International Journal of

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Background: Previous studies have shown that morphine consumption during pregnancy may delay embryo development or cause abnormal nervous system function.
Objective: The present study focused on the effect of maternal morphine consumption on development of placenta and blood corticosteron concentration in addictive pregnant mothers.
Materials and Methods: 24 female rats, 170-200g weight, were used. The experimental groups after pregnancy received an oral dose of 0.05 mg/ml of morphine by tap water while the control group received only tap water. On 10th and 14th day of pregnancy, rats were anesthetized and placenta removed surgically, 1ml blood was collected from each pregnant mother from retro-orbital sinus, the concentration of blood corticosteron was determined by corticosteron Elisa kit after centrifugation. The fixed tissue was processed, sectioned and stained with hematoxylin and eosin. Placenta was studied microscopically according to the thickness of layers, area of blood cisterns, and the number of cells.
Results: Comparing the plasma corticosteron concentration of the treatment and the control groups, not only a severe increase in the treatment group was detected, but also the thickness of maternal and embryonic portions of the placenta at day 10th and 14th of gestation was different significantly (p≤0.05). Furthermore, an increase in number of cells in maternal and embryonic portion of placenta and a decrease in blood cistern area were demonstrated in both the experimental and the control groups. Conclusion: The effects of morphine, including an increase in blood concentration of corticosteron, in dependent pregnant mothers were seen. Development of placenta in the experimental group was delayed.

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Background: Epigenetic reprogramming of differentiated cells can modify somatic cells into pluripotential state. Pluripotency can be induced in somatic cells by several approches. One of the easy ways to induce pluripotency is the exposure of the somatic cells to the embryonic stem cell (ESC) extract. Objective: The objective of this study was to increase the efficiency of reprogramming of granulosa cell as a differentiated cell into pluripotential state by using epigenetic modifier agents and extract.
Materials and Methods: The human granulosa cells were cultured in the medium containing 5-Aza-Deoxycytidine and trichostatin A. Then, the cells were exposed to mouse ESCs extract and co-cultured with mouse embryonic fibroblast in the presence of leukemia inhibitory factor (LIF). Alkaline phosphatase test and also immonohistochemistery staining for Oct4, Sox2 and Nanog were performed after 24 and 72 hours and 1 week.
Results: The granulosa cells showed the alkaline phosphatase activity after 24 hours and the enzyme activity maintained for 72 hours. They also expressed Oct4 after 24 hours. The cells also expressed Sox2 and Nanog, 72 hours after exposure to the ESCs extract. The expression of the pluripotency markers decreased after 1 week. It seems that the extract can induce dedifferentiation in granulosa cells and they can express the stem cell markers. Conclusion:   It seems that the inhibitors of the methyl transferase (5-Aza-Deoxycytidine) and histone deacetylase (trichostatin A) could delete the epigenetic markers and prepare the cells for reprogramming by administration of the extract.

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Background: Rupture of fetal membranes can occur at any gestational age. Premature rupture of membranes (PROM) means rupture of fetal membranes before the onset of labor.
Objective: The purpose of this study was to evaluate and compare the reliability of the vaginal washing fluid urea and creatinine for the diagnosis of PROM and to determine cut-off values.
Materials and Methods: A total of 179 pregnant women were recruited. All patients underwent different examinations. These included nitrazine paper test, fern test, amniotic fluid pooling, vaginal washing fluid urea and creatinine sampling. The one group consisted of 126 pregnant women between 14 and 41 weeks of gestation with the complaint of vaginal fluid leakage. Patients who had positive pooling, nitrazine paper test and fern test were considered as confirmed PROM group (group 1). On the other side, patients with pooling (-) and/or nitrazine paper test (-) and/or fern test (-) were taken as suspected unconfirmed PROM cases (group 2). The control group consisted of 53 pregnant women between 14 and 41 weeks of gestation without any complaint or complication. Weconducted one-way ANOVA test on the urea and creatinine measures and post-hoc comparison test. Cut-off value was determined by receiver operating characteristic (ROC) curve.
Results: Vaginal fluid concentrations of urea and creatinine were significantly different between the three groups (p<0.001). The sensitivity, specificity, positive and negative predictive values and accuracy were all 100% in detecting premature rupture of membranes by evaluation of vaginal fluid creatinine concentration with a cut-off value of 0.45 mg/dl, respectively.
Conclusion: This study demonstrates that of two markers investigated creatinine has the higher diagnostic power.

