Maternal loss-of-function of Nlrp2 results in failure of epigenetic reprogramming in mouse oocytes.

Background: NLRP2 belongs to the subcortical maternal complex (SCMC) of mammalian oocytes and preimplantation embryos. This multiprotein complex, encoded by maternal-effect genes, plays a pivotal role in the zygote-to-embryo transition, early embryogenesis, and epigenetic (re)programming. The maternal inactivation of genes encoding SCMC proteins has been linked to infertility and subfertility in mice and humans. However, the underlying molecular mechanisms for the diverse functions of the SCMC, particularly how this cytoplasmic structure influences DNA methylation, which is a nuclear process, are not fully understood. Results: We undertook joint transcriptome and DNA methylome profiling of pre-ovulatory germinal-vesicle oocytes from Nlrp2-null, heterozygous (Het), and wild-type (WT) female mice. We identified numerous differentially expressed genes (DEGs) in Het and Nlrp2-null when compared to WT oocytes. The genes for several crucial factors involved in oocyte transcriptome modulation and epigenetic reprogramming, such as DNMT1, UHRF1, KDM1B and ZFP57 were overexpressed in Het and Nlrp2-null oocytes. Absence or reduction of Nlrp2, did not alter the distinctive global DNA methylation landscape of oocytes, including the bimodal pattern of the oocyte methylome. Additionally, although the methylation profile of germline differentially methylated regions (gDMRs) of imprinted genes was preserved in oocytes of Het and Nlrp2-null mice, we found altered methylation in oocytes of both genotypes at a small percentage of the oocyte-characteristic hyper- and hypomethylated domains. Through a tiling approach, we identified specific DNA methylation differences between the genotypes, with approximately 1.3% of examined tiles exhibiting differential methylation in Het and Nlrp2-null compared to WT oocytes. Conclusions: Surprisingly, considering the well-known correlation between transcription and DNA methylation in developing oocytes, we observed no correlation between gene expression differences and gene-body DNA methylation differences in Nlrp2-null versus WT oocytes or Het versus WT oocytes. We therefore conclude that post-transcriptional changes in the stability of transcripts rather than altered transcription is primarily responsible for transcriptome differences in Nlrp2-null and Het oocytes.

Comparison of Four Protocols for In Vitro Differentiation of Human Embryonic Stem Cells into Trophoblast Lineages by BMP4 and Dual Inhibition of Activin/Nodal and FGF2 Signaling.

Human embryonic stem cells (hESCs) cultured in media containing bone morphogenic protein 4 (BMP4; B) differentiate into trophoblast-like cells. Supplementing media with inhibitors of activin/nodal signaling (A83-01) and of fibroblast growth factor 2 (PD173074) suppresses mesoderm and endoderm formation and improves specification of trophoblast-like lineages, but with variable effectiveness. We compared differentiation in four BMP4-containing media: mTeSR1-BMP4 only, mTeSR1-BAP, basal medium with BAP (basal-BAP), and a newly defined medium, E7-BAP. These media variably drive early differentiation towards trophoblast-like lineages with upregulation of early trophoblast markers CDX2 and KRT7 and downregulation of pluripotency markers (OCT4 and NANOG). As expected, based on differences between media in FGF2 and its inhibitors, downregulation of mesendoderm marker EOMES was variable between media. By day 7, only hESCs grown in E7-BAP or basal-BAP expressed HLA-G protein, indicating the presence of cells with extravillous trophoblast characteristics. Expression of HLA-G and other differentiation markers (hCG, KRT7, and GCM1) was highest in basal-BAP, suggesting a faster differentiation in this medium, but those cultures were more inhomogeneous and still expressed some endodermal and pluripotency markers. In E7-BAP, HLA-G expression increased later and was lower. There was also a low but maintained expression of some C19MC miRNAs, with more CpG hypomethylation of the ELF5 promoter, suggesting that E7-BAP cultures differentiate slower along the trophoblast lineage. We conclude that while all protocols drive differentiation into trophoblast lineages with varying efficiency, they have advantages and disadvantages that must be considered when selecting a protocol for specific experiments.

Loss of the Maternal Effect Gene Nlrp2 Alters the Transcriptome of Ovulated Mouse Oocytes and Impacts Expression of Histone Demethylase KDM1B.

