Srabani Mukherjee

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.

Proteomic analysis of human follicular fluid: a new perspective towards understanding folliculogenesis.

UNLABELLED Human follicular fluid is a complex body fluid that constitutes the microenvironment of developing follicles in the ovary. Follicular fluid contains a number of proteins that modulate oocyte maturation and ovulation. Information about the protein constituents of follicular fluid may provide a better understanding of ovarian physiology in addition to opening new avenues for investigating ovarian disorders. However, the composition of follicular fluid proteome remains poorly defined. In this study, we carried out SDS-PAGE, OFFGEL and SCX-based separation followed by LC-MS/MS analysis to characterize the proteome of human follicular fluid. We report high confidence identification of 480 proteins, of which 320 have not been described previously in the follicular fluid. The identified proteins belong to diverse functional categories including growth factor and hormones, receptor signaling, enzyme catalysis, defense/immunity and complement activity. Our dataset should serve as a resource for future studies aimed at developing biomarkers for monitoring oocyte and embryo quality, pregnancy outcomes and ovarian disorders. BIOLOGICAL SIGNIFICANCE Proteome analysis of human follicular fluid by multi-pronged approach of protein peptide fractionation revealed 480 proteins with high confidence. The identified protein may facilitate the understanding of folliculogenesis. This protein dataset should serve as a useful resource for development of biomarkers for oocyte quality, in vitro fertilization techniques and female infertility.

Efficient cryopreservation of testicular tissue: effect of age, sample state, and concentration of cryoprotectant

OBJECTIVE To study the effect of age and sample state on cryopreservation of testicular tissue, evaluate toxicity of commonly used cryoprotectants (CPs), and determine their optimal concentration for use. DESIGN Prospective experimental study. SETTING Academic research unit. PATIENT(S) Patients with prostate carcinoma undergoing orchidectomy. We also studied immature and adult male Holtzman rats. INTERVENTION(S) Toxicity of CPs before freezing, morphology, and relative viability after freezing were evaluated for rat testicular cell suspensions (CS) and tubular fragments (TUB). Relative viability of adult human testicular CS and TUB after thaw was evaluated. Human TUB were cultured after thaw for 48 hours in medium containing epidermal growth factor (EGF), and effects on viability, morphology, and gene expression were determined. MAIN OUTCOME MEASURE(S) Viability and ploidy were measured with flow cytometry, postthaw cryodamage of immature rat tissue was studied by transmission electron microscopy, cell proliferation and differentiation were evaluated by immunohistochemistry and by real-time polymerase chain reaction. RESULT(S) Immature testicular tissue was more susceptible to toxic assault by CP than adult tissue and displayed cell-specific sensitivity to CP, with glycerol, dimethyl sulfoxide and ethylene glycol being effective in protecting spermatid (1N), spermatogonia (2N) and spermatocyte (4N) populations respectively. Preservation as TUB may be preferred over CS and DMSO is an effective CP for immature and ethylene glycol for adult testicular tissue. CONCLUSION(S) Differential sensitivity of immature testicular tissue to CPs warrants judicious selection of CP on the basis of end application for prepubertal tissue.

A cross-sectional study of polycystic ovarian syndrome among adolescent and young girls in Mumbai, India

Introduction: Polycystic ovary disease is a common endocrine condition which is rapidly gaining epidemic proportions. No community based prevalence data is available for this syndrome in India. Materials and Methods: A cross-sectional community-based study was undertaken in a sampled census block of Mumbai to assess the prevalence of polycystic ovarian syndrome (PCOS) among 778 adolescents and young girls aged 15-24 years. Among them, 600 completed all clinical, ultrasonography (USG), and biochemical investigations. Results: The prevalence of PCOS among them was 22.5% by Rotterdam and 10.7% by Androgen Excess Society criteria. Nonobese comprised 71.8% of PCOS diagnosed by Rotterdam criteria. Mild PCOS (oligomenorrhea and polycystic ovaries on USG) was the most common phenotype (52.6%). History of oligomenorrhea had a positive predictive value of 93.3% and negative predictive value of 86.7% to detect a possible case of PCOS. Hyperinsulinemia (serum insulin >15 μlU/mL) was present among 19.2% of diagnosed PCOS cases. Obese girls with PCOS were more hirsute, hypertensive, and had significantly higher mean insulin and 2 h post 75 g glucose levels compared with nonobese PCOS. Conclusion: To our knowledge, this is the first urban community-based study diagnosing PCOS and phenotypes among adolescent and young girls in India. This study demonstrates that PCOS is an emerging disorder during adolescence and screening could provide opportunity to target the group for promoting healthy lifestyles and early interventions to prevent future morbidities.