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Background: Amniotic fluid is an indicator of placental function on the fetal development. The amniotic fluid index is the most commonly used method of measuring amniotic fluid.
Objective: The purpose of this study was to compare the pregnancy outcomes of a borderline versus normal AFI.
Materials and Methods: This cross-sectional study was carried out on a total of 235 pregnant women referred to Alzahra Medical Center between 2009-2011. Women with a singleton pregnancy in third trimester were enrolled into this study; of these subjects, 141 cases were in normal AFI group and 94 cases in borderline AFI group. Adequate information was obtained from the patients' medical record and the groups were compared on maternal and fetal complications. Data analysis was performed by using SPSS.
Results: The mean maternal age in borderline AFI group was 25.96±5.92 years and in normal AFI group was 27.88±6.5 years (p=0.023). Maternal outcomes such as preterm delivery and labor induction in women with borderline AFI were considerably higher than those in normal group (p=0.01 and p=0.001). There were no significant differences between the two groups in terms of high blood pressure, preeclampsia, diabetes and neonatal respiratory distress. The borderline AFI group had higher rate of neonatal complications such as Apgar score of less than 7 (p=0.004), IUGR (0.0001), LBW (0.001), and crucial need to NICU (0.003).
Conclusion: Findings indicated that there are statistical differences between adverse outcomes in borderline AFI group and normal group.

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Background: Creation of artificial gametes may provide a universal solution for these patients of lacking gametes. Stem cell technology may provide a way to obtain fully functional gametes. Retinoic acid (RA) can initiate meiosis. Several studies have demonstrated that RA can promote sperm cells differentiation from mouse embryonic stem cells (mESCs) and other cells from human embryonic stem cells (hESCs).
Objective: We sought to determine whether RA could promote differentiation of germ cells from hESCs.
Materials and Methods: hESCs were differentiated as embryoid bodies (EBs) in suspension with all-trans RA (atRA) or without atRA for 0, 1, 3, 5 and 7 days, and then compared the expression of VASA, SCP3, GDF9 and TEKT1 by real-time PCR. The statistical differences were evaluated by one way ANOVA.
Results: The expression of germ cell-specific markers including the gonocyte marker VASA, the meiotic marker SCP3, and postmeiotic markers, GDF9 and TEKT1, all increased in the presence and absence of RA as EB differentiation progressed. In addition, the expression of these markers increased an average of 9.3, 6.9, 7.2 and 11.8 fold respectively in the presence of RA, compared to the absence of RA, over 5 days differentiation.
Conclusion: Our results indicate that hESCs may have the potential to differentiate to primordial germ cells (PGCs) and early gametes. And RA can improve germ cells differentiation from hESCs.

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Background: HOXA11 and HOXA10 are expressed in endometrium throughout the menstrual cycle and show a dramatic increase during the mid-luteal phase at the time of implantation. The expression of these genes is decreased in women with myomas.
Objective: To determine whether myomectomy would reverse HOXA11 and HOXA10 expression, we evaluated the transcript levels of these genes in the endometria of patients before and after myomectomy.
Materials and Methods: Expression of HOXA11 and HOXA10 were examined prospectively during the midluteal phase in endometrium obtained from infertile women (n=12) with myoma before and three months after myomectomy. Endometrial HOXA11 and HOXA10 expression were evaluated using quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR).
Results: Endometrial HOXA11 and HOXA10 mRNAs expression levels (normalized to 18SrRNA) were increased insignificantly in endometrium of patients after myomectomy (p=0.7 and p=0.15 respectively).
Conclusion: The results suggest that the alteration in expression pattern of these genes could not account for some aspects of fertility after myomectomy.