The subcortical maternal complex (SCMC) is a multiprotein complex in oocytes and preimplantation embryos that is encoded by maternal effect genes. The SCMC is essential for zygote-to-embryo transition, early embryogenesis, and critical zygotic cellular processes, including spindle positioning and symmetric division. Maternal deletion of Nlrp2, which encodes an SCMC protein, results in increased early embryonic loss and abnormal DNA methylation in embryos. We performed RNA sequencing on pools of meiosis II (MII) oocytes from wild-type and Nlrp2-null female mice that were isolated from cumulus-oocyte complexes (COCs) after ovarian stimulation. Using a mouse reference genome-based analysis, we found 231 differentially expressed genes (DEGs) in Nlrp2-null compared to WT oocytes (123 up- and 108 downregulated; adjusted p < 0.05). The upregulated genes include Kdm1b, a H3K4 histone demethylase required during oocyte development for the establishment of DNA methylation marks at CpG islands, including those at imprinted genes. The identified DEGs are enriched for processes involved in neurogenesis, gland morphogenesis, and protein metabolism and for post-translationally methylated proteins. When we compared our RNA sequencing data to an oocyte-specific reference transcriptome that contains many previously unannotated transcripts, we found 228 DEGs, including genes not identified with the first analysis. Interestingly, 68% and 56% of DEGs from the first and second analyses, respectively, overlap with oocyte-specific hyper- and hypomethylated domains. This study shows that there are substantial changes in the transcriptome of mouse MII oocytes from female mice with loss of function of Nlrp2, a maternal effect gene that encodes a member of the SCMC.

DNA Methylation Dynamics in the Female Germline and Maternal-Effect Mutations That Disrupt Genomic Imprinting.

Genomic imprinting is an epigenetic marking process that results in the monoallelic expression of a subset of genes. Many of these 'imprinted' genes in mice and humans are involved in embryonic and extraembryonic growth and development, and some have life-long impacts on metabolism. During mammalian development, the genome undergoes waves of (re)programming of DNA methylation and other epigenetic marks. Disturbances in these events can cause imprinting disorders and compromise development. Multi-locus imprinting disturbance (MLID) is a condition by which imprinting defects touch more than one locus. Although most cases with MLID present with clinical features characteristic of one imprinting disorder. Imprinting defects also occur in 'molar' pregnancies-which are characterized by highly compromised embryonic development-and in other forms of reproductive compromise presenting clinically as infertility or early pregnancy loss. Pathogenic variants in some of the genes encoding proteins of the subcortical maternal complex (SCMC), a multi-protein complex in the mammalian oocyte, are responsible for a rare subgroup of moles, biparental complete hydatidiform mole (BiCHM), and other adverse reproductive outcomes which have been associated with altered imprinting status of the oocyte, embryo and/or placenta. The finding that defects in a cytoplasmic protein complex could have severe impacts on genomic methylation at critical times in gamete or early embryo development has wider implications beyond these relatively rare disorders. It signifies a potential for adverse maternal physiology, nutrition, or assisted reproduction to cause epigenetic defects at imprinted or other genes. Here, we review key milestones in DNA methylation patterning in the female germline and the embryo focusing on humans. We provide an overview of recent findings regarding DNA methylation deficits causing BiCHM, MLID, and early embryonic arrest. We also summarize identified SCMC mutations with regard to early embryonic arrest, BiCHM, and MLID.

Associations of insulin resistance, sex hormone-binding globulin, triglyceride, and hormonal profiles in polycystic ovary syndrome: A cross-sectional study.

