). The scaffolds were analyzed using FTIR, XRD, and microCT to observe surface topography and morphology. SSC were cultured and divided in to 2 culture groups: (SSC + basic medium), and (SSC + CA/GO 30 µg scaffold). Basic medium was DMEM-F12 with KSR 10%, consisting of Bmp4 40 ng/ml and Retinoic acid 10-6 M. The stem cells related markers for differentiation of SSCs (SYCP3 and TEKT1) were detected on all experimental groups by RT-qPCR and ICC.
Results: Incorporation of GO into CA matrix increased both crosslinking density as indicated by the reduction of crystalline peaks in the XRD patterns and polyelectrolyte ion complex as confirmed by the FTIR. MicroCT analyses indicate that the scaffold had a highly porous and interconnected pore structure with porosity of 81.56 %. The RT-qPCR results showed that the expression of SYCP3 and TEKT1 genes were higher after 14 days in the SSC + CA/GO group compared to control (p < 0.05). ICC assays results showed that the mean expression of SYCP3 for SSCs cultured on the CA/GO 30 µg scaffold after 14 days was 49.37 ± 6.20 while Its mean expression for SSCs cultured on the basic medium after 14 days was 35.97 ± 4.70. The mean expression of TEKT1 for SSCs cultured on the CA/GO 30 µg scaffold after 14 days was 73.12 ± 3.94 however this marker’s mean expression for SSCs cultured on the basic medium after 14 days was 53.46 ± 3.09. So, the expression of SYCP3 and TEKT1 for (SSC + CA/GO 30µg scaffold) had significant increase than the other scaffold group (p  <  0.05).
Conclusion: The most expression of differentiation markers was in CA/GO 30 µg group. This scaffold has biocompatibility and degradable properties and graphene oxide helps to strengthen the scaffold and thus improves cell culture. This scaffold will provide a more improved structural environment for increased differentiation of SSCs.
Key words: Spermatogonial stem cells, Chitosan-Alginate Scaffold, Graphene oxide nanocomposite, Differentiation.
 
P-36
Evaluation of antioxidant and anti-inflammatory effects of tannic acid on sperm survival and motility in sepsis-infected male rats
 
Pourmirzaei F¹, Seifi B¹, Kadkhodaee M¹, Ranjbaran M¹, Kianian F¹, Lorian K².

1. Department of Physiology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
2. Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: faa.pou.6.m@gmail.com
 
Background: One of the problems caused by infectious diseases is the reduction of sperm count and motility. In this study, sepsis model was used to investigate the oxidative stress and inflammation in testicular and sperm structure. Since tannic acid has antioxidant and anti-inflammatory effects on various organs of the body, in this study, the effect of tannic acid on the above-mentioned indices as well as testicular and sperm function and structure were investigated.
Objective: The main aim of this study was to investigate the protective effect of tannic acid on short-term infertility due to oxidative and inflammatory conditions.
Materials and Methods: Twenty-four male Wistar rats in the weight range of 300-250 g were randomly divided into 3 groups of 8: 1) sham 2) sepsis 3) tannic acid. In the sham group, the animals were anesthetized and then underwent laparotomy, but sepsis induction was not performed in this group. In the sepsis group, the animals underwent anesthesia and laparotomy to induce sepsis, then 30-40% of the end of the cecum was double-tied with a double layer of silk suture. Two needle holes were then made in the closed cecum area with a needle number 25 to allow the infection to enter the abdominal cavity. In the tannic acid group, the animals received tannic acid at a dose of 20 mg/kg at 6, 12, and 24 hr after sepsis induction. Thirty hr after induction of sepsis, the animals were anesthetized and testis was fixed in 10% formalin for histological examinations. The end of the epididymis was used to examine sperm motility and survival.
Results: The percentage of motile sperm and the percentage of sperm survival decreased significantly in the sepsis group. The use of tannic acid significantly improved the inflammatory and oxidative status of testicular tissues as well as improving sperm parameters.
Conclusion: The results of this study showed that the reproductive system as well as the sex cells of male rats are strongly affected by the conditions created during sepsis. Tannic acid as an antioxidant and anti-inflammatory agent improves short-term infertility caused by infection.
Key words: Short-term infertility, Sepsis, Oxidative stress, Inflammation, Tannic acid.
 
P-37
The association between fatty acids and steroids gene expression in abdominal subcutaneous fat depot: A comparison between pregnant PCOS and non-PCOS pregnant women
 
Emami N^{1, 2}, Alizadeh AR², Moini A^{3, 4, 5}, Yaghmaei P¹, Shahhosseini M^{6, 7, 8}.

1. Department of Biology, Faculty of Science, Science and Research Branch, Islamic Azad University, Tehran, Iran.
2. Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
3. Department of Endocrinology and Female Infertility, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
4. Breast Disease Research Center (BDRC), Tehran University of Medical Sciences, Tehran, Iran.
5. Department of Gynecology and Obstetrics, Arash Women’s Hospital, Tehran University of Medical Sciences, Tehran, Iran.
6. Reproductive Epidemiology Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
7. Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.
8. Department of Cell and Molecular Biology, School of Biology, College of Science, University of Tehran, Tehran, Iran.

Email: neda.emami58@gmail.com
 
Background: It was reported that steroid-related gene expressions in the adipose tissue (AT) of women differ between women affected with polycystic ovary syndrome (PCOS) (case) and non-PCOS (control). Although association between PCOS in mother and offspring’s health is a crucial issue, there are few studies focusing on effectiveness of AT profiles on steroids genes expression in pregnant women suffering from PCOS.
Objective: Our objectives were to assess association between fatty acid (FA) and genes related to steroids metabolism expression in abdominal subcutaneous AT of 12 PCOS (case) vs. 32 non-PCOS (control) age- and BMI-matched pregnant women.
Materials and Methods: Twelve pregnant women with PCOS (case) and thirty two non-PCOS pregnant women (control) (age- and BMI-matched) undergoing cesarean section were enrolled for the present study. Expressions of fifteen genes related to steriodogenesis in abdominal subcutaneous AT were investigated using quantitative real-time PCR. Fatty acids profiles assessed by gas chromatography. Linear regression was performed to determine the association of FA and gene expression in subcutaneous AT.
Results: Age and BMI were similar among two groups at delivery day. Current study showed that omega-3 fatty acids had the highest association with steroids gene expression rate (r = 0.500; p < 0.05).
Conclusion: It seems that fatty acids, both direct and by metabolites, can play a role in many diseases through extensive signaling pathways, specifically in exacerbating PCOS, although pregnancy can double the role of nutrition in exacerbating these effects.
Key words: Polycystic ovary syndrome, Subcutaneous adipose tissue, Sex steroid, Fatty acids.

The original full text of this abstract has been published in Adipocyte 2020 Jan 1; 9(1): 16-23. https://doi.org/10.1080/21623945.2019.1710021.
How to cite to this article: Emami N, Alizadeh A, Moini A, Yaghmaei P, Shahhosseini M. Differences in fatty acid profiles and desaturation indices of abdominal subcutaneous adipose tissue between pregnant women with and without PCOS. Adipocyte 2020; 9(1): 16-23.

 
P-38
Investigating the correlation between ubiquitination with motility, morphology, and DNA methylation in rat sperm
 
Razavi Sh¹, Khadivi F², Hashemi F¹.

1. Department of Anatomy, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
2. Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.

Email: razavi@med.mui.ac.ir
 
Background: There are various techniques for treatment of male infertility, nowadays. At the beginning, evaluations are performed to determine the cause of infertility and the treatment. These include advanced molecular evaluations and assessment of sperm parameters. There is no study investigating the relationship between sperm parameters as an elementary index of male infertility, and deoxyribonucleic acid (DNA) methylation as an important epigenetic mechanism with rat sperm ubiquitination, so far.
Objective: The aim of this study was to evaluate the motility, morphology, DNA methylation, and ubiquitination in rat sperm and to determine the relationship between them.
Materials and Methods: First, 10 male mature rats were kept in experimental condition for 9 weeks (one cycle of spermatogenesis). After sacrificing, their semen samples were used to determine the sperm parameters and smear preparation. Through prepared smear and immunofluorescence assay, percentage of ubiquitinationed and methylated sperm were determined and finally, the correlation coefficient between them was calculated.
Results: There was no significant correlation between the ubiquitination and DNA methylation. However, there was an inverse and significant correlation between the percentage of ubiquitination and morphological abnormalities in spermatozoa (p < 0.05). The percentage of ubiquitinized sperm and sperm motility showed no significant correlation.
Conclusion: Ubiquitination, as one of the important molecular processes, prevents the participation of defective sperm in fertilization, and transmission of disorders to next generation. DNA methylation and ubiquitination affect sperm chromatin, but these two processes act in an independent manner.
Key words: Sperm motility, DNA methylation, Infertility, Ubiquitination.

The original full text of this abstract has been published in Journal of Isfahan Medical School (I.U.M.S) 2017, 35(445), 1151-1155.
How to cite to this article: Razavi S, Khadivi F, Hashemi F. Investigating the correlation between ubiquitination with motility, morphology, and DNA methylation in rat sperm. Journal of Isfahan Medical School (I.U.M.S) 2017, 35(445), 1151-1155.

 
P-39
Protective effects of zinc on rat sperm chromatin integrity involvement: DNA methylation, DNA fragmentation and protamination after bleomycin etoposide and cis-platin treatment
 
Khadivi F¹, Razavi Sh², Hashemi F².

1. Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
2. Department of Anatomy, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

Email: razavi@med.mui.ac.ir
 
Background: Testicular cancer is the most common malignancy that threatens the male in their reproductive age. Combined treatment by bleomycin, etoposide, cis-platinum (BEP) is the most effective strategy for patients with testicular cancer, and these chemotherapeutic agents can increase the 5- years survival rate. BEP treatment has revealed negative side effects on different germ cells, finally impacting reproductive function and fertility. In addition to cancerous cells, oxidative stress due to BEP treatment can destroy testicular germ cells and induce changes in chromatin integrity.
Objective: We decided to investigate recovery effect of zinc (Zn) on chemotherapy-induced complications in rat chromatin integrity and protamination.
Materials and Methods: The male rats (n = 40) were treated with BEP at appropriate dose levels of BEP (0.75, 7.5, and 1.5 mg/kg) for 9 week, with or without Zn; Sperm DNA methylation through immunofluorescence, DNA fragmentation and protamination were evaluated through acridine orange staining and Chromomycin A3 staining.
Results: The mean percentage of global DNA methylation sperm was significantly reduced as compared with the control group (p < 0.001). In BEP+‏ Zn group, the mean percentage of global DNA methylation increased compared to BEP group. In Zn group, the mean percentage of global DNA methylation had no significant difference compared with the control group. Following BEP treatment, the mean sperm count that represented DNA fragmentation was significantly increased (p < 0.001). In addition, the mean percentage of DNA fragmentation was reduced in the BEP +‏ Zn group in comparison with the BEP group, but not as much as the control Group (p < 0.001). The mean percentage of DNA fragmentation in rats treated with Zn indicated no significant difference compared with the control group. Rats treated with BEP showed significantly increased protamine deficiency sperm as compared with the control group (p < 0.001). In the BEP + ‏Zn group, recovery in protamination was observed compared with the BEP group, but there is still significant difference in comparison with the control group (p < 0.05). The significant difference was observed between Zn and control group (p < 0.05).
Conclusion: Our findings confirm the recovery effects of Zn on rat sperm chromatin integrity following BEP consumption. It is suggested that Zn be utilized as an antioxidant following chemotherapy.
Key words: Spermatozoa, DNA methylation, Protamination.