Proteomics of follicular fluid from women with polycystic ovary syndrome suggests molecular defects in follicular development

CONTEXT Polycystic ovary syndrome (PCOS), a major cause of anovulatory infertility, is characterized by arrested follicular growth. Altered protein levels in the follicular fluid surrounding the ovum may reflect the molecular defects of folliculogenesis in these women. OBJECTIVE To identify differentially regulated proteins in PCOS by comparing the follicular fluid protein repertoire of PCOS with healthy women. METHODS The follicular fluid samples were collected from PCOS and normo-ovulatory women undergoing in vitro fertilization. Follicular fluid proteins were subjected to digestion using trypsin, and resultant peptides were labeled with isobaric tags for relative and absolute quantification reagents and analyzed by liquid chromatography tandem mass spectrometry. Differential abundance of selected proteins was confirmed by ELISA. RESULTS A total of 770 proteins were identified, of which 186 showed differential abundance between controls and women with PCOS. Proteins involved in various processes of follicular development including amphiregulin; heparan sulfate proteoglycan 2; tumor necrosis factor, α-induced protein 6; plasminogen; and lymphatic vessel endothelial hyaluronan receptor 1 were found to be deregulated in PCOS. We also identified a number of new proteins from follicular fluid, whose function in the ovary is not yet clearly established. These include suprabasin; S100 calcium binding protein A7; and helicase with zinc finger 2, transcriptional coactivator. CONCLUSIONS Proteins indispensable for follicular growth were found to be differentially expressed in follicular fluid of women with PCOS, which may in part explain the aberrant folliculogenesis observed in these women.

A comprehensive curated resource for follicle stimulating hormone signaling

BackgroundFollicle stimulating hormone (FSH) is an important hormone responsible for growth, maturation and function of the human reproductive system. FSH regulates the synthesis of steroid hormones such as estrogen and progesterone, proliferation and maturation of follicles in the ovary and spermatogenesis in the testes. FSH is a glycoprotein heterodimer that binds and acts through the FSH receptor, a G-protein coupled receptor. Although online pathway repositories provide information about G-protein coupled receptor mediated signal transduction, the signaling events initiated specifically by FSH are not cataloged in any public database in a detailed fashion.FindingsWe performed comprehensive curation of the published literature to identify the components of FSH signaling pathway and the molecular interactions that occur upon FSH receptor activation. Our effort yielded 64 reactions comprising 35 enzyme-substrate reactions, 11 molecular association events, 11 activation events and 7 protein translocation events that occur in response to FSH receptor activation. We also cataloged 265 genes, which were differentially expressed upon FSH stimulation in normal human reproductive tissues.ConclusionsWe anticipate that the information provided in this resource will provide better insights into the physiological role of FSH in reproductive biology, its signaling mediators and aid in further research in this area. The curated FSH pathway data is freely available through NetPath (http://www.netpath.org), a pathway resource developed previously by our group.

Aberrant expression of imprinted genes in post-implantation rat embryos.

AIM Imprinted genes are known regulators of embryo growth. Studies from our laboratory have demonstrated that treatment of adult male rats with tamoxifen increased post-implantation loss at around midgestation. Expression of insulin like growth factor 2 (Igf2), a paternally expressed imprinted gene was down-regulated in the resorbing embryos obtained at embryonic day 13. Hypomethylation of Igf2-H19 imprint control region was observed in the resorbing embryo sires and spermatozoa obtained from tamoxifen-treated rats thereby suggesting that errors in imprint acquisition during spermatogenesis can result in embryo loss. The present study aims at studying the expression of other imprinted genes, besides Igf2 in the embryos sired by tamoxifen-treated males. MAIN METHODS Gene expression profiles of resorbing versus normal embryos were assessed by microarrays. Real time quantitative RT-PCR for six imprinted genes and four genes involved in cell cycle was done to validate gene expression data. The affected pathways and functions were identified in the resorbing embryos and effect on cell cycle was confirmed by flow cytometry. KEY FINDINGS Aberrant expression of a number of imprinted genes was observed in the resorbing embryos when compared to the normal embryos at embryonic days 11 and 13. Down-regulation of Notch signaling, Wnt signaling and cell cycle pathway was observed in the resorbing embryos. SIGNIFICANCE The study suggests that exposure of male germ cells to tamoxifen during adulthood results in aberrant expression of imprinted genes and down-regulation of development associated pathways in the F(1) progeny thereby causing embryo loss.