BACKGROUND: Insulin resistance (IR) occurs in 50-70% of women with polycystic ovary syndrome (PCOS) and can be applied as a prediabetic feature in PCOS. OBJECTIVE: In this study, indirect methods including fasting blood sugar (FBS), fasting insulin (FI), FBS/FI ratio, and quantitative insulin sensitivity check index (QUICKI) were compared with the homeostasis model assessment of insulin resistance (HOMA-IR) as a standard technique. The association of IR to sex hormone-binding globulin (SHBG) and several hormones was also analyzed. MATERIALS AND METHODS: This cross-sectional study was performed on 74 PCOS women. Sensitivity and specificity of each IR method was calculated based on HOMA-IR. Hormonal profiles of the patients were compared between the groups with defined normal and abnormal values of IR. RESULTS: Triglyceride levels had a positive association with FBS and HOMA-IR (p = 0.002 and p = 0.01, respectively) with a negative association to QUICKI and SHBG (p = 0.02 and p = 0.02, respectively). SHBG showed a significant negative association with FBS (p = 0.001). Dehydroepiandrosterone sulfate showed a positive association with FI (p = 0.002). Seven PCOS women showed abnormal SHBG levels ( < 36 nmol/L) while expressed normal values of the rest of the studied variables. FI and QUICKI had the highest sensitivity while FBS/FI and QUICKI had the highest specificity when HOMA-IR was applied as a standard test. CONCLUSION: SHBG and triglyceride had a significant negative and positive association with IR, respectively. HOMA-IR followed by FI and QUICKI is the most sensitive test for the detection of IR. SHBG levels can be a helpful biomarker for the diagnosis of PCOS.

Glutathione-dependent enzymes in the follicular fluid of the first-retrieved oocyte and their impact on oocyte and embryos in polycystic ovary syndrome: A cross-sectional study.

BACKGROUND: Oxidative stress and GSH-dependent antioxidant system plays a key role in the pathogenesis of polycystic ovary syndrome (PCOS). OBJECTIVE: We compared glutathione peroxidase (GPx) and glutathione reductase activities and reduced glutathione (GSH) levels in serum and follicular fluid (FF) of the first-retrieved follicle and their impact on quality of oocyte and embryo in PCOS women undergoing IVF. MATERIALS AND METHODS: This cross sectional study was conducted on 80 pairs of blood samples and FF of the first-retrieved follicle from PCOS women, at the Infertility center of Ghadir Mother and Child Hospital. The mean activity of GPx and GR, also GSH levels in the serum and FF were compared to the quality of the first follicle and resultant embryo. RESULTS: Retrieved oocytes included 53 (66.25%) MII, 17 (21.25%) MI, and 10 (12.5%) germinal vesicles; after IVF 42 (52.50%) embryos with grade I and 11 (13.75%) with grade II were produced. The mean values for all three antioxidants were higher in the FF compared to serum (p < 0.001). Also all of the mean measured levels were significantly higher in the FF of the MII oocytes compared to that of oocytes with lower grades (p = 0.012, 0.006 and 0.012, respectively). The mean GPX activity and GSH levels were significantly higher in the serum (p = 0.016 and 0.012, respectively) and FF (p = 0.001 for both) of the high-quality grade I embryos. CONCLUSION: GSH-dependent antioxidant system functions more efficiently in the FF of oocytes and embryos with higher quality.

Prevalence of Cytomegalovirus in Semen of Male Partners of Infertile Couples and the Virus Impact on Sperm Parameters.

BACKGROUND: Genital tract infection is one of the causes of male infertility. Several studies have shown a role for human cytomegalovirus (CMV) in this context. In the present study, the prevalence of CMV in a population of male partners of infertile couples was estimated and the impact of CMV on sperm parameters was determined. METHODS: In this cross sectional study, CMV DNA and virus copy number were examined in the semen of 150 participants including 80 with normal semen analysis (SA) and 70 with abnormal SA, by quantitative Real-Time PCR. Sperm parameters were compared between CMV positive and negative groups. Comparisons with p- values under 0.05 were considered significant. Logistic regression was performed to control the effect of some variables with p<0.25 on sperm parameters. RESULTS: CMV DNA was detected in the semen of 28 (18.6%) individuals. 21 men (30%) with abnormal SA and 7 (8.8%) with normal SA were positive for CMV DNA (p=0.001). The mean virus copy number was 883.1±4662.01 for the men with abnormal SA and 2525.7±12680.9 for those with normal SA (p=0.001). Sperm count was (32.1±23.5) ×106 in CMV positive and (44.2±24.1) ×106 in CMV negative groups (p=0.022). Normal sperm morphology was 2.73±2.83% and 5.99±5.44% in CMV positive and negative groups, respectively (p<0.001). After controlling some variables, the sperm morphology remains the only statistically significant sperm parameter that was reduced by CMV. CONCLUSION: The higher CMV prevalence in the semen of males with abnormal SA compared to normal SA and significant reduction of sperm morphology in the presence of CMV, are in favor of the negative impact of CMV on male fertility.