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Background: Spermatogonial stem cell (SSC) is a self-renewing population of male adult stem cell. SSCs have a differentiation potential which are similar to embryonic stem cells. These Embryonic stem like (ES-like) cells can be a potential source for pluripotent cells for stem cell-based therapy.
Objective: This study presents an economical and simple co-culture system for pluripotent stem cells generation from neonatal mouse testis
Materials and Methods: Isolated testicular cells were cultured in DMEM/F12. Characteristics of the isolated cells and obtained ES-like cell were immune-cytochemically confirmed by examining the presence of PLZF, vimentin, Oct4 and Nanog protein. Expression of the pluripotency and germ-cell specific genes was analyzed by qPCR in derived ES-like colony and SSCs respectively.
Results: The experiment results indicated that our method of obtaining pluripotent ES-like cells from spermatogonial cells (SCs) is simpler than the described methods. ES-like cells were immunopositive for pluripotency markers. ES-like cell qPCR results indicated significant increase in pluripotency genes expression and significant decrease in germ cell-specific genes expression.
Conclusion: The results indicated that ES-like cell with pluripotency characteristic were generated from freshly isolated spermatogonial cells. The pluripotent stem cells provide a cellular reservoir usable for regenerative medicine instead of embryonic stem cells. 

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Background: Preterm birth is a leading cause of perinatal mortality and long-term morbidity as well as the long-term health consequences and cognitive outcomes.
Objective: Present study was conducted to determine prevalence and risk factors associated with preterm birth in Ardabil, Iran.
Materials and Methods: A case control study was conducted between Nov 2010 and July 2011 in all three maternal hospitals in Ardabil. All the live newborns during the study period were investigated. Of 6705 live births during the study period 346 births occurred in <37 weeks were taken as a case and 589 term neonates were taken as a control group. Data were obtained through review of prenatal and hospital delivery records. Univariate and multivariate logistic regression analysis were applied to obtain magnitude of association between independent variables and preterm birth.
Results: The prevalence rate of preterm birth was 5.1%. History of previous preterm birth (OR=12.7,CI: 3.9-40.4, p<0.001), hypertension (OR=7.3, CI:2.1-25.4, p=0.002), Oligohydramnios (OR=3.9, CI:1.6-9.5, p=0.002), spouse abuse (OR=3.7, CI:1.1-11.8, p=0.024), preeclampsia (OR=3.6, CI:1.3-10.3, p=0.014), premature rupture of membrane (OR=3.1, CI:1.9-4.9, p=0.000), bleeding or spotting during pregnancy (OR=2.0, CI:1.0-3.8, p=0.037), Hyperemesis Gravid arum (OR=2.0, CI: 1.1-3.8, p=0.015), urinary tract infection in 26-30 weeks , (OR=1. 8 CI:1.0-3.2, p=0.04), diastolic blood pressure ≤60 mmg (OR=1.5, CI: 0.99-2.2, p=0.049) were determined as significant risk factors for preterm birth.
Conclusion: Early detection and treatment of diseases or disorders among pregnant women especially hypertension, Oligohydramnios, preeclampsia, bleeding or spotting, Hyperemesis Gravid arum, urinary tract infection, and low diastolic blood pressure as well as the improving health care quality delivered to pregnant women may reduce preterm prevalence rate. 

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Background: Mouse embryonic stem (ES) cells are derived from the inner cell mass (ICM) of the preimplantation blastocysts. So it is suggested that ES and ICM cells should have similar cellular surface molecules and antiserum to ES cells can inhibit ICM development.
Objective: The objective of this study was to evaluate the effect of rabbit antiserum to ES cells on mouse preimplantation embryo development and chimera production.
Materials and Methods: Mouse 4-cell embryos were matured in vitro at 37.5oC, in humidified 5% CO2 atmosphere for 12-36 h. The embryos were cultured in KSOM medium with or without antiserum for 12-36 h. The ratios of in vitro embryo development of the blastocysts, cell division, attachment potential, alkaline phosphatase activity, post-implantation development, and chimera production were assessed and compared with the control group. P<0.05 was considered as significant.
Results: The rabbit antiserum to mouse ES cells showed delay in embryo compaction and induced decompaction at 8-cell stage. The development of 4-cell embryos in the presence of the antiserum for 36h did not lead to a reduced or absent ICM. These embryos still displayed positive alkaline phosphatase activity, normal cell division, embryo attachment, outgrowth formation, implantation and post-implantation development. In addition, decompaction induced by antiserum did not increase production and germline transmission of chimeric mice.
Conclusion: The results showed that antiserum to ES cells delayed embryo compaction and did not affect post-implantation development and chimera production.