The original full text of this abstract has been published in Theriogenology 2020 Jan 15; 142: 177-183. https://doi.org/10.1016/j.theriogenology.2019.09.039.
How to cite to this article: Khadivi F, Razavi S, Hashemi F. Protective effects of zinc on rat sperm chromatin integrity involvement: DNA methylation, DNA fragmentation, ubiquitination and protamination after bleomycin etoposide and cis-platin treatment. Theriogenology 2020; 142: 177-183.

 
P-40
Application of platelet-rich plasma increases in vitro proliferation of human spermatogonial stem cells in two-dimensional and three-dimensional culture systems
 
Khadivi F¹, Koruji M^{2, 3}, Akbari M¹, Jabari A⁴, Talebi A⁵, Ashouri Movassagh S⁶, Panahi Boroujeni A⁷, Feizollahi N¹, Nikmahzar A¹, Pourahmadi M⁸, Abbasi M¹.

1. Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
2. Stem Cell and Regenerative Medicine Research Center, Iran University of Medical Sciences, Tehran, Iran.
3. Department of Anatomy, Iran University of Medical Sciences, Tehran, Iran.
4. Department of Obstetrics and Gynecology, Moluod Infertility Center, Zahedan University of Medical Sciences, Zahedan, Iran.
5. School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran.
6. Human and Animal Cell Bank, Iranian Biological Resource Center, ACECR, Tehran, Iran.
7. Department of Anesthesiology, Shahrekord University of Medical Sciences, Shahrekord, Iran.
8. Department of Anatomy, Jahrom University of Medical Sciences, Jahrom, Iran.

Email: farnazkhadivi031@gmail.com, Abbasima@sina.tums.ac.ir
 
Background: According to the American Cancer Society, the annual incidence rate of childhood tumors aged birth to 19 years old is 186.6 per 1 million adolescents. Regarding the improvement in life expectancy through the efficient medical procedures, side effects of treatment such as life quality and infertility are of grown importance. Spermatogonial stem cells (SSCs) are very sensitive to chemotherapy and radiotherapy, so male infertility is a great challenge for prepubertal cancer survivors. Cryoconservation of testicular cells before cancer treatment can preserve SSCs from treatment side effects. Different two-dimensional (2D) and three-dimensional (3D) culture systems of SSCs have been used in many species as a useful technique to in vitro spermatogenesis.
Objective: Since there is no available data on the proliferative effect of platelet-rich plasma (PRP) on SSCs, this research focused on the self-renewing of adult human SSCs in two-dimensional and three-dimensional culture systems of PRP.
Materials and Methods: Human testes samples taken from four brain-dead donors at 17, 21, 25, and 26 years old from November 2018 to September 2019. Approval from the family of each donor was acquired by the Organ Procurement Unit (OPU) of Imam Khomeini Hospital affiliated to Tehran University of Medical Science. Testicular cells cultivated in 2D pre-culture system, characterization of SSCs performed by RT-PCR and flow cytometry analysis. PRP prepared and dosimetry carried out to determine the optimized dose of PRP. After preparation of PRP scaffold, SSCs cultivated into three groups: Control, 2D culture by optimized dose of PRP and PRP scaffold. Finally, the diameter and number of colonies measured.
Results: After 2D pre-culture of testicular cells a significant increase in expression of OCT4, Vimentin, and VASA observed in comparison to after digestion    (p < 0.01). Our results indicated that 16.2 % of all cells were positive for PLZF after enzymatic digestion, whereas after the 2D pre-culture significantly increased the purity of SSCs to 80.2%. After cultivation of SSCs in experimental groups, the number and diameter of colonies in the PRP-2D group increased significantly   (p < 0.01) as compared to the control group. Interestingly, in the PRP- scaffold group only the mean number of colonies increased significantly (p < 0.01) related to the control group.
Conclusion: Our results suggested that PRP scaffold can reconstruct a suitable structure to the in vitro self-renewal and proliferation of human SSCs. The cytokines and growth factors obtained.
Key words: Spermatogonial stem cell, Proliferation, Two dimensional culture system.

The original full text of this abstract has been published in Acta histochemica 2020; 122(8): 151627. https://doi.org/10.1016/j.acthis.2020.151627.
How to cite to this article: Khadivi F, Koruji M, Akbari M, Jabari A, Talebi A, Movassagh SA, Boroujeni AP, Feizollahi N, Nikmahzar A, Pourahmadi M, Abbasi M. Application of platelet-rich plasma (PRP) improves self-renewal of human spermatogonial stem cells in two-dimensional and three-dimensional culture systems. Acta histochemica 2020; 122(8): 151627.


 
P-41
The hormone-sensitive lipase polymorphism    (C-60G) is related with recurrent pregnancy loss in women with polycystic ovary syndrome
 
Vatannejad A.
Department of Comparative Biosciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.
Email: vatannejad@ut.ac.ir
 
Background: Lipid metabolism disruption is related to the development of polycystic ovary syndrome (PCOS) in women of reproductive age. Hormone-sensitive lipase (HSL) is an intracellular lipase that has a crucial role in normal lipid metabolism.
Objective: This study aimed to assess the frequency of C-60G polymorphism of HSL in healthy women and PCOS women, and its correlation with infertility and abortion in PCOS patients.
Materials and Methods: A total of 324 PCOS patients (including 199 infertile patients and 125 patients with a history of recurrent abortion) and 144 healthy controls enrolled in this study. Biochemical parameters were measured and the genotypes of C-60G polymorphism of the HSL gene were determined using PCR-restriction fragment length polymorphism techniques.
Results: There was no significant differences in the genotype and allele frequencies of C-60G polymorphism between PCOS, PCOS-infertile woman and non-PCOS subjects. However, a higher percentage of combined variants (CG+GG) and CG genotypes, as well as G allele was found in the PCOS-abortion group in comparison with non-PCOS women. The presence of the G allele conferred a 2.4-fold risk for abortion in women with PCOS (OR: 2.4, 95% CI [1.22-4.70], p = 0.011). Furthermore, a significant correlation between CG or GG genotype of HSL and the level of free testosterone was observed.
Conclusion: According to the obtained results, the C-60G polymorphism in the HSL promoter was associated with PCOS-related abortion, possibly through increasing the level of free testosterone. Therefore, the rs34845087 polymorphism may be considered as a promising prognostic biomarker for abortion in women with PCOS.
Key words: Polycystic ovary syndrome, Hormone-sensitive lipase, Infertility, Abortion.
 
 
P-42
Circulating level of plasma Complement C1q/tumor necrosis factor-related protein 15 in polycystic ovary syndrome
 
Vatannejad A.
Department of Comparative Biosciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.
Email: vatannejad@ ut.ac.ir
 
Background: Poly-cystic ovarian syndrome (PCOS) is one of the frequent metabolic and endocrine disorder in women population that has a close relation with parameters of metabolic syndrome and obesity. Studies have shown perturbation of adipokines levels in PCOS patients. Complement C1q/tumor necrosis factor-related protein 15 (CTRP15) is a prologue of adiponectin that indicated a close relation with insulin, glucose and lipids metabolism.
Objective: In the present study we sought to evaluate the levels of this adipokines and its relation with cardiometabomic data.
Materials and Methods: This case-control study carried out on 120 PCOS patients and 60 controls. Serum levels of adiponectin and CTRP15 were determined using ELISA technique.
Results: Serum levels of CTRP15 elevated in patients with PCOS compared to controls while adiponectin decreased considerably. In addition, CTRP15 indicated a relation with BMI, insulin resistance and FSH levels.
Conclusion: Elevated levels of CTRP15 could be a compensatory response in patients with PCOS in response to obesity and insulin resistance, however further studies are needed to dissect the possible underlying mechanism.
Key words: Polycystic ovary syndrome, Insulin resistance, CTRP15, Adiponectin.
 
P-43
Comparing the effect of reduced graphene and graphene-L-arginine on sperm fertilizing ability
 
Zare-Zardini H¹, Dehghanbaghi N², Soltaninejad H³.

1. Medical Nanotechnology and Tissue Engineering Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Department of Biology, Faculty of Sciences, Science and Arts University, Yazd, Iran.
3. Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.

Email: hadizarezardini@gmail.com
Background: Sperm cell maturation occurs for fertility with longevity in the female reproductive system. Inducing and maintaining the ability of in vitro fertility of sperm is an important and effective factor in the success of pregnancy techniques and in vitro fertilization. So, various strategies such as addition of calcium or creatine phosphate, can be used for enhancement of the fertilizing capacity of sperm during in vitro fertilization. The change of chemical agents in sperm membrane can alter its fertility. Graphene, an allotrope of carbon, has interesting physical and chemical properties such as high electron mobility, high surface area, stiffness, strength and toughness. This nanostructure and their derivatives have been used in different fields of sciences, especially in biological and medical sciences.
Objective: The aim of this study was the comparison of the effect of reduced Graphene and Graphene-L-Arginine on sperm fertilizing ability.
Materials and Methods: In this study, we synthesized reduced and L-arginine-functionalized graphene by microwave method and characterized by transmission electron microscopy, scanning electron microscope, fourier-transform infrared spectroscopy, and raman spectroscopy. Acquired sperm samples from healthy volunteers were treated with different concentrations of reduced Graphene and Graphene-L-Arginine (1, 2, 3, 4, 5, 8, 10, 12, and 15 µg/ml).
Results: Results showed that water solubility of Graphene-Arginine was higher than reduced Graphene. This increase in solubility facilitates the use of functionalized graphene in chemical and physiological fluids. Between reduced Graphene and Graphene- L-Arginine, Graphene- L-Arginine had more significant effects on increase of sperm fertilizing ability in same concentrations. On the other hand, reduced Graphene has more toxicity than same concentrations. In concentrations of 1, 2, 3, 4, 5, 8, and 10 µg/ml of Graphene- L-Arginine and 1, 2, 3, and 4 µg/ml of reduced Graphene, significant increase of fertilizing ability for sperm was occurred. In concentrations of 12 and 15 µg/ml of Graphene- L-Arginine and 5, 8, 10, 12, and 15 µg/ml of reduced Graphene, cell death, reduction of sperm motility and viability as well as membrane lysis was significantly observed. The reason for the higher activity and effect of L-Arginine-functionalized graphene is the synergistic effect of graphene (cholesterol extraction and enhancement of fertilizing ability) and L-Arginine (enhancement of nitric oxide synthesis and prevention of membrane lipid peroxidation) on increase sperm fertility.
Conclusion: Based on this study, L-Arginine-functionalized graphene in low concentrations can be used in assisted reproductive technology for in vitro increase of sperm fertilizing ability.
Key words: Assisted reproductive technology,Sperm fertilizing ability, Graphene, -Arginine, Nanostructure.
P-44
Relationship between leptin and its polymorphism (-2548 G/A) and recurrent pregnancy loss in women with polycystic ovary syndrome
 
Vatannejad A.
Department of Comparative Biosciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.
Email: vatannejad@ut.ac.ir
 
Background: Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders result in infertility and abortion in women. Leptin has a vital role in the regulation of body weight via interacting with its specific leptin receptor. Polymorphism of their related genes may play an important role in etiology and pathogenesis of PCOS related disorders.
Objective: This study investigate the relationship of leptin gene (LEP -2548 G/A) and its plasma level with the risk of infertility and recurrent pregnancy loss (RPL) in women with PCOS.
Materials and Methods: A total of 324 PCOS patients (including 199 infertile patients and 125 patients with a history of RPL) and 150 Non-PCOS enrolled in this study. Biochemical parameters were measured and the leptin gene (LEP -2548 G/A) was genotyped using PCR-restriction fragment length polymorphism (RFLP) techniques.
Results: There was a significant difference in GG genotype of leptin polymorphism in PCOS-infertile woman as compared to Non-PCOS subjects (p = 0.043, OR = 0.47, 95% CI = 0.22-0.97). Leptin level was significantly higher in PCOS-infertile (33.27 ± 8.45 ng/ml) and PCOS-RPL (36.47 ± 7.41 ng/ml) sub-groups compared to Non-PCOS group. Leptin level elevated the risk of PCOS (1.203, 95% CI [1.009-1.435]) as well as RPL related PCOS (1.267, 95% CI [1.054-1.522]) in females.
Conclusion: Our findings showed that high leptin level was associated with PCOS related disorders. Evidence suggests that high level of leptin increase the risk of RPL in PCOS women. However, more researches with large sample size is needed to find more leptin gene polymorphism in PCOS related disorders.
Key words: Polycystic ovary syndrome, Leptin, Infertility, Recurrent pregnancy loss.
 