Founder Effect of KHDC3L, p.M1V Mutation, on Iranian Patients with Recurrent Hydatidiform Moles.

BACKGROUND: Recurrent hydatidiform moles (RHMs) are an unusual pregnancy with at least two molar gestations that are associated with abnormal proliferation of trophoblastic tissue and a failure in the embryonic tissues development. Three maternal-effect genes, including NLRP7, KHDC3L, and PADI6 have been identified as the cause of RHMs. The present study aimed to understand the association of a founder mutation with the incidence and prevalence of a disease in different individuals of a population. METHODS: 14 unrelated Iranian patients with recurrent reproductive wastage, including at least two HMs, entered this study. In order to find a possible mutation in KHDC3L, all the 14 samples were Sanger sequenced. For haplotype analysis, three single nucleotide polymorphisms (SNPs) were selected with highest Minor Allele Frequency along KHDC3L. RESULTS: A common KHDC3L mutation with the same haplotype was identified in four out of 14 patients with RHM. Regarding the present study, c.1A>G is the highest reported mutation in KHDC3L so far and is also the first report of the homozygous state that has led to RHM. CONCLUSION: c.1A>G mutation in KHDC3L is the highest reported mutation around the world. Our data also demonstrated the presence of founder effects for this particular mutation in Iranian populations. These data suggest that the high frequency of this mutation is potentially responsible for a higher rate of RHM in Iran.

Pre-implantation genetic diagnosis in an Iranian family with a novel mutation in MUT gene.

BACKGROUND: Methylmalonic acidemia (MMA), which is an autosomal recessive metabolic disorder, is caused by mutations in methylmalonyl-CoA mutase (MUT) gene. As a result, the conversion of methylmalonyl-CoA to succinyl-CoA is impaired in this disorder, leading to a wide range of clinical manifestations varying from no signs or symptoms to severe lethargy and metabolic crisis in newborn infants. Since identification of novel mutations in MUT gene can help discover the exact pathogenesis of MMA and also use these disease-causing mutations in prenatal diagnosis, this study was conducted to uncover the possible mutations in an Iranian couple with a deceased offspring clinically diagnosed as having organic acidemia. Moreover, to prevent the occurrence of the mutation in the next pregnancy, we took the advantage of pre-implantation genetic diagnosis (PGD), which resulted in a successful pregnancy. CASE PRESENTATION: The affected individual was a 15-month-old boy who passed away due to aspiration pneumonia. The child presented at the age of 3 months with lethargy, protracted vomiting, hypotonia, and decreased level of consciousness. To find the mutated gene, Next Generation Sequencing (NGS) was performed as carrier testing for the parents and the results revealed a novel (private) heterozygous missense mutation in MUT gene (c.1055A > G, p.Q352R). After performing PGD on three blastomeres, one was identified as being homozygous wild-type that was followed by successful pregnancy. CONCLUSIONS: Our study identified a novel, deleterious, heterozygous missense mutation in MUT gene in a couple and helps to consider the genetic counselling and prenatal diagnosis more seriously for this family with clinical phenotypes of organic acidemia.

A KHDC3L mutation resulting in recurrent hydatidiform mole causes genome-wide DNA methylation loss in oocytes and persistent imprinting defects post-fertilisation.