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Background: Preterm premature rupture of membranes (PPROM) occurs in 3% of pregnancies and 30-40% of preterm labors are related to this problem. Early diagnosis of PPROM is very important due to its impact on pregnancy outcomes.
Objective: To determine the diagnostic value of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in vaginal fluid for the diagnosis of preterm premature rupture of membranes as a non-invasive and available test.
Materials and Methods:  A total of 148 pregnant women between the 26th-36th gestational weeks were enrolled in the study. 74 patients were in PROM group and 74 in control group. AST and ALT levels in vaginal fluid were measured in each group. Mann Whitney U-test was used to compare AST and ALT levels in each group.
Results:  The mean of AST level in vaginal fluid was 12.77±10.06 in PROM group vs. 6.91±10.92 in control group (p<0.001), while there were no significant difference between ALT levels in PROM group 1.51±3.17 and control group 0.89±1.15 (p=0.49). Optimal cut point of AST for the diagnosis of PROM was 4.5 IU/L in this study. The sensitivity, specificity, positive and negative predictive values were 82.4%, 63.5%, 69.32% and 78.33% respectively.
Conclusion:  According to the findings of this study, measurement of AST level in vaginal fluid can be used as a reliable test for diagnosis of PROM, but there is no good cut point for ALT level that can be practically used.

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Background: The discovery of circulating fetal DNA in maternal blood led to the discovery of new strategies to perform noninvasive testing for prenatal diagnosis.
Objective: The purpose of the study was to detect fetal aneuploidy at chromosomes 13, 18, 21, X, and Y by analysis of fetal cell-free DNA from maternal blood, without endangering pregnancy.
Materials and Methods: This retrospective study has been performed in Bucharest at Medlife Maternal and Fetal Medicine Department between 2013-2014. In total  201 women were offered noninvasive prenatal test. Maternal plasma samples were collected from women at greater than 9 weeks of gestation after informed consent and genetics counseling.
Results: From 201 patients; 28 (13.93%) had screening test with high risk for trisomy 21, 116 (57.71%) had advanced maternal age, 1 (0.49%) had second trimester ultrasound markers and the remaining 56 patients (27.86%) performed the test on request. Of those patients, 189 (94.02%) had a “low risk” result (<1/10,000). Of those who had a low risk result, 2 continued on to have amniocentesis with normal results.Five patients (2.48%) received “high risk” results (>99% risk) all for trisomy 21 (T21). T21 was confirmed by amniocentesis in 1 patient and the other 4 patients declined confirmation. The 7 remaining patients (3.48%) had a low fetal fraction of DNA.
Conclusion: It is probably that prenatal diagnosis using fetal DNA in maternal blood would play an increasingly role in the future practice of prenatal testing because of high accuracy.

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Background: Morbidity and mortality of preterm babies are important issues in perinatal medicine. In developed countries, preterm delivery is the cause of about 70% of mortality and 75% of morbidity in the neonatal period, respectively. Objective: The aim of this study was to determine the risk factors for preterm labor and the outcomes, in terms of perinatal mortality and morbidity at the time of discharge home, among preterm infants at less than 34 weeks gestation. Materials and Methods: A retrospective study was conducted and all infants with a gestational age of 24 to 33 weeks and 6 days who were born from November 1st, 2011 to March 31, 2012 were enrolled in this study. Results: From 1185 preterm infants were born during this period, 475 (40.08%) infants with less than 34 weeks gestational age were included in the study. Our study showed the major obstetrical risk factors for preterm labor were as follows: preeclampsia (21%), premature rupture of membranes (20.3%), abruption of placenta (10%), and idiopathic cases (48.7%). The neonatal mortality rate in less than 34 weeks was 9.05%. Significant perinatal morbidity causesd in less than 34 weeks were as follows: sepsis (46.94%), respiratory distress syndrome (41.47%), patent ductus arteriosus (21.47%), retinopathy of prematurity (3.57%), necrotizing entrocolitis (1.68%), intra-ventricular hemorrhage (9%), and broncho-pulmonary dysplasia (0.84%). Conclusion: Preterm birth is associated with adverse perinatal outcome. This situation needs to be improved by directing appropriately increased resources for improving prenatal health services and providing advanced neonatal care.