P-45
In vitro mouse spermatogenesis on artificial testis engineered by 3D printing of extracellular matrix
 
Bashiri Nahangi Z^{1, 2}, Amiri I^{3, 4}, Gholipourmalekabadi M^{5, 6}, Falak R⁷, Asgari H^{1, 2}, Koruji M^{1, 2}.

1. Stem Cell and Regenerative Medicine Research Center, Iran University of Medical Sciences, Tehran, Iran.
2. Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
3. Research Center for Molecular Medicine, Hamadan University of Medical Sciences, Hamadan, Iran.
4. Endometrium and Research Center, Hamadan University of Medical Sciences, Hamadan, Iran.
5. Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran.
6. Department of Tissue Engineering and Regenerative Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran.
7. Immunology Research Center (IRC), Institute of Immunology and Infectious Diseases, Iran University of Medical Sciences, Tehran, Iran.

Email: koruji.m@iums.ac.ir
 
Background: Male infertility accounts for about 50% of all infertility cases, and 25 % of infertile men are azoospermic. Due to the very small number of spermatogonia stem cells (SSCs) in testicular tissue biopsy specimens, SSCs culture for infertile patients can be important.
Objective: The proliferation of SSCs on printed scaffold derived from the extracellular matrix (ECM) of testicular tissues evaluated.
Materials and Methods: Ram testicular tissue was decellularized using hypertonic solution -Triton X-100 for 30 min. The extracted ECM (5% ratio) was used as a bio-ink for the fabrication of artificial testes along with alginate and gelatin. Testicular cells were then isolated from the testes of 3-7 days old neonate mice after enzymatic digestion. The nature of SSCs was confirmed by flow cytometry and RT-PCR for specific markers Plzf, Id4, Gfrα1, and Prm1. Finally, cell viability evaluated using MTT test and testicular cell proliferation process on printed alginate-gelatin scaffolds (group I) and ECM-alginate-gelatin scaffolds (group II) using immunocytochemistry, flow cytometry, and real-time PCR techniques was assessed.
Results: The MTT test indicated that the cell viability on the composite scaffold was significantly higher than the hybrid scaffolds and control group (p > 0.05). The results of 2 wk of proliferation on the printed system showed that the expression of Plzf, Id4, Gfrα1 gene using real-time PCR in group II was significantly higher than group I (p > 0.05). Flow cytometry analysis also showed that the number of Plzf-positive cells in group II was significantly higher than group I   (p > 0.05). Immunocytochemistry results confirmed that Plzf, Id4, and Gfrα1 markers were expressed in both groups, but their expression in group II was significantly higher than group I (p > 0.05).
Conclusion: We concluded that the culture of testicular cells on scaffolds containing ECM increases the viability, colonization, and proliferation of SSCs and achieves a high number of cells for differentiation in vitro. Therefore, 3D printing using the ECM of the testis can be an ideal strategy for the regeneration of seminiferous tubules.
Key words: Spermatogonia stem cells, Extracellular matrix, 3D printing, Proliferation.
 
P-46
In vitro spermatogenesis in artificial testis
 
Bashiri Nahangi Z^{1, 2}, Koruji M^{1, 2}, Zandieh Z^{2, 3}, Amjadi F^{2, 3}, Tanhaye Kalate Sabz F^{1, 2}, Asgari F^{1, 2}.

1. Stem Cell and Regenerative Medicine Research Center, Iran University of Medical Sciences, Tehran, Iran.
2. Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
3. Shahid Akbarabadi Clinical Research Development Unit (ShACRDU), Iran University of Medical Sciences, Tehran, Iran.

Email: koruji.m@iums.ac.ir
 
Background: As a valuable resource for cell therapy, human spermatogonial stem cells (hSSCs) have raised hopes for the treatment of male infertility. Various 3D methods have been developed to produce cellular aggregates and mimic the organization and function of the testis. The rate of progression and breakthrough in vitro spermatogenesis is lower than that of SSC transplantation, but newer methods are also being developed.
Objective: Therefore, this paper discusses the promising methods of artificial testis development, which can be used for sperm production in vitro.
Materials and Methods: The relevant articles were searched in PubMed, Google Scholar, and ScienceDirect databases.
Results: The production of an artificial reproductive organ capable of supporting SSC differentiation will certainly be a major step forward in male infertility. The mammalian extracellular matrix (ECM) increases proliferation, migration, and/or differentiation of different stem cells and can facilitate the survival of hSSC in the culture medium. The use of testicular ECM for culture of germ cells has recently been reported. Given the importance of testicular ECM, seeding stem cells onto such decellularized scaffolds to create artificial tissues and organs can be promising for the restoration, preservation, or improvement of tissue/organ function in clinical therapy. ECM hydrogels as substrates for cell culture can also be appropriate for hSSC culture. Organoids can be formed from pluripotent stem cells or under the support of a scaffold (generally matrigel) and tissue-specific growth factors and morphogens. Recently, few studies have been conducted on the development of dynamic culture systems including bioreactor and microfluidic systems in testicular tissue that the tissue is exposed to a continuous, controlled flow of fresh media. 3D bio-printing is a novel way of developing organs or functional structures that allow cells and tissues to accumulate with great accuracy. Bioprinting can support gamete differentiation in a matrix-rich 3D environment.
Conclusion: Fabrication of biofunctional testis can be one of the new options for in vitro sperm production, maintaining fertility, or transplantation without the risk of cancer cell infestation to restitute spermatogenesis in vivo.
Key words: Spermatogonial stem cell, Bioartificial testis, Differentiation.
P-47
Effect of intrauterine injection of platelet-rich plasma and the number of injections in increasing endometrial thickness and pregnancy rate in patients with thin endometrium: A clinical trial, before and after
 
Zamaniyan M^{1, 2}, Akrami F¹, Peyvani S¹, Moradi S³, Gordani N⁴.

1. Department of Obstetrics and Gynecology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.
2. Diabetes Research Center, Mazandaran University of Medical Sciences, Sari, Iran.
3. Psychiatry and Behavioral Sciences Research Center, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.
4. Mazandaran University of Medical Sciences, Sari, Iran.

Email: f.akrami1919@gmail.com
 
Background: Adequate endometrial growth is a principal factor for implantation and pregnancy. Thin endometrium is associated with lower pregnancy rate in assisted reproductive technology.
Objective: In this study we assessedthe effectiveness of intrauterine injection of platelet-rich plasma (PRP) and number of injections in increasing endometrial thickness (ET) and pregnancy rate in patients with thin endometrium.
Materials and Methods: In this clinical trial, 26 women that referred to infertility center of Imam Khomeini Hospital, Sari, Iran, from September 2019 to January 2020 participated. They had history of frozen-thawed embryo transfer cycle failure due to thin endometrium. ET was determined on the tenth day of cycle, and if ET was (< 7 mm), intrauterine injection of PRP was done on day 11-12 and it was repeated on day 13-14 until ET reached an optimal pattern, then embryo transfer was performed (p < 0.05 were considered as statistically significant).
Results: Mean age of women was 34.96 ± 3.86 yr. Mean of ET pre-PRP was 5.64 ± 0.79 mm which significantly (n = 26), increased to 7.20 ± 1.27 mm post-first PRP (n = 26) and increased to 7.65 ± 1.29 mm post-second PRP (n = 13) (p < 0.001). Chemical and clinical pregnancy rate was 23.1% and 19.2% respectively. One patient had ectopic pregnancy and five patient had normal ongoing intrauterine pregnancy. Of these 5 people, one had twin pregnancy that miscarriage at 19 wk of gestational age, and the other 4 had live birth at term.
Conclusion: Our findings showed that the PRP injection can be used as a method to increase ET and improvement the results of in vitro fertilization. To achieve the optimal results, PRP as a safe and low risk method can be performed twice, between 24 till 72 hr in in vitro fertilization cycles.
Key words: Platelet rich plasma, Endometrium, Fertilization in vitro, Pregnancy rate.
P-48
Effect of fennel supplementation along with high-protein, low-carbohydrate weight-loss diet on insulin resistance and percentage of fat and muscle mass in overweight/obese women with polycystic ovary syndrome
 
Hosseini Marnani E^{1, 2}, Ghadiri-Anari A³, Ramezani-Jolfaie N^{1, 2}, Mohammadi M^{1, 2}, Abdollahi N^{1, 2}, Namayandeh SM⁴, Mozaffari-Khosravi H², Salehi-Abargouei A^{1, 2}, Nadjarzadeh A^{1, 2}.

1. Nutrition and Food Security Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Department of Nutrition, School of Public Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
3. Department of Internal Medicine, Diabetes Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
4. Research Center of Prevention and Epidemiology of Non-Communicable Diseases, Shahid Sadoghi University of Medical Sciences, Yazd, Iran.

Email: elham_hosseini1993@yahoo.com
 
Background: Polycystic ovary syndrome (PCOS) is a common reproductive disorder with prevalence of 5-10% in premenopausal women, which is identified with hyperandrogenism and ovarian dysfunction.
Objective: The aim of this study was to investigate the effects of fennel supplementation with energy-restricted diets on body fat and muscle percentage and insulin resistance in women with PCOS.
Materials and Methods: Sixty-four overweight/obese women with PCOS were randomly allocated to 4 groups for 12 wk as follows: (1) standard diet + fennel (SDF), (2) high-protein, low-carbohydrate diet supplemented with fennel (HPF), (3) standard diet + placebo (SDP), and (4) high-protein, low-carbohydrate diet + placebo (HPP).
Results: After 12 wk of intervention, there were significant changes in the percentage of body fat and muscle in all groups. Decreasing in fasting insulin was −4.12 micIU/ml (p = 0.01) for HPF and −4.5 micIU/ml (p = 0.03) for SDP groups. In addition, HOMA-IR significantly decreased in HPF (p = 0.02) and SDP (p = 0.02) groups.
Conclusion: Energy-restricted diets independent of dietary composition improved the body fat and muscle percentage and insulin resistance indices in women with PCOS. High-protein diet and fennel compared with standard diet and placebo had no significant effect on insulin resistance, body fat and muscle.
Key words: High-protein diet, Standard diet, Fennel, Body fat percentage, Insulin resistance index.

The original full text of this abstract has been published in Journal of Functional Foods 2020; 67: 103848. https://doi.org/10.1016/j.jff.2020.103848.
How to cite to this article: Hosseini Marnani H, Ghadiri-Anari A, Ramezani-Jolfaie N, Mohammadi M, Abdollahi N, Namayandeh SM, Mozaffari-Khosravi H, Salehi-Abargouei A, Nadjarzadeh A. Effect of fennel supplementation along with high-protein, low-carbohydrate weight-loss diet on insulin resistance and percentage of fat and muscle mass in overweight/obese women with polycystic ovary syndrome. Journal of Functional Foods 2020; 67: 103848.

P-49
Which sperm preparation technique separate the best quality sperm?
 