BACKGROUND: Maternal effect mutations in the components of the subcortical maternal complex (SCMC) of the human oocyte can cause early embryonic failure, gestational abnormalities and recurrent pregnancy loss. Enigmatically, they are also associated with DNA methylation abnormalities at imprinted genes in conceptuses: in the devastating gestational abnormality biparental complete hydatidiform mole (BiCHM) or in multi-locus imprinting disease (MLID). However, the developmental timing, genomic extent and mechanistic basis of these imprinting defects are unknown. The rarity of these disorders and the possibility that methylation defects originate in oocytes have made these questions very challenging to address. METHODS: Single-cell bisulphite sequencing (scBS-seq) was used to assess methylation in oocytes from a patient with BiCHM identified to be homozygous for an inactivating mutation in the human SCMC component KHDC3L. Genome-wide methylation analysis of a preimplantation embryo and molar tissue from the same patient was also performed. RESULTS: High-coverage scBS-seq libraries were obtained from five KHDC3Lc.1A>G oocytes, which revealed a genome-wide deficit of DNA methylation compared with normal human oocytes. Importantly, germline differentially methylated regions (gDMRs) of imprinted genes were affected similarly to other sequence features that normally become methylated in oocytes, indicating no selectivity towards imprinted genes. A range of methylation losses was observed across genomic features, including gDMRs, indicating variable sensitivity to defects in the SCMC. Genome-wide analysis of a pre-implantation embryo and molar tissue from the same patient showed that following fertilisation methylation defects at imprinted genes persist, while most non-imprinted regions of the genome recover near-normal methylation post-implantation. CONCLUSIONS: We show for the first time that the integrity of the SCMC is essential for de novo methylation in the female germline. These findings have important implications for understanding the role of the SCMC in DNA methylation and for the origin of imprinting defects, for counselling affected families, and will help inform future therapeutic approaches.

A Novel Mutation in NLRP7 Related to Recurrent Hydatidiform Mole and Reproductive Failure.

BACKGROUND: Hydatidiform mole (HM) is an abnormal human pregnancy with excessive trophoblastic proliferation and abnormal embryonic development, dividing into two complete HM (CHM) and partial HM (PHM) groups. One subcategory of the CHMs is recurrent and familial, which is known as biparental HM (BiHMs) or recurrent HM (RHM). NLRP7, KHDC3L and PADI6 are maternal-effect genes involved in RHMs. NLRP7 is a major gene responsible for RHMs. This study was performed on patients with molar pregnancies and miscarriage. The aim of this study was to genetic screen for mutations in NLRP7 and KHDC3L genes in an affected woman with previous history of 5RHM and the sibling with history of miscarriage. MATERIALS AND METHODS: In this experimental study, DNA was extracted from blood samples. KHDC3L and NLRP7 were polymerase chain reaction (PCR) amplified. The PCR products were purified and Sanger sequenced. RESULTS: In this study, there is no mutation in KHDC3L gene but a novel mutation was identified in the NACHT domain of NLRP7 gene. Patient with five recurrent moles had this mutation in the homozygous state while her sister with one miscarriage and one normal child showed this mutation in the heterozygous state. CONCLUSION: In this study, we identified a new mutation in NLRP7 gene of a patient with recurrent HM. Following egg donation, this patient has a normal boy. The sister of this patient with heterozygous mutation has a spontaneous abortion and one normal child that confirm the impact of a defective allele of NLRP7 on reproductive wastage in a recent finding.

ADCY10 frameshift variant leading to severe recessive asthenozoospermia and segregating with absorptive hypercalciuria.

STUDY QUESTION: Can whole exome sequencing (WES) reveal a novel pathogenic variant in asthenozoospermia in a multiplex family including multiple patients? SUMMARY ANSWER: Patients were discovered to be homozygous for a rare 2-bp deletion in the ADCY10 coding region (c.1205_1206del, rs779944215). WHAT IS KNOWN ALREADY: ADCY10 encodes for soluble adenylyl cyclase (sAC), which is the predominant adenylate cyclase in sperm. It is already established that proper sAC activity and a constant supply of cAMP are crucial to sperm motility regulation, and knockout mouse models have been reported as severely asthenozoospermic. ADCY10 is a susceptibility gene for dominant absorptive hypercalciuria (OMIM#143870); however, no ADCY10 variations have been confirmed to cause human asthenozoospermia to date. STUDY DESIGN, SIZE, DURATION: This was a retrospective genetics study of a highly consanguineous pedigree of asthenozoospermia. The subject family was recruited in Iran in 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: The two patients were diagnosed as asthenozoospermic through careful clinical investigations. Both patients, respective parents, and an unaffected brother were subjected to WES. The discovered variant was validated by Sanger sequencing and segregated with the phenotype. To confirm the pathogenicity of the variant, sperm samples from both patients, 10 normozoospermic men and 10 asthenozoospermic patients not representing the variation, were treated with a cAMP analogue dissolved in human tubal fluid medium, followed by computer-assisted sperm analysis and statistical analyses. MAIN RESULTS AND THE ROLE OF CHANCE: The discovered homozygous variant occurs at 10 amino acids upstream of the ADCY10 nucleotide binding site leading to a premature termination (p.His402Argfs*41). Treatment of the patients' sperm samples with a cell-permeable cAMP analogue resulted in a significant increase in sperm motility, indicating the pathogenic role of the variant. Moreover, absorptive hypercalciuria, segregating within the family, was also associated with the same variant following a dominant inheritance. LIMITATIONS, REASONS FOR CAUTION: Though nonsense-mediated decay is highly likely to occur in the mutated transcripts, we were not able to confirm this due to low RNA levels in mature sperm. WIDER IMPLICATIONS OF THE FINDINGS: Our finding enlarges the phenotypic spectrum associated with the ADCY10 gene, previously described as a susceptibility gene for dominant absorptive hypercalciuria. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Royan Institute, Tehran, Iran, and San Raffaele Hospital, Milan, Italy. The authors have no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Origins of DNA methylation defects in Wilms tumors.