Polymorphisms and haplotypes of insulin-like factor 3 gene are associated with risk of polycystic ovary syndrome in Indian women.

BACKGROUND Insulin-like factor 3 (INSL3), secreted by the ovarian theca cells is involved in androgen production, follicular growth and oocyte maturation. Both androgens and INSL3 levels are reported to be elevated in women with polycystic ovary syndrome (PCOS), indicating that INSL3 could contribute to PCOS etiology. This case-control association study explored the impact of INSL3 polymorphisms on PCOS susceptibility and its related traits. METHODS Genotyping of exonic polymorphisms of INSL3 was performed in controls (n=333) and PCOS (n=405) women. Phenotyping (clinical, biochemical and hormonal parameters) was carried out in 205 controls and 301 PCOS women. Genotype, haplotype and genotype-phenotype associations were determined using statistical tests. RESULTS Three polymorphisms in exon 1-rs2286663 (G/A), rs1047233 (A/G), and rs6523 (A/G), and one in exon 3-rs1003887 (G/A), were present in our study subjects. The frequencies of rs6523 and AGAG haplotype were significantly increased in PCOS women. The rs6523 polymorphism showed significant association with increased cholesterol and HDL-C levels in PCOS women while in controls with decreased FBS, Bio-T and FAI, and increased SHBG levels. Significant association of, rs1047233 polymorphism with improved androgen related parameters in controls, rs2286663 polymorphism with decreased QUICKI in PCOS and rs1003887 polymorphism with increased insulin levels and HOMA-IR in controls were observed. CONCLUSIONS The rs6523 polymorphism and AGAG haplotype of INSL3 showed significant association with increased risk of PCOS. Additionally, INSL3 polymorphisms influenced metabolic and hyperandrogenemia related parameters in both controls and PCOS women. This is the first study to suggest that INSL3 may be a genetic predisposition factor in PCOS pathophysiology.

An overview of the oxytocin-oxytocin receptor signaling network

Oxytocin, a nine amino acid long neuropeptide hormone, is synthesized in the hypothalamus and stored and released from the neural lobe of the pituitary gland. Although commonly known for its central role in the regulation of parturition and lactation, oxytocin signaling also plays a key role in modulating social behavior, evoking contentment, initiating maternal behavior, inducing trust, generosity and bonding in humans and animals. Oxytocin signaling can prove to be of great importance in therapeutics and drug targeting because of its diverse range of actions. However, a well annotated map of oxytocin signaling pathway is currently lacking in the publicly available pathway resources. Therefore, we systematically curated the available signaling information of oxytocin from published literature and collated the data to develop a more complete map. We cataloged 66 molecules belonging to oxytocin signaling pathway, which included 9 protein-protein interactions, 39 post-translational modifications, 14 protein translocation events and 22 activation/inhibition events. Further, Oxytocin signaling network data is made freely available to academic fraternity by integrating this into NetPath (http://www.netpath.org/), a freely available human signaling pathway resource developed previously by our group.

PON1 polymorphisms are associated with polycystic ovary syndrome susceptibility, related traits, and PON1 activity in Indian women with the syndrome

OBJECTIVE To investigate the association of paraoxonase 1 (PON1) polymorphisms (L55M and Q192R) with polycystic ovary syndrome (PCOS) susceptibility and its related traits in Indian women. DESIGN Case-control study. SETTING Academic research institute, infertility, and endocrinology clinics. PATIENT(S) Controls (n = 326), women with PCOS (n = 482). INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Genotypic and allelic frequency distribution, genotype-phenotype association, different PON1 activities (lactonase, arylesterase, and paraoxonase). RESULT(S) The genotypic and allelic frequency distributions of the L55M polymorphism were significantly different between lean controls and lean women with PCOS, and this polymorphism reduced the risk of PCOS development in lean but not in obese Indian women. Furthermore, this polymorphism was significantly associated with decreased 2-hour glucose, apolipoprotein B, free and bioavailable T, and free androgen index concurrent with increased sex hormone-binding globulin (SHBG) and FSH levels only in lean women with PCOS. However, Q192R polymorphism showed comparable genotypic frequency distribution between controls and women with PCOS. PON1 lactonase and arylesterase activities were significantly decreased in women with PCOS compared with controls. PON1 polymorphisms were shown to influence its activities. CONCLUSION(S) Our study showed that L55M, but not Q192R, polymorphism is significantly associated with reduced PCOS susceptibility only in lean women and also impacts glucose metabolism, lipid parameters, and hyperandrogenemia in them. Our study therefore suggests the possibility of differential genetic pathophysiology of PCOS between lean and obese women.