Evidence for TET-mediated DNA demethylation as an epigenetic alteration in cumulus granulosa cells of women with polycystic ovary syndrome.

Peripheral and tissue-specific alterations in global DNA methylation (5-methylcytosine (5mC)) and DNA hydroxymethylation (5-hydroxymethylcytosine (5hmC)) profiles have been identified as both biomarkers for disease prediction and as hallmarks of dysregulated localized gene networks. Global and gene-specific epigenetic alterations in the 5mC profiles have shown widespread implications in the etiology of polycystic ovary syndrome (PCOS). However, there has been no study in PCOS that integrates the quantification of 5mC and 5hmC signatures alongside the expression levels of DNA methylating and demethylating enzymes as respective indicators of methylation and demethylation pathways. Having previously shown that the 5mC signatures are not substantially altered in PCOS, we assessed the global 5hmC levels in peripheral blood leukocytes and cumulus granulosa cells (CGCs) of 40 controls and 40 women with PCOS. This analysis revealed higher 5hmC levels in CGCs of PCOS women, indicating a more dominant demethylation pathway. Furthermore, we assessed the transcript and protein expression levels of DNA demethylating and methylating enzymes, i.e. ten-eleven translocation methylcytosine dioxygenases (TET1, TET2, TET3) and DNA methyltransferases (DNMT1, DNMT3A and DNMT3B), respectively, in CGCs. The relative transcript and protein expression levels of all three TETs were found to be higher in women with PCOS, and the TET mRNA expression profiles were positively correlated with 5hmC levels in CGCs. Also, all three DNMT genes showed altered transcript expression in PCOS, although only the downregulated DNMT3A transcript was correlated with decreasing 5mC levels. At the protein level, the expression of DNMT1 (maintenance methylation enzyme) was higher, while that of DNMT3A (de novo methylation enzyme) was found to be lower in PCOS compared to controls. Overall, these results indicate that DNA methylation changes in CGCs of PCOS women may arise partly due to intrinsic alterations in the transcriptional regulation of TETs and DNMT3A.

Chromosomal Analysis of Pre-implantation Embryos: Its Place in Current IVF Practice.

BACKGROUND: The intersection of ART and molecular genetic science is fast growing. It is now possible to utilize the advances in molecular genetics for clinical application to detect chromosomal aberrations in preimplanting embryos.As molecular genetic techniques have improved, it is now possible to test the complete characterization of human genome variation with reasonable accuracy. In this article, we have tried to summarize the common current indications of chromosomal analysis of preimplanting embryos in couples having various chromosomal dominant or chromosomal recessive heritable disorders leading to the birth of a new born baby with chromosomal aberrations or leading to repeated miscarriage. CONCLUSION: The currently available techniques of embryo biopsy have their advantages and shortcomings. Today, preimplantation genetic testing to diagnose a euploid embryo is widely used in clinical practice in couples undergoing IVF ET treatment. By eliminating the transfer of aneuploid embryos, the pregnancy rate improves per embryo transfer and it shortens the time of conception from the start of IVF treatment. We have also discussed the current scenario of the place of PGT-A for routine use in IVF treatment procedure in view of the possible risk of losing euploid embryos due to the shortcoming of the embryo biopsy procedure.

DNA methylome profiling of granulosa cells reveals altered methylation in genes regulating vital ovarian functions in polycystic ovary syndrome.

BACKGROUND: Women with polycystic ovary syndrome (PCOS) manifest a host of ovarian defects like impaired folliculogenesis, anovulation, and poor oocyte quality, which grossly affect their reproductive health. Addressing the putative epigenetic anomalies that tightly regulate these events is of foremost importance in this disorder. We therefore aimed to carry out DNA methylome profiling of cumulus granulosa cells and assess the methylation and transcript expression profiles of a few differentially methylated genes contributing to ovarian defects in PCOS. A total of 20 controls and 20 women with PCOS were selected from a larger cohort of women undergoing IVF, after carefully screening their sera and follicular fluids for hormonal and biochemical parameters. DNA extracted from cumulus granulosa cells of three women each, from control and PCOS groups was subjected to high-throughput, next generation bisulfite sequencing, using the Illumina HiSeq 2500® platform. Remaining samples were used for the validation of methylation status of some identified genes by pyrosequencing, and the transcript expression profiles of these genes were assessed by quantitative real-time PCR. RESULTS: In all, 6486 CpG sites representing 3840 genes associated with Wnt signaling, G protein receptor, endothelin/integrin signaling, angiogenesis, chemokine/cytokine-mediated inflammation, etc., showed differential methylation in PCOS. Hypomethylation was noted in 2977 CpGs representing 2063 genes while 2509 CpGs within 1777 genes showed hypermethylation. Methylation differences were also noted in noncoding RNAs regulating several ovarian functions that are dysregulated in PCOS. Few differentially methylated genes such as aldo-keto reductase family 1 member C3, calcium-sensing receptor, resistin, mastermind-like domain 1, growth hormone-releasing hormone receptor and tumor necrosis factor, which predominantly contribute to hyperandrogenism, premature luteolysis, and oocyte development defects, were explored as novel epigenetic candidates in mediating ovarian dysfunction. Methylation profiles of these genes matched with our NGS findings, and their transcript expression patterns correlated with the gene hypo- or hypermethylation status. CONCLUSION: Our findings suggest that the epigenetic dysregulation of genes involved in important processes associated with follicular development may contribute to ovarian defects observed in women with PCOS.