Vahidi N, Amjadi F, Tanhaye Kalate Sabz F, Zandie Z.
Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
Email: n.vahidi72@yahoo.com
 
Background: of all infertility cases, approximately 40-50% is due to male factor in fertility. One of the known causes of male infertility is associated with low sperm parameters and high level DNA fragmentation. Sperm preparation techniques in intracytoplasmic sperm injection procedures is used in order to obtain the best-quality sperm.
Objective: The present study was designed to compare Microfluidic, and Swim-up methods for sperm preparation and the effect of these methods on semen parameters and sperm DNA Integrity in Infertile men.
Materials and Methods: In this study, semen samples were collected from 25 infertile men. Each sample was divided into 2 groups, one part for preparing by Microfluidic method and the other one was prepared by swim up method. Then sperm count, viability, motility and morphology were assessed according to World Health Organization 2010. DNA damage were assessed by Sperm DNA Fragmentation assay.
Results: Sperm parameters including viability, motility, and morphology in the Microfluidic method were significantly improved and sperm DNA damage were significantly lower than the swim up method (p < 0.05).
Conclusion: Our results showed that Microfluidic method improved the sperm parameters and decreased sperm DNA damage, and it can be an effective way to improve sperm quality of infertile male compared to conventional preparation methods.
Key words: Microfluidic, Swim up, DNA fragmentation.
 
P-50
Prevalence of smoking in infertile men referred to the infertility ward of Ali ebn-e Abitaleb Hospital of Zahedan from 2017 to 2019
 
Javan S¹, Dehghan Haghighi J², Mirgaloyebayat Sh³, Sheybani Moghaddam F⁴, Farzaneh F^{5, 6}.

1. Faculty of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran.
2. Department of Community Medicine, Faculty of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran.
3. Department of Obstetrics and Gynecology, Faculty of Medicine, Endometriosis Research Center, Iran University of Medical Sciences, Tehran, Iran.
4. Department of Urology, Faculty of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran.
5. Department of Obstetrics and Gynecology, Faculty of Medicine, Infectious Disease and Tropical Medicine Research Center, Zahedan University of Medical Sciences, Zahedan, Iran.
6. Department of Obstetrics and Gynecology Endometriosis Research Center, Iran University of Medical Sciences, Tehran, Iran.

Email: Farahnaz1826@yahoo.com
Background: Infertility and its individual and social problems are one of the most important issues for couples. A significant problem with male infertility is that infertility is only detectable in 40% of cases and is not pathologically detectable in 60% of cases.
Objective: The aim of this study was to evaluate the frequency of smoking in male infertility referred to the infertility ward of Ali ebn-e Abitaleb Hospital of Zahedan from 2017 to 2019.
Materials and Methods: The present study was a cross-sectional study and included 200 infertile men with male factor referred to the infertility clinic of Ali ebn-e Abitaleb Hospital of Zahedan. The sampling method was easy or available and a questionnaire was used to collect information and SPSS software was used to analyze the data.
Results: The results showed that infertile patients with male factor that are non-smoking had the highest number with 174 (87%). Also, there was no significant relationship between smoking and sperm concentration in spermogram in infertile couples with male factor (p = 0.293). There was no significant relationship between smoking and sperm morphology in spermogram in infertile patients with male factor (p = 0.130). There was no significant relationship between smoking and sperm motility in spermogram in infertile patients with male factor (p < 0.05).
Conclusion: Although smoking as a risk factor can cause infertility, but in the present study, (the cross-sectional study), we were not able to show the cause-and-effect relationship between smoking and infertility.
Key words: Smoking, Male infertility, Semen analysis.
 
P-51
Evaluation of miRNAs involved in patients with endometriosis as diagnostic and therapeutic biomarkers using bioinformatics analysis
 
Bereimipour A^{1, 2}, Arabi M³.

1. Faculty of Sciences and Advanced Technologies in Biology, University of Science and Culture, Tehran, Iran.
2. Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
3. Young Researchers and Elite Club, Department of Genetic, Islamic Azad University, Tehran Medical Science, Islamic Azad University, Tehran, Iran.

Email: ahmadlta@yahoo.com
 
Background: Endometriosis is one of the most common reproductive diseases in women. About 10% of women are pregnant, and about 50% of women between the ages of 30 and 50 and between 30 and 50% of women experience pelvic pain or infertility due to endometriosis. There are several treatments available for people with endometriosis, but facilitating and optimizing early detection of the disease can help manage its treatment. miRNAs are important regulatory molecules at the cellular level that control the expression of many genes due to their inhibitory effect. miRNAs can be found in human secretions. Therefore, these regulatory molecules can be used as diagnostic biomarkers for endometriosis.
Objective: This study examined the ectopic and eutopic tissue data of patients with endometriosis and isolated the miRNAs in the extracellular matrix.
Materials and Methods: In this study, using bioinformatics analysis, we first isolated the appropriate data through the GEO database. We then uploaded the hypex gene to examine the signal path in the Enrichr database and the KEGG library. We loaded the genes involved in important pathways into the STRING database and measured their protein network. We then used the Targetscan database to obtain miRNAs.
Results: 700 low-expression genes were selected with LogFC < 2. These genes played a more prominent role in complement and coagulation cascades, cell adhesion molecules, TGF-beta, and Hedgehog signaling pathways. Of these pathways, 34 genes were involved in this pathways and extracellular matrix. After the examination, the hsa-miR-4800-3p, hsa-miR-4473, hsa-miR-614, hsa-miR-4671-3p, and hsa-miR-3659 miRNAs were identified more clearly.
Conclusion: Finally, this study showed that miRNAs, between ectopic and eutopic tissues, were significantly identified and could be found in the serum or plasma samples of patients with endometriosis.
Key words: Endometriosis, Biomarkers, Bioinformatics analysis, Micro RNAs.
 
P-52
“Protester Caregivers” semantic reconstruction of infertility outcomes in marital relationship of infertile women
 
Abooei A¹, Afshani S².

1. Department of Psychology, Science and Arts University, Yazd, Iran.
2. Department of Cooperation and Social Welfare, Social Sciences Faculty, Yazd University, Yazd, Iran.

Email: afshanialireza@yazd.ac.ir
 
Background: Spouses, who experience infertility, are much more overwhelmed by failure, anxiety, stress, and unpleasant marital relationships than other spouses. Infertility often has negative effects on the relationships between spouses. Infertile women are more likely to suffer from these unpleasant marital relationships.
Objective: By using a qualitative grounded theory approach, 21 women were selected and studied using theoretical sampling. Theoretical sampling continues until data saturation occurs. These qualitative interviews were conducted between January 2019 and January 2020. Data were collected and analyzed using open and axial coding.
Materials and Methods: The findings of this study included 7 main categories and one core category called Protester Caregivers. Consequently, conceptual tables, paradigm model and theoretical schema were presented. Determining the results in general indicates the phenomenon of “Protester Caregivers" in the target community.
Results: The findings of this study included 7 main categories and one core category called Protester Caregivers. Consequently, conceptual tables, paradigm model and theoretical schema were presented. Determining the results in general indicates the phenomenon of “Protester Caregivers" in the target community.
Conclusion: Based on the traditional culture of the target community, infertile women struggle with the men who play the role of "caregivers" as well as consistently protest their infertility. This paradoxical phenomenon leads to the emergence of contradictory strategies and outcomes, and make infertile women to rethink about their choices.
Key words: Infertile women, Marital relationship, Semantic reconstruction, Protester caregivers.
 
P-53
Comparison of GnRH-agonist+ vaginal progesterone and vaginal progesterone effects on luteal phase support in frozen–thawed embryo transfer cycles: An RCT
 
Askary E, Zareii A.
Shiraz University of Medical Sciences, Obstetrics and Gynecology Ward, Infertility Research Center, Shiraz, Iran.
Email: elliaskary_md@yahoo.com
 
Background: As it seems that the progesterone alone isn’t enough treatment for luteal phase support (LPS) specially in frozen embryo transfer (FET) cycles, so gonadotropin releasing hormone agonist (GnRH-a) was suggested as a adjuvant therapy with combination to progesterone for LPS.
Objective: This study aimed to evaluate the effects of the administration of a multiple doses of GnRH-a to routine LPS in FET cycles.
Materials and Methods: In this clinical trial study, 240 infertile women who were candidate for in vitro fertilization cycle were enrolled and divided into two groups (n = 120/each). Group 1 received 800 mg vaginal progesterone daily and group 2 received 0.1 mg dipherline in days 0, 3, and 6 of FET for LPS. Implantation rate, clinical pregnancy rate, ongoing pregnancy rate, and spontaneous abortion were checked and measured.
Results: Results showed that there was no significant difference between the mean age of women and also duration of infertility (p = 0.70, p = 0.60). There was no significant in term of implantation rate and rate of spontaneous abortion (p = 0.19, p = 0.31) respectively. In term of clinical pregnancy rate, significant difference were seen between groups (n = 37, 30.8% in group 1 and n = 57, 47.5% in group 2, p = 0.008). As a term of ongoing pregnancy rate (till 3 months after FET), significant difference between two groups were seen (p = 0.05).
Conclusion: The GnRH-a+cyclogest as opposed to cyclogest for LPS after FET cycles may be the superior choice.
Key words: FET, ART, LPS, Cyclogest.
P-54
Cytokines as biomarkers for embryo selection
 
Bahrami-Asl Z¹, Hajipour H², Rastgar Rezaei Y³, Novinbahador T⁴, Latifi Z⁵, Nejabati HR⁵, Farzadi L⁶, Fattahi A^{2, 6}, Nouri M², Dominguez F⁷.

1. Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
2. Department of Reproductive Biology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
3. Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
4. Department of Biology, Faculty of Natural Science, University of Tabriz, Tabriz, Iran.
5. Department of Biochemistry and Clinical Laboratories, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
6. Womenʼs Reproductive Health Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
7. Fundacion Instituto Valenciano de Infertilidad (FIVI), Instituto Universitario IVI (IUIVI), ISS LaFe, Valencia, Spain.

Email: yeganerastgar@gmail.com
 
Background: Studies have shown that the morphological assessments to select the best embryo for transfer could not provide satisfactory outcomes. Therefore, many studies have been conducted to find predictive biomarkers that can distinguish embryos with high implantation potential.
Objective: In the current study, we comprehensively reviewed the possibility of using embryo-secreted cytokines as potential biomarkers for embryo selection in assisted reproductive technology.
Materials and Methods: The present review involved published research articles that have investigated cytokines in the embryo secretome. A search in Google Scholar and PubMed was performed with no limitation on the date of publication using a combination of the following search terms: “secretome”, “culture media”, and “cytokine (s)”.
Results: It can be postulated that the embryo secretome can well reflect the embryo condition. Since the immune system has an indubitable role in implantation and also the immunological factors are involved in the embryo-endometrial crosstalk, the embryo-secreted cytokines can be used as potential biomarkers.
Conclusion: In conclusion, the following three points should take into consideration while using embryo-secreted factors as biomarkers: 1) The culture media should be evaluated at a certain stage of embryo development (e.g. cleavage and blastocyst), 2) The measurement method should be able to detect very small levels of factors, and 3) Changing in the concentration of several embryo-secreted factors in combination should be evaluated to propose an appropriate embryo selection method.
Key words: Embryo, Cytokines, Implantation, Secretome, Culture media.

The original full text of this abstract has been published in American Journal of Reproductive Immunology 2020. https://doi.org/10.1111/aji.13385.
How to cite to this article: Bahrami‐Asl Z, Hajipour H, Rastgar Rezaei Y, Novinbahador T, Latifi Z, Nejabati HR, Farzadi L, Fattahi A, Nouri M, Dominguez F. Cytokines in embryonic secretome as potential markers for embryo selection. American Journal of Reproductive Immunology 2020: e13385.

 
P-55
Effect of dietary polyunsaturated fatty acids on the status of uterine prostaglandins during the window of pre-implantation
 
Rastgar Rezaei Y¹, Latifi Z², Nikanfar S², Nouri M³, Fattahi A⁴.

1. Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
2. Department of Biochemistry and Clinical Laboratories, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
3. Department of Reproductive Biology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
4. Womenʼs Reproductive Health Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

Email: yeganerastgar@gmail.com
 
Background: The role of prostaglandins (PGs) in embryo implantation can be influenced by polyunsaturated fatty acids supplementation.
Objective: Therefore, the present study was conducted to investigate the effects of dietary omega-3 and -6 fatty acids on uterine PGs and their relevant receptors during the pre-implantation period in mice.
Materials and Methods: Twenty female mice were randomly divided into three groups and fed a standard pellet (control group), standard pellet +10% (w/w) fish oil, and +10% (w/w) soybean oil. The uterine levels of PGI2, PGD2, and PGF2α, the mRNA expression of PG I, D, and F synthesis enzymes (PGIS, PGDS, and PGFS, respectively), and protein expression of their receptors (PI, PD, and PF, respectively) were evaluated in uterine tissues of all treated groups at days 1-5 of pregnancy.
Results: Our results showed that the uterine levels of PGI2, PGD2, and PGF2α and expression of their synthesis enzymes were markedly high on the 5^th day of pregnancy, while protein expression showed significant elevation only for PF and PI during this day (p < 0.05). Omega-6 significantly raised uterine levels of all three PGs on the fifth day of pregnancy compared to mice received omega-3 (p < 0.05). Furthermore, the omega-6 group showed higher expression of PGDS and PGFS than the omega-3-supplemented group on days 5 and 4 of pregnancy, respectively. In addition, we found positive correlations between the implantation rate and expression levels of PGIS, PGFS, IP, and FP and the PGI2 uterine levels.
Conclusion: Our study showed the positive effect of omega-6 PUFA supplementation on PGFS and PGDS expression together with the uterine levels of PGI2, PGD2, and PGF2α which makes PGs status as a possible indicator of successful implantation.
Key words: Embryo implantation, Pre-implantation window, Polyunsaturated fatty acids, Omega-3, Prostaglandins.
 
P-56
Effects of bacteria on male fertility: Spermatogenesis and sperm function
 
Oghbaei H¹, Rastgar Rezaei Y², Nikanfar S³, Zarezadeh R³, Sadegi M⁴, Latifi Z³, Nouri M⁵, Fattahi A^{6, 7}, Ahmadi Y⁸, Bleisinger N⁷.

1. Department of Physiology, Tabriz University of Medical Sciences, Tabriz, Iran.
2. Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
3. Department of Biochemistry and Clinical Laboratories, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
4. Department of Molecular Medicine, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
5. Department of Reproductive Biology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
6. Womenʼs Reproductive Health Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
7. Department of Obstetrics and Gynecology, Erlangen University Hospital, Friedrich-Alexander University of Erlangen-Nürnberg, Erlangen, Germany.
8. Department of Urology, Sina Hospital, Tabriz University of Medical Sciences, Tabriz, Iran.

Email: amirfattahi@gmail.com
 
Background: Interestingly, bacteria can induce different damages on sperm cells such as DNA fragmentation, cell membrane peroxidation, and acrosome impairment. Such negative effects can be mediated by bacteria-secreted toxins and metabolites or by direct attachment of bacteria on the sperm cells and subsequent activation of signaling pathways related to oxidative stress, apoptosis, and inflammation.
Objective: In this study, we reviewed the impact of male urogenital bacteria on spermatogenesis and sperm functions as well as the underlying mechanisms by which the bacteria can damage sperm.
Materials and Methods: The present review involved all published research articles that have investigated the effect of bacteria on spermatogenesis and sperm function. Combinations of the following terms were searched in Google Scholar, PubMed, and Science Direct databases with no limitation on the date of publication: “sperm”, “spermatozoa” “spermatogenesis“, “bacteria”, and “bacterium”.
Results: Interestingly, bacterial contamination of semen, which ordinarily originates from the urinary tract or by the partner through sexual intercourse, can induce DNA fragmentation, cell membrane peroxidation, acrosome impairment, vacuolization, and mitochondrial damage in sperm cells. Bacteria can exert these effects by toxins or by direct attachment to the sperm cells and subsequent activation of signaling pathways related to oxidative stress, apoptosis, and inflammation. These bacteria-induced changes in the sperm can impair semen parameters including concentration, motility, morphology, viability, and fertilization capacity, and subsequently cause infertility.
Conclusion: Given the significant destructive effect of some bacteria on sperm cells, the type of bacterial contamination in the patient’s genital tract should be diagnosed and its potential negative effects on male fertility, and the underlying mechanisms should be taken into account in the treatment of bacteria-induced subfertile men. Furthermore, future studies are recommended to investigate possible therapeutic strategies to inhibit bacteria-induced sperm damage based on the type of bacterium and its potential damages on sperm cells.
Key words: Bacteria, Sperm, Fertility, DNA fragmentation.

The original full text of this abstract has been published in Life Sciences 2020; 256: 117891. https://doi.org/10.1016/j.lfs.2020.117891.
How to cite to this article: Oghbaei H, Rastgar Rezaei Y, Nikanfar S, Zarezadeh R, Sadegi M, Latifi Z, Nouri M, Fattahi A, Ahmadi Y, Bleisinger N. Effects of bacteria on male fertility: Spermatogenesis and sperm function. Life Sciences 2020; 256: 117891.

 
P-57
Effect of one-way blunt testis on sperm parameters in acute and chronic periods after injury in mice
 
Pourentezari M², Dehghan M¹, Ashoorzadeh S³, Talebi AR^{1, 4}.

1. Department of Biology and Anatomical Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Anatomy Department, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
3. Afzalipour Clinical Center for Infertility, Kerman University of Medical Sciences, Kerman, Iran.
4. Department of Reproductive Biology, Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd. Iran.

Email: m.pourentezari@gmail.com
 
Background: So far, the effects of blunt trauma on sperm parameters and reproductive capacity have not been firmly established and diverse reports have been presented.
Objective: The aim of this study was to investigate the effect of unilateral blunt testis on sperm parameters in acute and chronic periods after injury in mice.
Materials and Methods: In this study, 40 adult male NMRI mice with a weight of 35-30 gram were selected randomly and divided into 3 groups: control, sham (Mice in this group were only surgically treated) and experimental (Mice were surgically treated in this group and the blunt was hit by their left testes). Sampling was performed in two acute (48 hours after surgery) and chronic (1 month and 2 months after surgery), after anesthesia the tail of epididymis was separated and placed in Ham's F10 solution. Then sperm samples were examined microscopically in terms of motility, number (with 40x lens), viability (eosin stain color and hypoosmotic swelling).
Results: In the case of rapid sperm motility, the control group with acute experimental, one and two month chronic, acute sham group with sham one month, acute and chronic experimental one and two months, and sham group two months with acute experimental groups and sham one and two months were significant. Regarding the sperm viability, the control group with one month acute and chronic sham, the acute sham group with one month sham groups, an acute and chronic one month experimental, and a one month chronic group with acute and chronic two month experimental groups were significant. Regarding sperm count, a one month chronic group with control groups, acute sham, one to two months sham and an acute experimental with one month chronic group was significant.
Conclusion: The testicular blunt has affected sperm parameters. In other words, the mean number of sperm (ml /ml) and the percentage of sperm survival and motility (progressive, non-progressive, and inactivity) were significant between the control and sham groups and the experimental group, which was not effective on fertility.
Key words: Blunt, Testis, Sperm parameters, Mice.

The original full text of this abstract has been published in J Shahid Sadoughi Uni Med Sci 2020; 27(10): 1968-1980. https://doi.org/ 10.18502/ssu.v27i10.2404.
How to cite to this article: Pourentezari M, Dehghan M, Ashoorzadeh S, Talebi AR. Effect of one-way blunt testis on sperm parameters in acute and chronic periods after injury in mice. J Shahid Sadoughi Uni Med Sci 2020; 27(10): 1968-1980.


 
P-58
The effects of morphine abuse on sperm parameters, chromatin integrity and apoptosis in men consuming morphine
 
Ghasemi-Esmailabad S^{1, 2}, Talebi AH³, Talebi AR^{1, 4, 5}, Amiri S¹, Moshrefi M^{1, 6}, Pourentezari M⁵.

1. Research and Clinical Center for Infertility, Yazd Reproductive Science Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2. Abortion Research Center, Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
3. Department of English Language and Literature, Faculty of Humanities and Social Sciences, Yazd University, Yazd, Iran.
4. Andrology Research Center, Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
5. Department of Biology and Anatomical Scineces, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
6. Medical Nanotechnology and Tissue Engineering Research Center, Yazd Reproductive Science Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: m.pourentezari@gmail.com
 
Background: Morphine is one of the major psychoactive chemicals in opium that can increase the production of free radicals and thus can negatively affect spermatogenesis.
Objective: The purpose of this survey was to demonstrate the effect of morphine consumption on sperm parameters, DNA integrity and apoptosis in men taking morphine.
Materials and Methods: In this case-control study, 30 man abusing morphine (cases) and 30 healthy men (controls) were compared for sperm parameters (count, motility and morphology) and sperm chromatin quality, with aniline blue, toluidine blue and Chromomycin A3 stainings. The participants were matched for age, weight, amount and duration of cigarette smoking.
Results: In men with morphine dependency, sperm progressive and total motility (p = 0.038 and p < 0.001 respectively) showed significant decreasing compared to control group. Regard to morphine abusing, although morphine can decrease the sperm chromatin condensation and increases the rate of sperm apoptosis, but these differences were not statistically significant.
Conclusion: According to our result morphine dependence can reduce male fertility by affecting sperm parameters and also it may affect sperm chromatin/DNA integrity.
Key words: Morphine, Sperm parameters, Chromatin, Human.
 
P-59
Comparison of the Betatrophin level and its association with metabolic and inflammatory parameters in PCOS and non-PCOS infertile women condidated for IUI
 
Keikha F¹, Shahrokh Tehraninejad E¹, Rakhshkhorshid M², Afiat M³, Hagholahi F¹, Ghasemi F¹.

1. Health Reproductive Research Center, Imam Khomeini Hospital, Tehran University of Medical Sciences, Tehran, Iran.
2. Department of Midwifery, Mashhad University of Medical Sciences, Mashhad, Iran.
3. Department of Obstetrics and Gynecology, Milad Infertility Center, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.

Email: malihehafiat0@gmail.com
 
Background: Betatrophin may be associated with metabolic diseases.
Objective: To investigate the betatrophin level and its association with metabolic and inflammatory parameters in infertile women with polycystic ovary syndrome (PCOS) and other infertile women during the intrauterine insemination cycle.
Materials and Methods: This case control study was conducted on 90 infertile women (45 PCOS and 45 non-PCOS) chosen by convenience sampling method, in Tehran. Participants were interviewed to obtain age, body mass index, reproductive history. Fasting brachial venous blood samples were obtained on the 3^rd day of the menstrual cycle in order to measure the betatrophin, fasting blood sugar, insulin, luteinizing hormone, follicle-stimulating hormone, low-density lipoprotein cholesterol, estradiol, and high-sensitivity C-reactive protein. To analyze the data, SPSS 25 software and statistical tests such as independent Student's t test, chi-square test, and multiple linear regression were used.
Results: The results showed that the level of betatrophin in women with PCOS was significantly higher than in the control group (p = 0.05). Based on multiple linear regression analyses, the effects of metabolic and inflammatory parameters on betatrophin were not significant (p > 0.05). The results showed no significant difference between groups in folliculogenesis (p = 0.57).
Conclusion: According to the results, betatrophin levels were higher in PCOS infertile women than in infertile non-PCOS women. Indirectly, one might argue that there is a potential association between an increase in betatrophin and a possible increase in the incidence of PCOS syndrome. Further studies with a larger sample size are needed to investigate the role of betatrophin in insulin resistance, lipid metabolism, and its effects on infertility treatment outcomes.
Key words: Betatrophin protein, Human, Infertility, Polycystic ovarian syndrome, Iran.
 