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Background: Blighted ovum is one of the most common reasons for abortion during the first three months of pregnancy. Manganese superoxide dismutase (MnSOD) is an important antioxidant enzyme in the human immune system. The gene is located on 6q25 chromosome and acts on mitochondrial matrix. In the case of mutation or inactivity of this enzyme, mitochondrial and nuclear DNA will severely be destructed. The most common polymorphism of its gene is Val16Ala.
Objective: The aim was to investigate a possible mutation in pregnant women who had abortion during the first trimester of pregnancy due to blighted ovum.
Materials and Methods: In this case-control study, 34 women were entered as the case and control groups, respectively. Genome DNA was extracted from saliva samples and its genotype was determined using Tetra-primer amplification refractory mutation system polymerase chain reaction technique.
Results: In the case group, 16 (48%) cases had Val/Val genotype, 17 (50%) were heterozygote and had Val/Ala genotype, and 1 (2%) had Ala/Ala genotype. Among controls, 7 (22%) items had Val/Val genotype, 6 (17%) had Val/Ala genotype, and 21 (61%) had Ala/Ala genotype. The frequency of TT, CT, and CC genotypes was 48%, 50%, and 2% in case group and 22%, 17%, and 61% in control group, respectively. Statistical analysis revealed a significant relationship between Val16Ala polymorphism of MnSOD gene and blighted ovum (p= 0.0003).
Conclusion: It has concluded that a significant relationship exists between Val16Ala polymorphism of MnSOD gene and blighted ovum.

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Background: Preeclampsia is associated with maternal and neonatal complications. It has been indicated that increased uric acid might have a predictive role on preeclampsia.
Objective: We aimed to investigate the relationship between the level of uric acid with maternal and neonatal complications in women with preeclampsia.
Materials and Methods: In this cross-sectional study, 160 singleton preeclamptic women at more than 28 wk gestational age were included. Hemoglobin, hematocrit, platelet count, liver and uric acid tests, and maternal and neonatal complications were assessed. The severity of preeclampsia, placental abruption, preterm labor, thrombocytopenia, elevated alanine aminotransferase and aspartate aminotransferase (ALT and AST), HELLP syndrome, eclampsia and required hospitalization in the ICU was considered as the maternal complication. Fetal complications were: small for gestational age (SGA), intrauterine fetal death, hospitalization in the neonatal intensive care unit, and Apgar score <7 at five minutes.
Results: Of our participants, 38 women had severe preeclampsia (23.8%). The mean level of uric acid in women with severe preeclampsia was significantly higher than non-severe preeclampsia (p=0.031), also in those with an abnormal liver test (p=0.009). The mean level of uric acid in women with preterm delivery was significantly higher than women with term delivery (p=0.0001). Also, the level of uric acid had no effect on neonatal hospitalization in neonate invasive care unit. Based on logistic regression, the incidence of severe preeclampsia not affected by decreased or increased serum levels of uric acid.
Conclusion: With higher level of uric acid in server preeclampsia we can expected more complications such as hepatic dysfunction and preterm delivery. Thus serum uric acid measurement can be helpful marker for severe preeclampsia.

