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Bisphenol A (BPA) is a widely used chemical that has been detected in follicular fluid and associated with adverse reproductive effects. Granulosa cells have an important role in follicular growth and oocyte maturation, however, little is known about the biological mechanisms of BPA toxicity on human granulosa cells. In this study, we exposed primary granulosa cells to different concentrations of BPA (0, 20, 200, 2000, and 20 000 ng/ml) and used quantitative polymerase chain reaction to measure the expression levels of miRNAs enriched in extracellular vesicles (EV-enriched miRNAs), and cellular levels of selected target genes of differentially expressed EV-enriched miRNAs. We found that exposure to 20 000 ng/ml BPA was associated with decreased levels of EV-miR-27b-3p (FC = 0.58, p = .04) and increased levels of its biologically relevant target genes FADD (FC = 1.22, p = .01), IGF1 (FC = 1.59, p = .06), and PPARG (FC = 1.73, p = .001) as compared with the control. In addition, we observed that under the same exposure conditions, the expression levels of miR-27b-3p in granulosa cells were also downregulated (FC = 0.65, p = .03) as compared with the control. Our findings suggest that both cellular and extracellular changes in gene expression may mediate BPA toxicity in granulosa cells., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Phenols and phthalates are potential endocrine disrupting chemicals (EDCs) that are associated with adverse health outcomes. These EDCs dysregulate a number of biomolecules and pathways, including microRNAs. MicroRNAs can be carried in transport systems called extracellular vesicles (EVs) that are present in most biofluids. EVs in the follicular fluid, which fills the ovarian follicle and influences oocyte developmental competency, carry microRNAs (EV-miRNAs) that have been associated with In Vitro Fertilization (IVF) outcomes. However, it remains unclear whether EDCs affect EV-miRNAs in follicular fluid., Objectives: This study sought to determine whether urinary concentrations of phenols and phthalates biomarkers are associated with EV-miRNAs expression in follicular fluid collected from women undergoing IVF treatment., Methods: This cross-sectional study included 130 women recruited between January 2014 and August 2016 in a tertiary university-affiliated hospital. Participants provided urine samples during ovarian stimulation and on the day of oocyte retrieval. We assessed urinary concentrations of five phenols, eight phthalate metabolites, and one phthalate alternative metabolite. EV-miRNAs were isolated from follicular fluid and their expression profiles were measured using the TaqMan Open Array® Human microRNA panel. We fitted multivariable linear regression models and principal component analysis to examine associations between individual and molar sums of exposure biomarkers and EV-miRNAs., Results: Of 754 miRNAs tested, we detected 133 EV-miRNAs in the microRNA array which expressed in at least 50% of the follicular fluid samples. After adjusting for multiple testing, we identified eight EV-miRNAs associated with individual phenols and phthalate metabolites, as well as molar ΣDEHP that met a q < 0.10 false-discovery rate (FDR) threshold. Hsa-miR-125b, hsa-miR-106b, hsa-miR-374a, and hsa-miR15b was associated with mono(2-ethylhexyl) phthalate concentrations, hsa-let-7c with concentrations mono-2-ethyl-5-hydroxyhexyl phthalate (MEHHP), mono-2-ethyl-5-oxohexyl phthalate (MEOHP), mono-2-ethyl-5-carboxypentyl phthalate (MECPP), and the sum of metabolites of di(2-ethylhexyl) phthalate, hsa-miR-24 with mono-n-butyl phthalate concentrations, hsa-miR-19a with cyclohexane-1,2-dicarboxylic acid monohydroxy isononyl ester (MHiNCH), and hsa-miR-375 with ethyl paraben concentrations. Using Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, gene targets and pathways of these EV-miRNAs were predicted in silico and 17 KEGG FDR-significant pathways related to follicular development and oocyte competence were identified., Conclusions: Our results show that urinary concentrations of select phenol and phthalate metabolites are correlated with altered EV-miRNAs expression in follicular fluid. These findings may provide insight regarding the molecular mechanisms underlying adverse effects of phenol and phthalate exposure on female fertility., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Mounting evidence suggests possible adverse effects of intrauterine exposure to certain phenols and phthalates, two classes of endocrine disruptor chemicals, on the developing fetus, with consequences into later