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Mande P.V.,National Health Research Institute | Parikh F.R.,Jaslok Hospital | Hinduja I.,INKUS IVF Clinic | Zaveri K.,INKUS IVF Clinic | And 3 more authors.
Reproductive BioMedicine Online | Year: 2011

Antibodies to multiple ovarian antigens have been proposed as markers of ovarian autoimmunity. The role of ovarian autoantibodies has been widely discussed in the pathophysiology of premature ovarian failure and unexplained infertility, but the autoantigens are yet to be identified. Three immunodominant ovarian autoantigens, α-actinin 4 (αACTN4), heat shock 70 protein 5 (HSPA5) and β-actin (ACTB), have been identified using anti-ovarian antibody-positive sera from women with idiopathic premature ovarian failure (n = 50) and women undergoing IVF (n = 695), using mass spectrometry. These autoantigens were subsequently validated using Western blot, immunohistochemistry and enzyme-linked immunosorbent assay. These autoantigens are localized to different components of the ovary such as the ooplasm of the oocyte, theca, granulosa, corpus luteum and zona pellucida. All the above antigens were found to be expressed in the ooplasm throughout follicular development. All the autoantigens are expressed specifically in the oocyte except αACTN4. The three autoantigens could contribute to the array of biomarkers to be used for developing specific and sensitive tests for diagnosis of women at risk of premature ovarian failure and IVF failure due to ovarian autoimmunity and could give an insight into the molecular mechanisms involved in the pathophysiology of these conditions. Anti-ovarian antibodies (AOA) have been reported in women with premature ovarian failure and unexplained infertility. Among women with infertility, those with evidence of ovarian autoimmunity appear to have poorer IVF-embryo transfer outcomes. Diagnosis of an autoimmune mechanism in these pathologies has relied for a long time on the detection of AOA. Little is known about the precise ovarian antigenic targets in terms of its molecular and cellular identities that are recognized by the antibodies and immune cells in autoimmune diseases of the ovary. In the present study, we observed that 31% of the total women recruited under an IVF-embryo transfer programme (group I) and 46% of women with premature ovarian failure (group II) tested positive for AOA. Three immunodominant ovarian autoantigens, namely non-muscle α-actinin 4, heat shock 70 protein 5 and cytoplasmic β-actin, were identified using mass spectrometry and validated and characterized using AOA-positive sera from women from both groups. Further investigation of the identified targets could give us an insight into the molecular mechanism involved in the pathophysiology of human ovarian autoimmunity. © 2011, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Ambekar A.S.,National Health Research Institute | Nirujogi R.S.,Institute of Bioinformatics | Nirujogi R.S.,Pondicherry University | Srikanth S.M.,Institute of Bioinformatics | And 10 more authors.
Journal of Proteomics | Year: 2013

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. © 2013 Elsevier B.V.

Ambekar A.S.,National Health Research Institute | Kelkar D.S.,Institute of Bioinformatics | Pinto S.M.,Institute of Bioinformatics | Pinto S.M.,Manipal University India | And 8 more authors.
Journal of Clinical Endocrinology and Metabolism | Year: 2015

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 isobarictags 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 lymphaticvessel endothelial hyaluronan receptor 1 werefound 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. Copyright © 2015 by the Endocrine Society.

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