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.

Compromised Cumulus-Oocyte Complex Matrix Organization and Expansion in Women with PCOS.

The cumulus-oocyte complex (COC) matrix plays a critical role in the ovulation and fertilization process and a major predictor of oocyte quality. Proteomics studies of follicular fluid showed differential expression of COC matrix proteins in women with polycystic ovary syndrome (PCOS), indicating altered COC matrix in these women. In the present study, we aimed to understand COC matrix gene induction in humans and its probable dysfunction in women with PCOS. Animal studies have shown that amphiregulin (AREG) and growth differentiation factor-9 (GDF-9) are important in the induction of COC matrix genes which are involved in cumulus expansion. The effects of AREG and GDF-9 on expression of tumor necrosis factor alpha induced protein 6 (TNFAIP6) and hyaluronan synthase 2 (HAS2) on human cumulus granulosa cells (CGCs) and murine COC expansion were evaluated. Further time-dependent effects of growth factor supplementation on these gene expressions in CGCs from PCOS and control women were compared. Follicular fluid from PCOS showed reduced COC matrix expansion capacity, using murine COCs. Expression of COC matrix genes TNFAIP6 and HAS2 were significantly reduced in CGCs of PCOS. Treatment of CGCs with AREG and GDF-9 together induced expression of both these genes in controls and could only restore HAS2 but not TNFAIP6 expression in PCOS. Our results suggest that the reduced potential of follicular fluid to support COC expansion, altered expression of structural constituents, and intrinsic defects in granulosa cells of women with PCOS may contribute to the aberrant COC organization and expansion in PCOS, thus affecting fertilization.

Genetic variation in exon 17 of INSR is associated with insulin resistance and hyperandrogenemia among lean Indian women with polycystic ovary syndrome.

OBJECTIVE: Polycystic ovary syndrome (PCOS) is a multigenic disorder, and insulin resistance is one of its hallmark features. Polymorphisms in exon 17 of insulin receptor (INSR) gene are reported to be associated with PCOS. We investigated this association in Indian women and its putative relationship with PCOS associated traits, which has not been explored so far. METHODS: In this case control study, the polymorphisms were investigated by direct sequencing in 180 women with PCOS and 144 age matched controls. Clinical, anthropometric, biochemical, and hormonal parameters were also estimated. RESULTS: The silent C/T polymorphism at His1058 in exon 17 of INSR was found to be present in our study population. The polymorphic genotype (CT+TT) was significantly associated with PCOS in lean women (chi(2)=8.493, df=1, P=0.004). It showed association with higher fasting insulin levels (P=0.02), homeostasis model assessment of insulin resistance (P=0.005), free androgen index (P=0.03), and lower quantitative insulin sensitivity check index (P=0.004) in lean PCOS women. No other novel or known polymorphism was identified in exon 17 in this cohort. CONCLUSIONS: The study shows significant association of C/T polymorphism at His1058 of INSR with PCOS in the lean rather than obese Indian women. Its association with indices of insulin resistance and hyperandrogenemia is also seen in the same group. The findings strengthen the concept that pathogenesis of PCOS is different in lean and obese women.

Granulosa cell apoptosis induced by a novel FSH binding inhibitory peptide from human ovarian follicular fluid.

Pituitary gonadotropins, follicle-stimulating hormone and luteinizing hormone, are the key regulators of ovarian folliculogenesis; these are known to be directly or indirectly modulated by many intraovarian factors. Our group has identified and studied one such novel peptide from human ovarian follicular fluid. Its partial N-terminal eight amino acid sequence has been deduced, referred to as octapeptide (OP). OP induces follicular atresia in mice and interferes with normal ovarian function in non-human primates, this action being similar to the native peptide. Thus, in this study, an attempt has been made to elucidate the mechanism of action of the synthetic OP by studying the pathway of follicular atresia in mouse ovary. Changes in granulosa cells were studied using various apoptotic markers by flow cytometry and immunohistochemistry. An increase in apoptotic cell population in atretic- and peptide-treated groups was observed compared with normal controls. Interestingly, both these groups exhibited differences in the apoptotic pathway. Results showed that the mitochondrial pathway was predominant in the atretic group, whereas the Fas-FasL pathway was predominant in the peptide-treated groups. The ultrastructural study also showed apoptotic changes in the OP-treated and atretic groups; the pattern of apoptosis differed at the subcellular level.

Production of murine monoclonal antibody to fetal hemoglobin.

Fetal hemoglobin (Hb F) is a major hemoglobin (Hb) component at birth. Hb F levels are markedly elevated in a number of inherited and acquired disorders. Measurement of Hb F levels is usually carried out by alkali denaturation which is not very accurate for low and high values. An accurate estimation of Hb F, and also of F cells, is desired in many hematological disorders like sickle cell disease, in monitoring the efficacy of hydroxyurea (HU) therapy, to assess feto-maternal hemorrhage (FMH) during pregnancy and in the postpartum period. We have raised a murine monoclonal antibody to human Hb F, that accurately measures the number of F cells by flow cytometry. The antibody was found to be potent and specific for F cells.