Wilms tumor is an embryonic renal cancer that typically presents in early childhood and accounts for 7% of all paediatric cancers. Different genetic alterations have been described in this malignancy, however, only a few of them are associated with a majority of Wilms tumors. Alterations in DNA methylation, in contrast, are frequent molecular defects observed in most cases of Wilms tumors. How these epimutations are established in this tumor is not yet completely clear. The recent identification of the molecular actors required for the epigenetic reprogramming during embryogenesis suggests novel possible mechanisms responsible for the DNA methylation defects in Wilms tumor. Here, we provide an overview of the DNA methylation alterations observed in this malignancy and discuss the distinct molecular mechanisms by which these epimutations can arise.

Effect of low-dose aspirin on the development of ovarian hyperstimulation syndrome and outcomes of assisted reproductive techniques in the women with PCOS, a randomized double-blinded clinical trial.

OBJECTIVE: Ovarian hyperstimulation syndrome (OHSS) is a major complication of assisted reproductive technologies (ART). Polycystic ovary syndrome (PCOS) is a risk factor for OHSS. The aim of this randomized clinical trial (RCT) was to study the effect of low-dose aspirin (LDA) on the development of OHSS and ART outcomes in PCOS during ART. MATERIALS AND METHODS: This double-blinded placebo controlled RCT was performed on 232 PCOS infertile women in their first ART cycles during 2010-2016. LDA and placebo capsules were prepared, packed and specified by code numbers in similar shapes. One package was given to every woman and asked to take one capsule/day since the 21st day of her cycle prior to the gonadotropin stimulation. Gonadotropin releasing hormone agonist long protocol and triggering by human chorionic gonadotropin were used. Development of moderate to severe OHSS and their ART outcomes were documented then the codes were broken and data analyzed. Chi-square and Mann-Whitney U tests were used for the statistical analyses. RESULTS: Eighteen cases that did not follow the study design were excluded. 214 cycles remained for the final analyses with 109 cases in LDA and 105 in the placebo group. Rate of the moderate to severe OHSS in LDA group was 34.9% compared to 30.5% in placebo group (P = 0.494). Fertilization rate was 71.8% vs 65.1% (P = <0.001) and the mean number of grade III embryos were 3.28 ± 3.53 vs 1.46 ± 1.42 (P = 0.014) in LDA and placebo groups, respectively. The mean number of the oocytes in different grades, total and frozen embryos also implantation and clinical pregnancy rates were not different between the groups. CONCLUSION: Moderate to Severe OHSS was not decreased but fertilization rate and the mean number of poor quality embryos were increased in LDA arm. REGISTRATION NUMBER: IRCT 201105216541N1.

The Impact of Methylenetetrahydrofolate Reductase (MTHFR) Sperm Methylation and Variants on Semen Parameters and the Chance of Recurrent Pregnancy Loss in the Couple.