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Background: Bone morphogenetic protein 4 (BMP4) is a significant signaling molecule that involves in initiating of differentiation and performs multifunctional effects on embryonic stem cells (ESCs) and embryos.
Objective: The goal of the present study was to evaluate an in vitro differentiation model of mouse embryonic stem cells into germ cells, using BMP4.
Materials and Methods: in this experimental study, we used Oct4-GFP mouse ESCs to form embryoid body (EB) aggregations for two days. Then, single cells from EB were cultured for four days with BMP4. Using MTT assay and gene expression levels for evaluation of Mvh and Riken by real-time RT-PCR of six concentrations, 12.5 ng/ ml BMP4 was determined as an optimized dose. Then, the expression level of Fkbp6, Mov10l1, 4930432K21Rik, Tex13, Mvh, Scp3, Stra8, Oct4 were evaluated. Flow cytometry and immunostaning were used to confirm the findings of the real-time RT-PCR.
Results: In the +BMP4 group, the genes encoding Riken (p≤0.001) and Mvh (p≤0.001) were found to be increased with significant differences compared with the control group. Mov10l1 (p=0.22), Tex13 (p=0.10), Fkbp6 (p=0.90), Scp3 (p=0.61) and Stra8 (p=0.08) were up-regulated without significance differences compared with control group. Flow cytometry analysis showed that the mean number of Mvh-positive cells in the +BMP4 group was greater when compared with ESCs, -BMP4 and EB groups (p=0.03, p≤0.001, p=0.02, respectively).
Conclusion: Down-regulation of Oct4, expression of germ cells genes and meiosis markers expression raise this hypothesis that ESCs were differentiated by BMP4, and may be introduced into the first meiosis as germ cell-like cells.

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Abstract
Background: Concentrations of plasma lipids levels during pregnancy clearly increases. According to some studies, dyslipidemia is effective in the incidence of preeclampsia and insulin resistance.
Objective: This study aimed to examine the relationship between hyperlipidemia and maternal and neonatal outcomes in pregnant women.
Materials and Methods: This is a cross-sectional study which was conducted on two groups of pregnant women with hyperlipidemia and normal ones to assess maternal and neonatal outcomes. Maternal data including gestational age, mother's age, body mass index, and maternal weight gain during pregnancy, gestational diabetes mellitus, preeclampsia, cholestasis, and delivery method. Also, birth weight and Apgar score were gathered as the neonatal outcomes.
Results: The results showed that the prevalence of abnormal lipid parameters increased with increasing gestational age. In pregnant women with dyslipidemia in combination with increased triglyceride, cholesterol and Low-density lipoprotein, and decreased High-density lipoprotein, the incidence rates of gestational diabetes (p< 0.001), preeclampsia (p< 0.001), cholestasis (p= 0.041), fetal growth retardation (p< 0.001), and macrosomia (p< 0.001) were statistically higher.
Conclusion: Dyslipidemia was associated with some adverse effects of pregnancy and harmful fetal outcomes. Therefore, it seems that adding laboratory assessment of lipid profiles before and during pregnancy can be effective in early diagnosis of dyslipidemia.

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Background: Coronavirus disease 2019 (COVID-19) pandemic has raised concerns about the susceptibility amongst different groups of the population. Pregnant women are one such group. This study was conducted to investigate the effect of COVID-19 on pregnancy and maternal/neonatal outcomes.
Case presentation: This case series was conducted on 16 pregnant women with COVID-19 from March 21 to May 11, 2020. Clinical characteristics, pregnancy complications, medication used, maternal/neonatal outcomes, and fatality rate were investigated through this study. The mean age of the patients was 30.06 yrs. Patients from all three trimesters were included (1 in first, 5 in second, and 10 in the third trimesters). The most common clinical symptoms were shortness of breath (n = 10), dry cough (n = 10), myalgia (n = 8), and chills (n = 7). Also, three cases had papulosquamous skin lesions with fissuring. The most common laboratory results were leukocytosis (n = 8), increased liver enzymes (n = 6), elevated CRP (n = 5), and thrombocytopenia (n = 4). There was one case of maternal mortality, five of premature labor pain (PLP), two of preeclampsia, and two of placenta accreta. Twelve pregnancies were terminated (nine cesarean sections, three vaginal deliveries). Among neonates, we had 6 cases of preterm labor. All neonates had negative PCR results.

Conclusion: Clinical manifestations and paraclinical results were similar to non-pregnant patients. There was no evidence of vertical transmission. PLP and premature rupture of membranes (PROM) were the most common complications in the second and third trimesters of pregnant COVID-19 women, which can lead to rupture of the uterus. Termination and delivery should be planned individually.

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