Examining the Use of Magnesium Sulfate to Treat Pregnant Women with Preeclampsia and Eclampsia: Results of a Program Assessment of Emergency Obstetric Care (EmOC) Training in India.

BACKGROUND: The aim of this study is to examine rates of magnesium sulfate utilization by emergency obstetric care trainees to treat preeclampsia-eclampsia in India. Secondarily, structural barriers are identified which limit the use of magnesium sulfate, highlighting limitations of emergency obstetric care training, which is a commonly implemented intervention in resource-poor settings. METHODS: Trainees' curriculum specified magnesium sulfate treatment for eclampsia and severe preeclampsia. Case records were analyzed for preeclampsia-eclampsia diagnosis, magnesium sulfate utilization, delivery route, and maternal and neonatal outcomes from 13,238 reported deliveries between 2006 and 2012 across 75 district hospitals in 12 Indian states. RESULTS: Of 1320 cases of preeclampsia-eclampsia, 322 (24.4%) had eclampsia. Magnesium sulfate was given to 12.9% of preeclamptic and 54.3% of eclamptic women, with lower usage rates in rural communities. Among the 1308 women with preeclampsia-eclampsia, only 24 deaths occurred (1.8%). In contrast, among the 17,179 women without preeclampsia-eclampsia, there were 95 reported deaths (0.6%). Both maternal mortality ratios were found to be much higher than the Millennium Development Goal target of 0.15%. Magnesium sulfate administration was associated with a higher death rate in preeclamptic but not eclamptic women, representing possible confounding by severity. CONCLUSION: To optimize resources spent on emergency obstetric care training, the consistent availability of magnesium sulfate should be improved in India. Increasing drug availability, implementing clinical guidelines around its administration, and training health-care providers on the identification and treatment of preeclampsia-eclampsia could lead to notable improvements in maternal and infant mortality.

LINE1 CpG-DNA Hypomethylation in Granulosa Cells and Blood Leukocytes Is Associated With PCOS and Related Traits.

Context: Altered global DNA methylation is indicative of epigenomic instability concerning chronic diseases. Investigating its incidence and association with polycystic ovary syndrome (PCOS) is essential to understand the etiopathogenesis of this disorder. Objectives: We assessed global DNA methylation differences in peripheral blood leukocytes (PBLs) and cumulus granulosa cells (CGCs) of controls and women with PCOS; and their association with PCOS and its traits. Design, Setting, Participants, Main Outcome Measure: This study included a total of 102 controls and women with PCOS. Forty-one women undergoing controlled ovarian hyperstimulation (COH) and 61 women not undergoing COH were recruited from in vitro fertilization (IVF) and infertility clinics. DNA methylation was measured by ELISA for 5'-methyl-cytosine content and bisulfite sequencing of 5'-untranslated region (5'-UTR) of long interspersed nucleotide element-1 (LINE1/L1). Results: Total 5'-methyl-cytosine and L1 methylation levels in PBLs and CGCs were similar between controls and women with PCOS. Methylation assessed at CpG sites of L1 5'-UTR revealed a single CpG-site (CpG-4) to be consistently hypomethylated in PBLs of both PCOS groups and CGCs of stimulated PCOS group. In unstimulated women, hypomethylation at CpG-4 was strongly associated with PCOS susceptibility, whereas in stimulated group it showed strong associations with PCOS and its hormonal traits. Furthermore, CGCs demonstrated consistent global and CpG-DNA hypomethylation relative to PBLs, irrespective of normal or disease states. Conclusion: Our study revealed strong association of single hypomethylated CpG-site with PCOS. Identification and characterization of more such methyl-CpG signatures in repetitive elements in larger study populations would provide valuable epigenetic insights into PCOS.

