Abdallah Mansur1, Ariel Israel1,2, Catherine M H Combelles3, Michal Adir1, Catherine Racowsky4, Russ Hauser5, Andrea A Baccarelli6, Ronit Machtinger7. 1. Infertility and IVF Unit, Department of Obstetrics and Gynecology, Chaim Sheba Medical Center and Sackler School of Medicine, Tel-Aviv University, Tel Hashomer 52561, Israel. 2. Department of Family Medicine, Clalit Health Services, Jerusalem, Israel. 3. Biology Department, Middlebury College, Middlebury, VT, USA. 4. Department of Obstetrics, Gynecology, and Reproductive Biology, Brigham & Women's Hospital and Harvard Medical School, Boston, MA, USA. 5. Departments of Environmental Health and Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MA, USA. 6. Department of Environmental Health Sciences, Columbia University Mailman School of Public Health, New York, NY, USA. 7. Infertility and IVF Unit, Department of Obstetrics and Gynecology, Chaim Sheba Medical Center and Sackler School of Medicine, Tel-Aviv University, Tel Hashomer 52561, Israel Ronit.Machtinger@sheba.health.gov.il.
Abstract
STUDY QUESTION: Does bisphenol-A (BPA) affect gene expression in human membrana granulosa cells (MGC)? SUMMARY ANSWER: In vitro, short exposure to supra-physiological concentrations of BPA alters human MGC gene expression. WHAT IS KNOWN ALREADY: Exposure to BPA may interfere with reproductive endocrine signaling. In vitro studies, mostly in animal models, have shown an inverse correlation between exposure to BPA and follicular growth, meiosis, and steroid hormone production in granulosa cells. STUDY DESIGN, SIZE, DURATION: Primary cultures of MGC obtained from 24 patients undergoing IVF (for PGD, male factor infertility or unexplained infertility) were exposed to various concentrations of BPA (0, 0.02, 0.2, 2 or 20 µg/ml) for 48 h. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study was conducted in a university-affiliated hospital. Microarray analysis was used to identify genes exhibiting expression changes following BPA exposure. Genes significantly altered were identified based on changes greater than 2-fold relative to the control group (not treated by BPA) and a Student's t-test P-value <0.05. Statistical significance was adjusted for multiple comparisons using the Benjamini-Hochberg method. Alterations in the expression of genes that are involved in the enriched functional annotations altered by BPA at the concentration of 20 µg/ml were confirmed by real-time PCR. MAIN RESULTS AND THE ROLE OF CHANCE: A distinct pattern of gene expression was observed in primary cultures of MGC exposed to the highest BPA concentration compared with untreated cells. We identified 652 genes that exhibited at least 2-fold differences in expression after BPA exposure (all P < 0.05 versus untreated). These genes were significantly enriched for annotations related to cell cycle progression, segregation of chromosomes, steroid metabolism, apoptosis, lipid synthesis, oocyte maturation and chromosomal alignment. No significant changes in gene expression were found at the lower doses of BPA most relevant to human exposure. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Human exposure to BPA in vivo occurs over long periods of time. In this in vitro model, cells were exposed to the chemical for 48 h only. Thus, the effects of BPA on the human follicle might be underestimated. WIDER IMPLICATIONS OF THE FINDINGS: As BPA exposure is ubiquitous, understanding the effects of the chemical on the ovary, specifically in women of reproductive age, has public health significance. The clinical evidence to date points to an association between BPA exposure and impaired IVF outcome, although not all studies have shown negative effects. Our study adds valuable mechanistic information showing that exposure to BPA alters granulosa cell gene expression at high and supra-physiological doses. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grant number 1936/12 from the ISF. The authors have nothing to disclose.
STUDY QUESTION: Does bisphenol-A (BPA) affect gene expression in human membrana granulosa cells (MGC)? SUMMARY ANSWER: In vitro, short exposure to supra-physiological concentrations of BPA alters human MGC gene expression. WHAT IS KNOWN ALREADY: Exposure to BPA may interfere with reproductive endocrine signaling. In vitro studies, mostly in animal models, have shown an inverse correlation between exposure to BPA and follicular growth, meiosis, and steroid hormone production in granulosa cells. STUDY DESIGN, SIZE, DURATION: Primary cultures of MGC obtained from 24 patients undergoing IVF (for PGD, male factor infertility or unexplained infertility) were exposed to various concentrations of BPA (0, 0.02, 0.2, 2 or 20 µg/ml) for 48 h. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study was conducted in a university-affiliated hospital. Microarray analysis was used to identify genes exhibiting expression changes following BPA exposure. Genes significantly altered were identified based on changes greater than 2-fold relative to the control group (not treated by BPA) and a Student's t-test P-value <0.05. Statistical significance was adjusted for multiple comparisons using the Benjamini-Hochberg method. Alterations in the expression of genes that are involved in the enriched functional annotations altered by BPA at the concentration of 20 µg/ml were confirmed by real-time PCR. MAIN RESULTS AND THE ROLE OF CHANCE: A distinct pattern of gene expression was observed in primary cultures of MGC exposed to the highest BPA concentration compared with untreated cells. We identified 652 genes that exhibited at least 2-fold differences in expression after BPA exposure (all P < 0.05 versus untreated). These genes were significantly enriched for annotations related to cell cycle progression, segregation of chromosomes, steroid metabolism, apoptosis, lipid synthesis, oocyte maturation and chromosomal alignment. No significant changes in gene expression were found at the lower doses of BPA most relevant to human exposure. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Human exposure to BPA in vivo occurs over long periods of time. In this in vitro model, cells were exposed to the chemical for 48 h only. Thus, the effects of BPA on the human follicle might be underestimated. WIDER IMPLICATIONS OF THE FINDINGS: As BPA exposure is ubiquitous, understanding the effects of the chemical on the ovary, specifically in women of reproductive age, has public health significance. The clinical evidence to date points to an association between BPA exposure and impaired IVF outcome, although not all studies have shown negative effects. Our study adds valuable mechanistic information showing that exposure to BPA alters granulosa cell gene expression at high and supra-physiological doses. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grant number 1936/12 from the ISF. The authors have nothing to disclose.
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