P-60
Investigation and comparison of the effect of TGF-β3, kartogenin and Avocado/Soybean unsaponifiables on the in-vitro and in-vivo chondrogenesis of human adipose derived stem cells on fibrin scaffold
 
Hashemibeni B¹, Izadi MA¹, Valiani A¹, Esfandiari I¹, Bahramian H¹, Dortaj H², Pourentezari M³.

1. Department of Anatomical Sciences and Molecular Biology, Isfahan University of Medical Sciences, Isfahan, Iran.
2. Department of Tissue Engineering and Applied Cell Science, Shiraz University of Applied Medical Science and Technologies, Shiraz, Iran.
3. Department of Biology and Anatomical Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: m.pourentezari@gmail.com
 
Background: There are lack of suitable therapeutic approaches to cartilage defect.
Objective: This study was designed to determine the effect of TGF-β3, avocado/soybean (ASU) and Kartogenin (KGN) on chondrogenic differentiation in human adipose-derived stem cells (hADSCs) on fibrin scaffold.
Materials and Methods: hADSCs seeded in fibrin scaffold and cultured in chondrogenic media. These cells divided in to 4 groups (control, TGF-β3, ASU and KGN). Cell viability was estimated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, or MTT assay, differentiated cells evaluated by histological and immunohistochemical techniques. Expression genes [sex determining region Y-box 9 (SOX9), Aggrecan (AGG), type II collagen (Coll II) and type X collagen (Coll X)] assessed by real-time PCR. For study on animal model, differentiated cells in fibrin scaffolds were subcutaneously transplanted in rats. Histological and immunohistochemistry was done in animal model.
Results: The results of the real-time PCR indicated that SOX9, AGG and Col II genes expression in TGF-β3, KGN and ASU groups were significantly higher (p < 0.01) compared to the control group, Col X gene expression only in TGF-β3 group was significantly higher (p < 0.01) compared to the control group. The glycosaminoglycan (GAG) deposition was higher in TGF-β3, KGN and ASU groups compared to the control group. The immunohistological analysis showed the distribution of collagen type X in the extracellular matrix in fibrin scaffold TGF-β3 group was significantly higher in control, KGN and ASU groups, (p < 0.001).
Conclusion: ASU, and in particular KGN was suitable for successful chondrogenic differentiation of hADSCs and a suppressor of the consequent hypertrophy.
Key words: TGFβ3, Avocado/Soybean, Kartogenin, Human adipose-derived stem cells.
 
P-61
An overview of application of stem cells as a resource for male infertility treatment
 
Fereidooni H.
Department of Biology, Developmental Biology, Fars (Tehran) Research and Science University, Shiraz, Iran.
Email: fereidooni_hamed@yahoo.com
 
Background: Male infertility due to decreased semen quality is a growing global problem.
Objective: Commonly used strategies for treating infertility include medication, intrauterine insemination, and in vitro fertilization. In recent years, mesenchymal stem cells have created new opportunities to treat a variety of disorders, including infertility and new expectations for managing reproductive disabilities. Stem cells are undifferentiated cells that are able to regenerate and proliferate and are also able to produce specialized cells under appropriate conditions. They are present in all stages of the fetus, embryo and adult and can multiply in different cells.
Materials and Methods: We searched the articles published in English from 1985-2020 using the keywords nanoparticles, male infertility, imaging, and toxicity in Scopus and PubMed databases. While many questions remain about stem cells, stem cells have undoubtedly opened up new avenues for infertility treatment.
Results: In summary, most studies have shown that mesenchymal stem cells can be used as a viable option for the treatment of azoospermia in men. However, there is a need for further evaluation of the effectiveness of these cells in treating infertility.
Conclusion: In this review, we discuss and summarize different stem cell approaches to the treatment of male infertility to provide updates on stem cell therapy research.
Key words: Stem cells, Male infertility, Treatment, Transplantation.
P-62
Investigation the effect of avocado soybean unsaponifiables and icariin on the chondrogenesis of human adipose derived stem cells on poly (Lactic-Co-Glycolic) acid/fibrin hybrid scaffold
 
Hashemibeni B¹, Pourentezari M², Mardani M¹, Dourtaj H³, Valiani A¹, Yadegari M², Rajabi A², Izadi S², Zakizadeh F², Ghaderi N², Shahedi A².

1. Department of Anatomical Sciences and Molecular Biology, Isfahan University of Medical Sciences, Isfahan, Iran.
2. Department of Biology and Anatomical Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
3. Department of Tissue Engineering and Applied Cell Science, Shiraz University of Applied Medical Science and Technologies, Shiraz, Iran.

Email: abbasshahedi1355@gmail.com
 
Background: Avocado soybean unsaponifiables (ASU) and icariin (ICA) components are described to have a chondroprotective.
Objective: The aim of this study was to investigate the effect of ASU and ICA on the chondrogenesis of human adipose derived stem cells (hADSCs) on poly (lactic-co-glycolic) acid (PLGA)/fibrin hybrid scaffold.
Materials and Methods: hADSCs seeded in PLGA/fibrin scaffold and cultured in chondrogenic media. These cells divided into 5 groups (control, TGFβ-3, ASU, ICA and ASU/ICA). After 14 days, the viability of cells in all groups were calculated by MTT. The gene expression of chondrogenic was quantified by real time PCR. Protein expression levels were evaluated by Western blotting.
Results: The cell viability in ASU/ICA group significantly increased in comparison with the TGF-β3 group. Genes expression levels of type II collagen (Col II) and SOX9 significantly increased in all groups in comparison with the control group. Aggrecan (AGG) gene significantly increased in TGF-β3, ASU and ASU/ICA groups in comparison with the control group. Type X collagen (Col X) gene significantly increased in TGF-β in comparison with the all groups. Genes expression levels of type X collagen (Col I) significantly increased in TGF-β3 group in comparison with the ASU/ICA group. Protein levels of Col II significantly increased in all groups in comparison with the control group. Protein levels Col X significantly decreased in the groups of ASU, ICA and ASU/ICA in comparison with TGF-β3.
Conclusion: Using ASU, ICA and particularly synergist form can induce chondrogenesis in hADSCs in PLGA/Fibrin composite scaffold.
Key words: Human adipose-derived stem cells, Avocado/Soybean, Icariin.
 
P-63
Association of soluble leptin receptor level and its polymorphism (rs1137101) with infertility and abortion in Iranian women with polycystic ovary syndrome
 
Kheirollahi A.
Department of Comparative Biosciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.
Email: kheirolahi_asma@ut.ac.ir
Background: Leptin is an adipocyte-derived adipokine that plays a crucial role in metabolic and reproductive functions via interacting with its specific leptin receptor (LEPR). A form of LEPR binds to leptin in the circulation and modulates its level in plasma. It has been indicated that the level LEPR and also rs1137101 polymorphisms of the LEPR gene are associated with metabolic disorders.
Objective: This study was to investigate the levels of the soluble LEPR, and also the frequency of rs1137101 polymorphism in subjects with polycystic ovary syndrome (PCOS) and those without PCOS.
Materials and Methods: A total of 324 PCOS patients (including 199 infertile patients and 125 patients with a history of recurrent pregnancy loss) and 150 non-PCOS were included in this study. Biochemical parameters and plasma level of soluble LEPR were measured and the genotype of rs1137101 polymorphism was determined using PCR-restriction fragment length polymorphism techniques.
Results: There was a significantly lower level of LEPR in PCOS (58.13 ± 24.3 ng/ml), PCOS-infertile (58.74 ± 24.04 ng/ml), and PCOS-abortion (57.62 ± 24.67 ng/ml) compared to the non-PCOS group (72.95 ± 22.95 ng/ml). Our data also shown that there was significant differences in allelic (G) and genotypic (GG) frequencies for the LEPR rs1137101 polymorphism in PCOS women when compared with the non-PCOS subjects (p = 0.033, OR = 0.67, 95% CI = 0.46-0.96 and p = 0.02, OR = 0.39, 95% CI = 0.18-0.86, respectively). The analysis of LEPR rs1137101 polymorphism gene revealed significant differences in GG genotype and G allele in PCOS-infertile women as compared to non-PCOS subjects.
Conclusion: According to the results, the levels of soluble LEPR were associated with PCOS, and rs1137101 polymorphism was correlated to PCOS-related infertility. Thus, this polymorphism may be considered as a prognostic biomarker of infertility in PCOS women.
Key words: Polycystic ovary syndrome, Leptin receptor, Polymorphism, Infertility.
 
P-64
Polymorphism of hormone-sensitive lipase C-60G in obese and non-obese polycystic ovary syndrome patients
 
Kheirollahi A.
Department of Comparative Biosciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.
Email: kheirolahi_asma@ut.ac.ir
 
Background: Polycystic ovary syndrome (PCOS) is a common endocrine-metabolic disorder in women of reproductive age. Since most PCOS patients are obese, abnormal lipid metabolism has an essential role in the pathological development of PCOS. Hormone-sensitive lipase (HSL) is an intracellular lipase that has a crucial role in normal lipid metabolism.
Objective: This study aimed to assess the frequency of C-60G polymorphism of HSL in healthy women and PCOS women.
Materials and Methods: 324 women with PCOS and 144 healthy controls were enrolled in this study. All subjects were further divided into Non-PCOS and body mass index (BMI) ≥ 25 (n = 72), PCOS and BMI ≥ 25 (n = 197), Non-PCOS and BMI < 25 (n = 67) and PCOS and BMI < 25 (n = 117) subgroups. Biochemical parameters were measured and the genotypes of C-60G polymorphism of the HSL gene were determined using PCR-restriction fragment length polymorphism techniques.
Results: Age, BMI, Insulin, HOMA-IR, FT, and follicle-stimulating hormone levels were significantly different between subgroups. Our results have shown that there was no significant difference between fasting blood sugar, triglyceride, serum total cholesterol, luteinizing hormone low-density lipoprotein, and high-density lipoproteins levels in cases and controls. The genotypic and allelic frequencies of HSL showed no significant differences between PCOS – with BMI ≥ 25 and/or BMI < 25- and non-PCOS subjects.
Conclusion: According to the obtained results, the C-60G polymorphism in the HSL promoter was not associated with PCOS and BMI.
Key words: Polycystic ovary syndrome, Hormone-sensitive lipase, Polymorphism.
 
P-65
The protective effects of omega3 on ubiquitination and protamination of rat sperm after bleomycin, etoposide, and cisplatin treatment
 
Hashemi F¹, Razavi Sh¹, Khadivi F².

1. Department of Anatomy, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
2. Department of Anatomy, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran.