Functional characterization of two naturally occurring mutations (Val514Ala and Ala575Val) in follicle-stimulating hormone receptor.

CONTEXT: Inactivating mutations have been reported in subjects with primary/secondary amenorrhea, whereas activating mutations are rare and seen only in women with ovarian hyperstimulation syndrome (OHSS). In the present study, we describe the functional characterization of the two mutations Val(514)Ala (novel mutation) and Ala(575)Val in FSH receptor (FSHR) identified in women with OHSS developed during in vitro fertilization and primary amenorrhea, respectively. OBJECTIVE: The objective of the investigation was to study the effect of mutations (514 and 575) on FSHR activity by in vitro functional studies. SETTING: The study was conducted at an academic research institute and a private in vitro fertilization clinic. METHODS: The site-directed mutagenesis was carried out to generate the mutations at position 514 and 575 in pSG5-FSHR construct. Stable cell lines expressing wild type or each of the mutant receptor were generated using Chinese hamster ovary cells. Functional characteristics of both the mutant receptors were assessed by a radioreceptor assay and a cAMP assay. RESULTS: The mutant receptor 514 showed increased cell surface expression as compared with the wild-type (WT) receptor. Although the hormone binding characteristics were similar to the WT receptor, its signaling activity was distinctly higher at lower dose of FSH as monitored by a cAMP assay. On the other hand, the mutant receptor 575 showed lower cell surface expression and higher internalized hormone receptor complex. Additionally, a dose-dependent increase in the cAMP accumulation was not observed in the case of this mutant as compared with WT. CONCLUSION: OHSS and primary amenorrhea observed in the two affected women, respectively, could be attributed to the functional characteristics of respective mutant FSHR.

Association of allelic combinations of FSHR gene polymorphisms with ovarian response.

During an IVF protocol, exogenous FSH is administered to women for ovulation induction. The ovarian response to gonadotrophin stimulation is variable and unpredictable in these women. The FSHR is the most studied gene in relation to ovarian response. The association of a FSHR gene polymorphism at position 680 (p.Asn680Ser) with ovarian response has been well documented. Recently, a polymorphism at position -29 in the 5'-untranslated region of FSHR (g.-29G>A) has been reported to be associated with poor ovarian response and reduced FSHR expression. The present study evaluated the combined effect of the polymorphisms at positions -29 and 680 of FSHR with type of ovarian response and receptor expression. The two FSHR gene polymorphisms together formed four discrete haplotypes and nine allelic combinations. Various clinical parameters revealed that 75% of the subjects with A/A-Asn/Asn genotype were poor ovarian responders (odds ratio 7.92; P=0.009). The relative FSHR mRNA expression in granulosa cells indicated that subjects with A/A-Asn/Asn genotype express significantly lower level of FSHR as compared to the subjects with G/G-Asn/Ser genotype (P=0.029). These results indicate that A/A-Asn/Asn genotype could be used as a potential marker to predict poor ovarian response. The action of FSH is mediated by its receptor (FSHR) present on the granulosa cells in the ovary. Any alterations in the hormone or its receptor are likely to disrupt its normal function, thus causing infertility. Several alterations (mutations/polymorphisms) of the FSHR gene have been reported in women with primary or secondary amenorrhoea. It has also been reported that FSHR gene polymorphisms are associated with variable ovarian response to FSH stimulation during IVF. Women may show poor or normal or hyperovarian response to FSH stimulation. It is well documented that the level of FSHR expression has a great effect on FSH action and is associated with ovarian response. In the present study, we screened normally menstruating women undergoing IVF due to tubal/male factor or unexplained infertility. We analysed two polymorphisms of FSHR, g-29G>A and p.Asn680Ser, in these women. In the subjects studied, 75% women with A/A-Asn/Asn genotype were observed to be poor ovarian responders to FSH stimulation. FSHR expression at the transcript level was observed to be significantly lower in women with A/A-Asn/Asn genotype as compared to women with G/G-Asn/Ser genotype. We also observed that women with A/A-Ser/Ser genotype were not present in the study population. These findings indicate the significance of A/A-Asn/Asn genotype as a predictive marker for poor ovarian response to FSH stimulation.

Endometrial epithelial cell modifications in response to embryonic signals in bonnet monkeys (Macaca radiata).