BACKGROUND: Recurrent pregnancy loss (RPL) defined as three or more consecutive spontaneous miscarriages before the 20th week of gestation is caused by different factors including genetic and epigenetic background. However the involvement of paternal background on RPL is an interesting novel argument, which is not well studied. The main focus of the present study was to investigate for the association of paternal methylenetetrahydrofolate reductase (MTHFR) epigenotypes with sperm parameters and RPL. Moreover, the frequency of two of MTHFR Single Nucleotide Polymorphisms (SNPs) in males was assessed. METHODS: This is a case-control study. Methylation Specific PCR (MSP) was used to evaluate the methylation status of MTHFR promoter on sperm DNA of 25 male partners of RPL and 25 male partners of non-RPL couples. PCR-RFLP method was used to analyze 1,298 A>C (rs1801131) and 677 C>T (rs1801133) polymorphisms. RESULTS: No significant difference was observed in frequency of methylated MTHFR epigenotype between RPL and non-RPL males. Furthermore, methylated MTHFR epigenotype was more frequent (but not statistically significant) among men with abnormal sperm parameters compared to normal-sperm men. Among studied polymorphisms, only the mutated allele of C677T showed statistically higher prevalence among RPL males. CONCLUSIONS: Although our results do not establish any connection between MTHFR epigenotypes and RPL they do highlight the impact of C677T in the pathology.

Assessment of oxytocin level, glucose metabolism components and cutoff values for oxytocin and anti-mullerian hormone in infertile PCOS women.

OBJECTIVE: Comparing oxytocin level and some other parameters between infertile women with or without polycystic Ovary Syndrome (PCOS), to evaluate the correlation between oxytocin with anti-mullerian hormone (AMH), Body Mass Index (BMI) and insulin resistance (IR). MATERIALS AND METHODS: This cross-sectional study was performed on 80 PCOS and 81 non-PCOS women as the control group. Oxytocin, various hormones, Oral glucose tolerance test (OGTT) and Homeostatic model assessment of insulin resistance (HOMA-IR) were compared between two groups. Correlations between parameters were assessed by the spearman's rank correlation coefficient. Cutoff values for oxytocin and AMH in PCOS were calculated by the ROC-Curve and DeLong method. RESULTS: The mean oxytocin level was statistically lower in the case group (p ≤ 0.001). The mean BMI, AMH, HOMA-IR, fasting insulin and insulin 2-h after 75-g glucose were significantly higher in the PCOS group. Oxytocin was negatively correlated to AMH when evaluated for all participants or only among controls. Moreover oxytocin was negatively correlated to HOMA-IR among all participants. However the relationship between oxytocin and BMI was not statistically significant. The calculated cutoff value for oxytocin was 125 ng/L and for AMH was 3.6 ng/mL in the PCOS group. CONCLUSION: The mean oxytocin level in the PCOS infertile women was lower than non-PCOS women. Oxytocin showed a significant reverse correlation with AMH and HOMA-IR.

Ovarian Hyperstimulation Syndrome: A Narrative Review of Its Pathophysiology, Risk Factors, Prevention, Classification, and Management.

Ovarian hyperstimulation syndrome (OHSS) is a serious complication of ovulation induction that usually occurs after gonadotropin stimulation, followed by human chorionic gonadotropin administration, for infertility treatment. The existing knowledge about the pathophysiology, risk factors, and primary and secondary methods for the prevention of OHSS is reviewed in this manuscript. The clinical manifestations and characteristics of mild, moderate, severe, and critical forms of the syndrome are defined. The methods of handling affected cases as outpatient or in-hospital management methods as well as indications for hospitalization are summarized in this review. The clinical and biochemical routes of assessing and monitoring hospitalized patients with OHSS, various drugs and medical treatment strategies including indications for aspiration of the ascitic fluid and pleural effusion, and also rare indications for surgery are briefly explained in this article. Severe OHSS, which two decades ago was considered an iatrogenic life-threatening condition, can now be effectively prevented or managed during the early stages. An OHSS-free clinic can be established nowadays by carefully considering the endocrinology of ovulation and using appropriate and dose-adjusted pharmaceutical agents, which are summarized and discussed in this review.

Detection of Y Chromosome Microdeletions and Hormonal Profile Analysis of Infertile Men undergoing Assisted Reproductive Technologies.