Email: razavi@med.mui.ac.ir
 
Background: Generation of oxidative stress during chemotherapy leads to imbalance between oxidant and antioxidant in different organs, that consequencely, increase the risk of male infertility. Bleomycin, etoposide, cisplatin (BEP) are popular agents for testicular cancer treatment. Chemotoxic effects of BEP on reproductive organ including: weight loss, significant decrease in sperm concentration and motility. Also mentioned abnormal chromatin condensation induced by BEP can influence histone and reduced translation of protamine1. Ubiquitin is a small chaperone protein that ubiquitinated Histone2A protein during chromatin remodeling. Histone modification plays critical role for regulating nucleosome stability in order to control gene transcription and DNA repairment. During spermiogenesis, 95% of somatic histones replaced with protamine that results chromatin condensation. Every abnormalities in chromatin remodeling can lead to infertility. Omega3 (polyunsaturated fatty acids) has antioxidant, antiapoptotic and anti-inflammatory properties. Also, omega3 inhibits generation of reactive oxygen species that protects against oxidative damage and lipid peroxidation in testis.
Objective: The purpose of this study is evaluation the protective effect of omega3 on rat sperm protamination and ubiquitination after treatment with BEP drugs.
Materials and Methods: In this present study, 40 male rats were divided into four groups: Control, BEP, omega3 and BEP+omega3. Sperm protamination and ubiquitination were assessed using chromomycin A3 and immunofluorescence staining respectively.
Results: The mean percentage of ubiquitinated sperm in BEP group was significantly increased relative to control group (p < 0.001). But, the mean percentage of sperm protamination significantly decreased in BEP group relative to control group (p < 0.001). Rats in BEP+omega3 group showed a significantly decreased in the mean percentage of sperm ubiquitination as compared to BEP group (p < 0.05) while, sperm protamination increased significantly relative to BEP group (p < 0.001). Administration of omega3 after chemotherapy showed an improvement in sperm ubiquitination and protamination.
Conclusion: Our data indicated that omega3 after chemotherapy may be beneficial for chromatin remodeling during spermatogenesis following BEP treatment.
Key words: Chemotherapy, Ubiquitination, Protamination, Rat sperm.

The original full text of this abstract has been published in Nutrition and Cancer 2018; 70(8): 1308-1314. https://doi.org/10.1080/01635581.2018.1521438.
How to cite to this article: Hashemi F, Razavi S, Khadivi F. The protective effects of Omega3 on ubiquitination and protamination of rat sperm after bleomycin, etoposide, and cisplatin treatment. Nutrition and Cancer 2018; 70(8): 1308-1314.

 
P-66
The role of the SYCE1 gene in the male and female fertility: A literature review
 
Ghasemi A^{1, 2}, Alavi A¹.
1 Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran.
2 Student Research Committee, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran.
Email: afaghalavi@gmail.com, af.alavi@uswr.ac.ir
 
Background: Infertility is described as the inability to become pregnant after at least a year of unprotected intercourse. It affects approximately 10-15% of couples worldwide and can involve males and females. There are different risk factors for infertility. Genetic factors are one of the most important among those and some of them affect synaptonemal complex (SC). It is a protein set that facilitates synapsis formation and crossing over during meiotic division in males (spermatogenesis) and females (oogenesis).
Objective: Here, we try to summarize the roles of SC especially, the synaptonemal complex central element-1 (SYCE1) in fertility.
Materials and Methods: We used the search term TITLE-ABS-KEY (SYCE1) from article names, keywords, and abstracts to search the article in PubMed. In addition, we screened all references to related papers for extra research.
Results: The SC comprises several compartments: lateral elements (LEs), which are located on both sides of homologous chromosome axes, and a central region containing a central element (CE), and transverse filaments (TFs). The protein components of the mammalian SC include synaptonemal complex protein-2 (SYCP2), synaptonemal complex protein-3 (SYCP3) in the LEs, synaptonemal complex central element protein-1 (SYCE1), synaptonemal complex central element protein-2-testis-expressed protein-12 (SYCE2-TEX12), synaptonemal complex central element protein-3(SYCE3), and Six6 opposite strand transcript 1 (SIX6OS1) in the CE and synaptonemal complex protein-1(SYCP1) in the TFs. All components have been associated with meiosis division, so mutations in those can be associated with abnormalities of gametogenesis. Mutant mice have been associated with infertility for all SC proteins, except for female mutants in Sycp2 and Sycp3. Functional studies revealed that the male Syce1-knockout mice had excessive primary spermatocytes with maturation arrest, and the females had small ovaries with almost complete lack of follicles. In humans, the function of the SYCE1 gene also appears to be as important as in mice. To date, homozygous mutations of SYCE1 affecting infertility have been reported in three families worldwide. In 2014, a nonsense homozygous mutation (c.613C>T; p.Gln205*) in two sisters affected with primary ovarian insufficiency (POI) was reported. Thereafter, in 2015 and recently in our study in 2020 by whole exome sequencing (WES), two multi affected families were described with non-obstructive azoospermia (NOA), who carried splice-site mutations c.197-2A>G and c.375-2A>G, respectively. Whereas, the C-terminal of the SYCE1 protein is essential for interaction with SYCE3 and thereby assembly of SC, all three aforementioned mutations produce the truncated proteins without C-terminal of the normal protein. It can be possible that nonsense mediated decay (NMD) causes no expression of the mutated mRNA.
Conclusion: To date, all three reported families with mutations in SYCE1 have originated from the Middle East; Israeli-Arab, Iranian-Jewish, and Iranian families. Therefore, screening of SYCE1 mutations among the large cohorts of infertile males and females in Iran and other Middle East countries is recommended.
Key words: Synaptonemal complex, SYCE1, Infertility, Iranian population.
 
P-67
Evaluation the remote organ functions in polycystic ovary syndrome-induced by estradiol valerat in rats
 
Abdi A, Seifi B, Kadkhodaee M, Asadi B, Vaziripour M.
Department of Physiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Email: a-abdi@razi.tums.ac.ir
Background: Polycystic ovarian syndrome (PCOS), Metabolic and heterogeneous disorder, prevalence in women between 5-10%. PCOS presents itself by a numerous clinical manifestation that may affect remote organs such as brain, liver and kidney as well as ovaries.
Objective: The main aim of this study is to investigate the effects of PCOS on remote organs (brain, liver, and kidney).
Materials and Methods: Twelve female rats were randomly assigned to 2 experimental groups: 1) Sham and 2) PCOS. In the Sham group PCOS induction was not performed. In the PCOS group animals received 4 mg/kg estradiol valerate in 0/2 mg sesame oil as a single dose intra muscular. After 31 days, animals were anesthetized and blood, liver, and brain tissues were collected for evaluation of kidney function markers, Urea and plasma creatinine, and liver enzymes, Alanine transaminase and Aspartate transaminase, and measurement of oxidative stress (Malondialdehyde and superoxide dismutase) in liver and brain.
Results: PCOS altered kidney function and liver enzymes significantly as well as the oxidative stress markers in liver and brain as malondialdehyde levels increased and superoxide dismutase activity decreased.
Conclusion: The current study showed that PCOS may affect other organs like kidney, liver, and brain via oxidative stress. Thus notice to remote organs in PCOS is as important as reproductive organs.
Key words: PCOS, Oxidative stress, Remote organs, Brain, Liver.
 
P-68
Evaluation the effects of polycystic ovarian syndrome-induced by estradiol valerat on ovaries function, oxidative stress and histological changes in female rats
 
Asadi B, Seifi B, Rakhshan K, Kadkhodaee M, Abdi A, Vaziripour M.
Department of Physiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Email: b-asadi@razi.tums.ac.ir
 
Background: Polycystic ovarian syndrome (PCOS) is the most prevalent endocrine disorder of women of reproductive age usually accompanied by polycystic ovarian morphology, hyperandrogenism  and anovulation or oligomenorrhea.
Objective: The aim of this study is to clarify tissue changes and oxidative stress status in PCOS rats.
Materials and Methods: The PCOS rat model was developed by the once intra muscular injection of Estradiol valerat. Twelve wistar female rats were randomly assigned to two control and PCOS groups. Control group was without any manipulation; PCOS group was administered with Estradiol valerat (4 mg/kg) dissolved in sesame oil (0.2cc); Ovary functional parameters (folliculogenesis, corpora lutea, stage of follicles), SOD activity and MDA levels were measured in ovary samples.
Results: Significant changes were observed in ovary functional parameters, ovarian SOD activity and MDA levels in compared to control group.
Conclusion: This study showed that due to oxidative stress in ovary, the growth of follicles in the preantral stage, folliculogenesis and the number of corpora lutea were changed in PCOS. Therefore, in PCOS the chance of fertility may reduce.
Key words: Estradiol valerat, Polycystic ovarian syndrome, Oxidative stress.
 
P-69
Inhibition effect of gamma-aminobutyric acid ergic system on oxidative stress in the dorsal hippocampus in an experimental model of polycystic ovary syndrome induced by morphine
 
Jamshidi Z¹, Karami M², Khalili MA³, Roghani M³.

1. Department of Biology, Faculty of Basic Sciences, Shahed University, Tehran, Iran.
2. Department of Biology, Faculty of Basic Sciences, Shahed University, Tehran, Iran.
3. Department of Physiology, Faculty of Medicine, Shahed University, Tehran, Iran.

Email: karami@shahed.ac.ir
 
Background: Because of noticeable occurrence of endocrine disorders in women such as polycystic ovary syndrome, researchers have conducted extensive experimental studies to detail the mechanism of the disease by using the animal model that simulates this type of complication in the model.
Objective: We used the baclofen as a GABAB receptor agonist to reduce oxidative stress induced of polycystic ovary syndrome (PCOS) in active brain regions such as the dorsal hippocampus.
Materials and Methods: For this experiment, 48 female Wistar rats (in the diestrous phase) were randomly divided into seven groups: control (saline), morphine (5 mg/kg), baclofen alone (at doses of 10, 20, and 30 mg/kg), and pre-injection of baclofen doses to the morphine. 24 hr after the experiment, the animals' brains were removed and the hippocampus was isolated on ice for histological and oxidative stress studies. The results were analyzed by analysis of variance (ANOVA) with an error coefficient of 0.05.
Results: In this morphine-induced experimental model of PCOS, the level of dorsal hippocampustissue stress was significant compared to the control group, but in the groups pretreated with baclofen, stress in the dorsal hippocampus decreased.
Conclusion: Reproductive difficulties such as PCOS cause oxidative stress in the active brain areas such as the dorsal hippocampus. The use of baclofen as an agonist of the gamma-aminobutyric acid ergic system shows a protective effect in this complication. Therefore, the gamma-aminobutyric acid ergic system may involve in stress inhibition circuits in this area.
Key words: Polycystic ovary syndrome, Morphine, Baclofen, Oxidative stress, Dorsal hippocampus.
P-70
The stereological evaluation of testis structure on protective effect of quercetin against lead acetate toxicity
 
Khodabandeh Z¹, Dolati P², Zamiri MJ², Mehrabani D^{1, 3}, Bordbar H^{4, 5}, Alaee S⁶, Jamhiri I¹, Azarpira N⁷.

1. Stem Cells Technology Research Center, Shiraz University of Medical Science, Shiraz, Iran.
2. Department of Animal Science, College of Agriculture, Shiraz University, Shiraz, Iran.
3. Li Ka Shing Center for Health Research and Innovation, University of Alberta, Edmonton, AB, Canada.
4. Histomorphometry and Stereology Research Center, Shiraz University of Medical Science, Shiraz, Iran.
5. Department of Anatomy, Shiraz University of Medical Sciences, Shiraz, Iran.
6. Department of Reproductive Biology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran.
7. Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.

Email: Jahromi@sums.ac.ir
 
Background: Exposure to environmental pollutants tightly impacts on male fertility sometimes are irretrievable.
Objective: In the present study, we studied the toxic effects of lead acetate (Pb) on testicular structure, and the possible effect of quercetin on qualifying these effects.
Materials and Methods: Experimental groups, including the Pb, quercetin (QE), (Pb + QE), and control mice, were treated at least one spermatogenic cycle. The fixed testes were dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin wax. Serial sections were prepared using Cavalieri method in a series of equal parallel planes (5 and 20-μm thickness).Then the samples were evaluated by stereological methods.
Results: Testicular weight, both absolute and relative, was higher in Pb-exposed mice in comparison with the control and Pb-quercetin groups. The increase in the size of testis was related to the lumen and connective tissue in this group. Lead acetate induced different patterns in testicular cell number; as spermatogonia, spermatocyte, and Sertoli cells number did not affect in lead acetate exposed group, while the total number of round spermatids and long spermatids significantly reduced.
Conclusion: In conclusion, Pb administration adversely impacted on the cellular organization and activation of the apoptotic pathways in the testis; on the other hand, quercetin co-administration with lead partially ameliorated these adverse effects.
Key words: Antioxidant, Heavy metal, Male fertility.