life. These findings have contributed to the replacement of some chemicals, such as di‑2‑ethylhexyl phthalate (DEHP) and bisphenol A (BPA), in consumer products. For the current study we quantified urinary concentrations of biomarkers of exposure among 50 pregnant women in Israel to several phthalates, bisphenols and personal care product chemicals, as well as DEHP and BPA alternatives. We detected 14 of the 31 biomarkers in more than 90% of the women. We detected biomarkers of 1,2‑cyclohexane dicarboxylic acid, diisononyl ester (DINCH), bisphenol S, and bisphenol F not as frequently (27-56%). This study is the first to evaluate exposure to triclosan, bisphenols, parabens, and phthalates and BPA alternatives among Israeli pregnant women., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Bisphenol A (BPA) is detectable in follicular fluid. However, the effect of BPA exposure on human cumulus cells (CC) that surround the oocyte and are crucial for oocyte competence has been largely unexplored. We exposed primary cultures of CC to increasing concentrations of BPA [0,0.002, 0.02 and 20μg/mL] and tested the effects of BPA on the expression of genes associated with apoptosis using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR); we also assessed the effect of BPA on apoptosis by staining with anti-caspase 3. Exposure to 20μg/mL BPA led to significantly decreased expression of CDC20, BUB1B and HAS2 (p<0.03), increased expression of TRIB3 and LUM (p≤0.005), and increased frequency of cells positive for anti-CASP3 (p=0.03), compared to control. Our results imply that BPA may lead to ovarian toxicity by increasing CC apoptosis and provide an important molecular mechanism for the effect of BPA on human CC in vitro., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Study Question: Does Bisphenol A (BPA) impair steroid hormone production in human luteinized granulosa cells in vitro?, Summary Answer: At supra-physiological concentrations, BPA alters progesterone and estradiol synthesis in vitro and significantly reduces the mRNA and protein expression levels of three genes encoding steroidogenesis enzymes., What Is Known Already: In IVF patients, the effects of BPA exposure on cycle outcome are controversial. Previous animal studies have shown that granulosa cell steroid hormone synthesis is compromised after BPA exposure, but their findings have been difficult to replicate in humans due, in part, to the low availability of discarded biological material., Study Design, Size, Duration: Luteinized granulosa cells obtained from 44 fertile and infertile patients undergoing in vitro fertilization were cultured for 48 h with different concentrations of BPA (0, 0.2, 0.02, 2.0, 20 µg/ml)., Participants/materials, Setting, Methods: Culture medium and total RNA extracted from the luteinized granulosa cells were examined for estradiol and progesterone levels as well as mRNA and protein expression of steroidogenesis enzymes, using enzyme immunoassays, real-time PCR and western blots., Main Results and the Role of Chance: Treatment of granulosa cells with 2 or 20 µg/ml BPA for 48 h resulted in significantly lower progesterone biosynthesis (P < 0.005 and <0.001, respectively). Estradiol production was significantly altered only after incubation with 20 µg/ml of BPA (P < 0.001). These concentrations also significantly reduced the mRNA levels of 3β-hydroxysteroid dehydrogenase (3β-HSD), CYP11A1 and CYP19A1 without affecting StAR and 17β-hydroxysteroid dehydrogenase mRNA expression. Similarly, 3β-HSD, CYP11A1 and CYP19A1 protein levels were reduced after administration of 20 µg/ml BPA. Lower BPA concentrations similar to, and up to 100 times, the concentrations measured in human follicular fluid, serum and urine did not alter steroidogenesis in primary granulosa cell cultures., Limitations, Reasons for Caution: This was an in vitro study investigating the effects of acute exposure (48 h) of BPA on discarded material. As such, the model may not accurately reflect the effect of BPA on the physiological events of follicular steroid hormone synthesis in vivo., Wider Implications of the Findings: Our results show that in vitro exposure to BPA at low doses does not affect granulosa cells steroidogenesis. Combined with recent in vivo studies, these data can be reassuring but further studies are needed to assess the effects of chronic exposure to BPA on ovarian steroidogenesis., Study Funding and Competing Interests: This study was supported by grant number 1936/12 from the Israeli Science Foundation (ISF). The authors have no conflict of interest., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)