BACKGROUND: Y chromosome deletions (YCDs) in azoospermia factor (AZF) region are associated with abnormal spermatogenesis and may lead to azoospermia or severe oligozoospermia. Assisted reproductive technologies (ART) by intracytoplasmic sperm injection (ICSI) and testicular sperm extraction (TESE) are commonly required for infertility management of patients carrying YCDs. The aim of this study was to estimate the frequency of YCDs, to find the most frequent variant in infertile men candidate for ART and to compare YCD distribution with a control fertile group. The semen parameters, hormonal profiles and ART outcomes of the infertile group were studied. MATERIALS AND METHODS: This case-control study consisted of 97 oligozoospermic or non-obstructive azoospermic (NOA) infertile men, who had undergone ART, as the case group and 100 fertile men as the control group. DNA samples were extracted from blood samples taken from all 197 participants and YCDs were identified by multiplex polymerase chain reaction (PCR) of eight known sequence-tagged sites. The chi-square test was used to compare the mean values of hormone and sperm parameters between the two groups. P<0.05 was considered statistically significant. RESULTS: No YCD was detected in the control group. However, 20 out of 97 (20.6%) infertile men had a YCD. AZFc, AZFbc and AZFabc deletions were detected in 15 (75%), four (20%) and one (5%) YCD-positive patients. No fertilization or clinical pregnancy was seen following ICSI in this sub-group with YCD. The mean level of FSH was significantly higher in the group with YCD (28.45 ± 22.2 vs. 4.8 ± 3.17 and 10.83 ± 7.23 in YCD-negative patients with and without clinical pregnancy respectively). CONCLUSION: YCD is frequent among NOA men and YCD screening before ART and patient counseling is thus strongly recommended.

Clinical and genetic-epignetic aspects of recurrent hydatidiform mole: A review of literature.

Hydatidiform Mole (HM) is the most common form of Gestational Trophoblastic Disease (GTD), defined by hyper-proliferation of trophoblastic cells. HM is typified as abnormal proliferation of extraembryonic trophoblastic (placental) tissues and failure of embryonic tissues development and is the only GTD with Mendelian inheritance, which can reoccur in different pregnancies. Moles are categorized into Complete Hydatidiform Moles (CHM) or Partial Hydatidiform Moles (PHM) and a rare familial trait, which forms a CHM and despite having androgenetic pattern, shows normal biparental inheritance, conceived from one sperm and egg. Recessive maternal-effect mutations in NLRP7 (NACHT, leucine rich repeat and PYD containing 7) and KHDC3L (KH Domain Containing 3-Like) genes have been shown to be responsible for Recurrent Hydatidiform Moles (HYDM1 MIM# 231090 when is caused by mutation in the NLRP7 gene and HYDM2 MIM#614293 when is caused by mutation in the KHDC3L gene). Methylation aberration in multiple maternally imprinted genes is introduced as the cause of Recurrent HYDM pathology. The current article reviews the histopathology, risk factors, and genetic and epigenetic characteristics of Recurrent HYDMs.

ZFP57 maintains the parent-of-origin-specific expression of the imprinted genes and differentially affects non-imprinted targets in mouse embryonic stem cells.

ZFP57 is necessary for maintaining repressive epigenetic modifications at Imprinting control regions (ICRs). In mouse embryonic stem cells (ESCs), ZFP57 binds ICRs (ICRBS) and many other loci (non-ICRBS). To address the role of ZFP57 on all its target sites, we performed high-throughput and multi-locus analyses of inbred and hybrid mouse ESC lines carrying different gene knockouts. By using an allele-specific RNA-seq approach, we demonstrate that ZFP57 loss results in derepression of the imprinted allele of multiple genes in the imprinted clusters. We also find marked epigenetic differences between ICRBS and non-ICRBS suggesting that different cis-acting regulatory functions are repressed by ZFP57 at these two classes of target loci. Overall, these data demonstrate that ZFP57 is pivotal to maintain the allele-specific epigenetic modifications of ICRs that in turn are necessary for maintaining the imprinted expression over long distances. At non-ICRBS, ZFP57 inactivation results in acquisition of epigenetic features that are characteristic of poised enhancers, suggesting that another function of ZFP57 in early embryogenesis is to repress cis-acting regulatory elements whose activity is not yet required.