The original full text of this abstract has been published in Biological Trace Element Research 2020. https://doi.org/10.1007/s12011-020-02454-8.
How to cite to this article: Khodabandeh Z, Dolati P, Zamiri MJ, Mehrabani D, Bordbar H, Alaee S, Jamhiri I, Azarpira N. Protective effect of quercetin on testis structure and apoptosis against lead acetate toxicity: An stereological study. Biological Trace Element Research 2020: 1-1.

P-71
Evaluation of H2S level changes and oxidative stress in the ovary in the polycystic ovary syndrome in rat model
Vaziripour M, Seifi B, Kadkhodaee M, Faghihi M, Abdi A, Asadi B.
Department of Physiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Email: vaziripour_m@razi.tums.ac.ir
Background: Polycystic ovary syndrome (PCOS) is one of the common endocrine and metabolic disorders and occurs in reproductive-aged women. Recent findings have shown that hydrogen sulfide, as one of the gaseous transmitters, is involved in the process of egg maturation and follicogenesis.
Objective: In this study, we investigated changes in H2S levels and their relationship with changes in oxidative stress index levels in this disease.
Materials and Methods: Twelve female rats are randomly selected and divided into 2 groups of 6: 1) control 2) PCOS. In order to induce the polycystic ovary, we dissolve 4 mg of estradiol valerate in 0.2 ml of sesame oil , then inject it intramuscularly in a single dose. Ovarian tissue samples were taken after 21 days to measure the level of oxidative stress indices and determine the level of H2S.
Results: In this study, there were observed that after induction of PCOS, the level of H2S and SOD activity in ovarian tissue reduced and the MDA concentration increased compared with the control group.
Conclusion: This study showed that there is a relationship between H2S level and polycystic ovarian syndrome. Measurement of this parameter may be considered as a reliable diagnostic test for patients with PCOS.
Key words: H2S, Ovary, Oxidative stress.
 
P-72
Protective effect of Sophora pachycarpa root extract on testicular histopathology and sex hormones level in acid in carbon tetrachloride-intoxicated in male rats
 
Banan Khojasteh SM¹, Javanmard Khameneh R², Hoursfand M², Dehghan Gh¹, Heidari R², Iranshahi M³.

1. Department of Animal Biology, Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran.
2. Department of Biology, Faculty of Sciences, University of Urmia, Urmia, Iran.
3.  

1. Department of Pharmacognosy, Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran.

Email: Javanmard_96@mihanmail.ir
 
Background: Carbon tetrachloride (CCl4) is an industrial solvent that causes liver, kidneys, lungs, testicular and brain damage as well as in blood diseases by generating free radicals. Alterations in the spermatogenic cycle and degeneration in seminiferous tubules has been induced with CCl4 in rat. Previous studies on the chemical composition of Sophora pachycarpa (S.pachycarpa) have shown the presence of antioxidant compounds such as flavonoids.
Objective: The purpose of this study was to investigate the protective effects of S.pachycarpa roots extracts on testicular histopathology and serum level of sex hormones in carbon tetrachloride-intoxicated in male rats.
Materials and Methods: Thirty six male wistar rats (195-200 g) were selected and randomly divided into 6 groups (n = 6): pre-treatment groups I, II, III received S.pachycarpa extract at doses 50 mg/kg/day, 100 mg/kg/day and 250 mg/kg/day by gavage for 21 days prior to intraperitoneal injection of CCl4 500 µl/kg on 21^st day, control group, CCl4 group received 500 µl/kg CCl4 on the 21^st day, post-treatment group received extract at doses 100 mg/kg/day for 10 day at 12 h after CCl4 250 µl/kg injection. At the end of the treatment, blood was collected by cardiac puncture from all of the animals and serum levels of Follicle Stimulating Hormone, Luteinizing Hormone and Testosterone were assessed, also the testis tissues were harvested for histological examination.
Results: Serum levels of testosterone and follicle stimulating hormone were significantly increased in serum of pre-treatment group III and serum level of luteinizing hormone in serum of pre-treatment group III compared to CCl4 was significantly increased (p < 0.05). treatment of S.pachycarpa extract (250 mg/kg) showed noticeable improvement in histopathalogical changes induced by CCl4 in testis sections.
Conclusion: From the results it is suggested that S.pachycarpa extract can partly ameliorate toxic effects of CCl4  in male reproductive system, possibly through antioxidant effects of its bioactive compounds.
Key words: Sophora pachycarpa, Carbon tetrachloride, Testis, Sex hormones, Male rat.

The original full texts of this abstract have been published in:

• Zahedan J Res Med Sci 2018; 20(8): e64950. https://doi.org/10.5812/zjrms.64950.
•  

• J Med Plants 2016; 15(60) :94-100. http://jmp.ir/article-1-1003-fa.html.

How to cite to thess articles:

• Banan Khojasteh SM, Javanmard Khameneh R. Sophora pachycarpa root extract improves testicular damage in carbon-tetrachloride intoxicated rats, Zahedan J Res Med Sci 2018; 20(8): e64950. doi: 10.5812/zjrms.64950.
• Banan Khojasteh SM, Javanmard khameneh R, Houresfand M, Dehghan G, Heidari R, Iranshahi M. Investigation in protective effects of Sophora pachycarpa extracts on serum level of sex hormones, urea and uric acid in carbon tetrachloride-intoxicated in male rats. J Med Plants 2016; 15(60): 94-100.

P-73
Insulin resistance defects in male fertility
 
Asefy Z¹, Kayati Sh², Hoseinnejhad S¹, Ceferov Z³, Abbasi Oshaghi E⁴.

1. Maragheh University of Medical Sciences, Maragheh, Iran.
2. Azad Islamic University of Medical Sciences, Maragheh, Iran.
3. Baku State University, Baku, Azerbaijan.
4.  

1. Hamadan University of Medical Sciences, Hamadan, Iran.

Email: zasefy@gmail.com
Background: Insulin resistance (IR) in men with unexplained infertility can be a possible cause of hypogonadism and idiopathic oligozoospermia along with other metabolic abnormalities.
Objective: In this study we investigate IR and following that inflammation effects in male infertility.
Materials and Methods: 45 men with IR deficiency (case group) and 30 men without IR deficiency (control group) were enrolled in this study. Body mass index, testicular volume, semen samples, serum hormone/lipid profiles and high sensitive C-reactive protein (hsCRP) were compared in two groups.
Results: Both case and control groups have shown no significant differences in terms of age, testicular volume, serum hormone, and lipid profiles and body mass index. Nevertheless, HOMA-IR was associated with hsCRP levels (r = 0.92, p < 0.0001).
Conclusion: Lifestyle management is an essential aspect of IR on men's health and fertility  which  include, nutrition therapy, physical activity, smoking cessation. It is assumed  that  male infertility pathophysiology discovery should be effective in therapeutic interventions.
Key words: Infertility, Dyslipidemias, Insulin resistance, C-reactive protein.
 
P-74
Bacteriospermia and its association with seminal fluid parameters and infertility
 
Heidari Pebdeni P¹, Ahmadrajabi R¹, Saffari F¹, Ashoorzadeh S², Mirshekari T², Taheri Soodejani M³.

1. Department of Microbiology and Virology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran.
2. Afzalipour Clinical Center for Infertility, Kerman University of Medical Sciences, Kerman, Iran.
3.  

1. Department of Epidemiology, School of Public Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: paras2_hp@yahoo.com
 
Background: Chlamydia trachomatis (C. trachomatis), Ureaplasma urealyticum, Ureaplasma parvum (U. parvum), Mycoplasma hominis (M. hominis) and Mycoplasma genitalium (M. genitalium) are among the most prevalent sexually transmitted bacteria. The impact of these bacteria on semen quality and their role in male infertility remains a controversial issue.
Objective: This study was conducted to determine the prevalence of bacteriospermia in infertile and fertile men and evaluate the correlation between the presence of these bacteria with infertility and semen quality.
Materials and Methods: In this cross-sectional study, 100 infertile and 100 fertile men attending to the research and clinical centers for ifertility in Kerman, Iran were enrolled from July to December 2019. Semen analysis was performed using the methods outlined by the World Health Organization. Polymerase chain reaction was used for detection of C. trachomatis, Ureaplasma urealyticum, U. parvum, M. hominis, and M. genitalium. Then the correlation between the presence of mentioned bacteria with infertility and the semen quality was evaluated.
Results: There was a significant difference in the presence of M. genitalium and C. trachomatis between infertile and fertile men (p = 0.003). The mean values of volume, progressive motility, non- progressive motility, sperm concentration, total progressive motility and viability were significantly lower in infertile men than in fertile ones (p ˂ 0.05). Statistically significant correlations were observed between the presence of M. genitalium and progressive sperm motility (p = 0.04), the presence of M. hominis and semen volume (p = 0.03), contamination with U. parvum and the normal form of sperm (p = 0.02) and finally between the presence of C. trachomatis and the progressive motility of sperm as well as sperm viability (p = 0.03). Logistic regression analysis showed that the presence of M. genitalium (OR = 8.06, p = 0.007) and C. trachomatis (OR = 16, p = 0.016) was significantly associated with infertility in men.
Conclusion: According to these results, clinician should consider C. trachomatis and M. genitalium in men with decreased sperm progressive motility and viability during the infertility assessment.
Key words: Chlamydia trachomatis, Ureaplasma, Mycoplasma hominis, Mycoplasma genitalium, Infertility, Sperm quality.
 
P-75
Development and characterization of a novel PEGylated liposome-encapsulated Ceratonia siliqua to improved sperm parameters
 
Hemati M¹, Nikukar H¹, Sadeghian-Nodoushan F¹, Abdoli AM², Haghirosadat BF¹.

1. Medical Nanotechnology and Tissue Engineering Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2.  

1. Research and Clinical Centre for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Email: fhaghirosadat@gmail.com
 
Background: Ceratonia silique is found to have antioxidative properties that may inactivate free radicals and minimize the oxidative stress inside the testis cells in infertile male. Also, it is reported as a protective pharmacological agent against several diseases. In order to increase the drug stability, bioavailability, and to deliver the drug in the targeted tissue, it can be incorporated in a nano-sized vesicle of liposome formulations.
Objective: The aim of this study was to optimize the PEGylated liposome-containing carob to enhance the therapeutic response.
Materials and Methods: Polyethylene glycol (Lipoid PE 18:0/18:0-PEG2000, DSPE-mPEG 2000) and          1, 2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were purchased from Lipoid GmbH (Ludwigshafen, Germany). Cholesterol and all chemicals, solvents, and salts were bought from Sigma Chemical Company in St. Louis: USA. In this study, we synthesized liposome formulations containing cholesterol: DPPC: DSPE-PEG2000 at a molar ratio of 70:30:5 and thin-film evaporation method was used for the preparation of stealth controlled- release liposomal the carob. Outcome parameters were mean size of the vesicle, zeta potential, entrapment efficiency and in vitro drug release.
Results: The formulation of liposomal carob demonstrated that the optimum nano-vesicle size with an average diameter of below 100 nm. The vesicles have a suitable negative charge that are stable without aggregation at 4°C. The entrapment efficiency (EE%) for carob was above 80%. Moreover, the release profile of carob during 72 h was slow and continuous with an initial rapid release period followed by a slower release phase.
Conclusion: In conclusion, we successfully developed a stealth liposomal carob with a high potential for systemic delivery. The sustained-release properties of liposomal carob as a successful lipid-based nano-carriers that improves in vivo stability of the drug and leads to pharmaceutical benefits increase and loss of the drugs followed by the high-dose drug.
Key words: Liposome, Ceratonia silique, Infertility, Sperm.