The present investigation reports embryo-induced modifications in the epithelial cells of the endometrium in a primate species. In vivo, epithelial cell response to the embryonic signals was assessed at the embryo attachment stage in the gestational uterus of bonnet monkeys (Macaca radiata) and in vitro response was investigated by treating human endometrial epithelial cell line (Ishikawa) with human embryo conditioned media (CM). Endometrial epithelial (EE) cells at the embryo attachment stage in bonnet monkeys revealed higher proliferation accompanied by significant up regulation (p < 0.05) in the expression of estrogen receptor (ER)α and down regulation (p < 0.05) in ERß expression. Further gestational EE cells showed higher (p < 0.001) expression of mucin-1, except in the embryo attachment site. Also, observed were significantly higher expression (p < 0.05) and altered cytoplasmic distribution of α(v) and ß(3) integrins, when compared to non-pregnant animals. In pregnant animals, the embryo attachment zone showed differential expression of immunoreactive integrins as compared to the non-attachment zone. This suggested the role of embryo secreted factors in modulation of the epithelial cell profile. In vitro studies partially supported this assumption. Significantly higher proliferation (p < 0.05), as well as increased expression of ERα, integrin ß(3) and mucin-1 (p < 0.05) were observed in Ishikawa cells, on stimulation with CM. Taken together, these results indicated the proliferation and modulation in the expression of estrogen receptors and cell adhesion molecules in the EE cells; at the embryo attachment stage in bonnet monkeys. Further it is likely that embryo secreted factors contribute to some of these modifications in EE cells. This report is the first account of discrete cellular events, which occur in the uterine epithelium, at the embryo attachment stage in a primate species.

Follicle-stimulating hormone receptor polymorphism (G-29A) is associated with altered level of receptor expression in Granulosa cells.

CONTEXT: Polymorphisms of the FSHR gene are associated with variable ovarian response to FSH stimulation in subjects undergoing in vitro fertilization (IVF) treatment. The type of ovarian response is correlated with the level of FSH receptor (FSHR) expression on granulosa cells. OBJECTIVE: We investigated whether the polymorphism at position -29 in the promoter of the FSHR gene may contribute in altered receptor expression. DESIGN AND PATIENTS: FSHR polymorphism at position -29 was studied in 100 subjects undergoing IVF treatment. Association of this polymorphism with level of FSHR expression was retrospectively analyzed. SETTING: The study was conducted at an academic research institute and private IVF clinic. METHODS: The genotype at position -29 of the FSHR gene was studied in IVF subjects by PCR-restriction fragment length polymorphism. Total RNA and protein was extracted from granulosa cells. The relative FSHR mRNA expression was carried out by real-time PCR. The receptor protein expression was evaluated by Western blot and confocal microscopy. RESULTS: The clinical and endocrinological parameters revealed that almost 72% of subjects with the AA genotype at position -29 of FSHR gene were poor ovarian responders (odds ratio 8.63, 95% confidential interval 1.84-45.79; P = 0.001). The lower cleavage intensity predicted by in silico analysis for A allele as compared with the G allele suggest the difference in the DNA-protein binding affinity. The relative expression of FSHR at mRNA and protein level was significantly reduced in subjects with AA genotype as compared with the GG genotype. CONCLUSION: Poor ovarian response observed in subjects with the AA genotype at position -29 of the FSHR gene is due to reduced receptor expression.

Selection of viable spermatozoa from testicular biopsies: a comparative study between pentoxifylline and hypoosmotic swelling test.

OBJECTIVE: To compare fertilization, cleavage, and pregnancy outcome using pentoxifylline and a hypoosmotic swelling (HOS) test to select viable spermatozoa from testicular biopsy specimens. DESIGN: Open, comparative, prospective study. SETTING: G.S. Medical College and Fertility clinic, Mumbai, India. PATIENT(S): A total of 50 couples enrolled for infertility treatment having a male factor indication of nonobstructive azoospermia. INTERVENTION(S): Assessment of viable spermatozoa using pentoxifylline and using an HOS test from a population of nonmotile spermatozoa obtained from testicular biopsies. MAIN OUTCOME MEASURE(S): Comparison of fertilization, cleavage, and clinical pregnancy rates using viable sperms recovered using pentoxifylline and an HOS test. RESULT(S): Viable spermatozoa were obtained in both the study groups. Significantly higher fertilization rates (pentoxifylline 62.05% vs. HOS 41.07%) and clinical pregnancy rates (pentoxifylline 32% vs. HOS 16%) were observed. There was no significant difference in cleavage rates among both groups. CONCLUSION(S): We found that obtaining viable spermatozoa using pentoxifylline was more effective in terms of fertilization and pregnancies than obtaining it through an HOS test.

Poor ovarian response to gonadotrophin stimulation is associated with FSH receptor polymorphism.

Similarities in the phenotype observed in women with FSH receptor mutation and in FSH receptor knockout mice have clearly established a critical role of this protein in normal gonadal function. Two common single nucleotide polymorphisms in the exonic region of the FSH receptor gene have been shown to be associated with altered ovarian response in subjects undergoing gonadotrophin treatment. Recent in-vitro studies have shown that the A allele at the -29 position in the 5 untranslated region of the FSH receptor gene is associated with impaired transcriptional activity. Differential expression of the FSH receptor and its function may be one of the factors responsible for altered ovarian response. These observations prompted a study of the association between FSH receptor genotype at the -29 position and ovarian response in women undergoing gonadotrophin treatment. Analysis of the data revealed that the subjects with AA genotype at the -29 position required the highest amount of exogenous FSH for ovulation induction, and oestradiol concentrations before the day of human chorionic gonadotrophin administration were significantly lower (P = 0.015) compared with the GA genotype. The number of pre-ovulatory follicles and retrieved oocytes were lowest in the subjects with AA genotype. These results indicate that the AA genotype at position -29 may be associated with the poor ovarian response.

Follicle-stimulating hormone receptor polymorphism (Thr307Ala) is associated with variable ovarian response and ovarian hyperstimulation syndrome in Indian women.

OBJECTIVE: To evaluate the association of FSH receptor polymorphism and ovarian response. DESIGN: Retrospective study. SETTING: Academic research institute and private IVF clinic. PATIENT(S): Fifty women were recruited in an assisted reproductive technology program (ART) and 100 proven fertile women of Indian origin. INTERVENTION(S): Polymerase chain reaction, restriction fragment-length polymorphism for detecting polymorphisms at T(307)A and N(680)S. MAIN OUTCOME MEASURE(S): FSH receptor polymorphisms, serum FSH, and estradiol levels, amount of FSH administered, occurrence of ovarian hyperstimulation syndrome (OHSS). RESULT(S): Prevalence of polymorphism at 307 position was 24%, 53%, and 23% in controls and 24%, 62%, and 14% in ART subjects for TT, TA, and AA, respectively, whereas at position 680, it was 31%, 56%, and 13% in controls and 42%, 46%, and 12% in ART subjects for NN, NS, and SS, respectively. The amount of FSH required for ovulation induction was low in AA compared with TT and TA subjects; the estradiol levels before and on the day of hCG administration were significantly higher. Eighty-five percent of the subjects with AA genotype developed OHSS. CONCLUSION(S): In Indian women, the subjects with AA genotype require low amounts of FSH for ovarian stimulation and have an increased risk of developing OHSS.

Derivation and characterization of two genetically unique human embryonic stem cell lines on in-house-derived human feeders.

This study describes the successful derivation of two human embryonic stem (hES) cell lines using 53 frozen and 18 fresh "slow-growing" surplus embryos, obtained from collaborating in vitro fertilization clinics, on in-house-derived human feeder layers. The cell lines have been derived by whole embryo culture followed by further expansion of manually dissected inner cell mass from the surrounding trophoectodermal cells. Immunocytochemical localization of cell surface markers like SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81, staining for alkaline phosphatase and reverse transcriptase polymerase chain reaction (RT-PCR) analysis of pluripotency state markers viz. Oct-4, TERT, Nanog, Rex1, and Sox2 indicate that both cell lines possess typical features of embryonic stem cells. Both cell lines exhibit normal female karyotype after 40 passages in culture. Pluripotent nature of the cell lines was confirmed both in vitro and in vivo. Embryoid bodies, formed in suspension culture, express markers for all three lineages as indicated by RT-PCR analysis for SOX 1 (ectoderm), HAND 1 (mesoderm), AFP (endoderm), and CDX2 (trophoectoderm). Teratoma formed in vivo in severe combined immunodeficient mice revealed cells of all the three embryonic germ layers. Comparison of the STR and human leukocyte antigen profiles of these cell lines with the existing human ES cell lines indicate that they are genetically distinct. The addition of our hES cell lines contributes usefully to the globally restricted repertoire of human ES cell lines.