The Nmechanisms Of Bisphenol A And DNA Methylation Induced Hyperandrogenism In Polycystic Ovary Syndrome

PART ? The association between bisphenol-A and hyperandrogeneismObjective:To analyze the bisphenol-A(BPA)levels in the follicular fluid(FF)of PCOS patients and the correlations between BPA and androgen levels;to identify the phenotypes induced by BPA in Sprague-Dawley(SD)rats.Materials and methods:Infertile women with PCOS or with tubal blockage(control)undergoing in vitro fertilization(IVF)from Women Hospital,Zhejiang University were recruited for this study.We examined the BPA in the FF by HPLC-MS/MS.And the concentration of total testosterone(TT),free androgen index(FAI),sex hormone-binding globulin(SHBG),and androstenedione(A)in FF.We established a neonatally BPA exposed femle rats model.The estrous cycle,ovarian morphology and endocrine alterations were examined.Results:BPA levels were significantly higher in the FF of PCOS patients compared with the normal ovulating peers.The BPA levels in the FFs were positively correlated with the TT,FAI and A levels in all enrolled subjects,and,the assocication is stronger in the PCOS groups.BPA-induced rat model showed consistent phenotypes with PCOS patients:1)persistent estrous cycles;2)polycystic ovary;3)elvated androgen levels and the ratio of LH to FSH in serum.Conclusion:PCOS patients have higher concentrations of BPA in FF.The BPA levels in FF were tightly correlated with androgen levels,especially for the PCOS ones.BPA induces the phenotypes of PCOS in rats.PART ? Androgen receptor mediated BPA induced aromatase repression and abnormal androgen accumulationObjective:To explore the mechanisms in which BPA induces hyperandrogenism.Materials and methods:The expressions of aromatase were determined in granulosa cells(GCs)by quantitative Real-time PCR and western blot.The concentrations of testosterone(T),A,estradiol(E2)and estrone(E1)in the supernant of culture medium were determined by ELISA.GCs were pretreated with estrogen receptor(ER)/androgen receptor(AR)blocker,or AR siRNA,and then the expression of the aromatase detected by quantitative Real-time PCR.The transcriptional capacities of AR on the androgen response element(ARE)were illustrated by luciferase assay and the binding of AR induced by BPA on ARE were performed by chromatin immunoprecipitation(ChIP).Results:The expression and bioactivity of aromatase were repressed by BPA in a dose-dependent manner,and the repression was inhibited by either AR blocker-flutamide or AR knockdown.The transcriptional activity of AR elited by T was suppressed by BPA,and BPA interfered the capacity of T binding on AR and the precipitation induced by T on CYP19A1 promoter.Conclusion:BPA disrupts the normal process of the aromatization of androgens into estrogens on the transcriptional level,thus resulting in abnormal accumulation of androgen in FF and subsequent hyperandrogenism.PART ? Aberrant expression and DNA methylation of lipid metabolism genes in PCOS:a new insight into its pathogenesisObjective:To identify the differentially expressed genes(DEGs)in GCs from PCOS women with elevated BP A levels in the FFs and provide an epigenetic insight related to the pathogenesis of PCOS.Materials and methods:71 PCOS women and 50 women with normal ovulatory cycles(control)undergoing IVF from Women Hospital,Zhejiang University participated in the study.3 GCs from women with elevated BPA levels in the FFs and 3 GCs from control women were selected for RNA-Sequencing(RNA-Seq).RNA-Seq was performed using Illumina Hiseq 2000.The transcriptome results for selected genes were confirmed by real-time quantitative PCR.5-methycytosine analysis was employed to determine the global methylation level of genomic DNA and MassArray EpiTYPER quantitative DNA methylation analysis was introduced to determine the methylation level of and each CpG site or unit in the promoters of related genes.Results:92 DEGs were identified in GCs of PCOS women with higher BPA levels in FF(fold change?1.5,P<0.05,false discovery rate<0.05)in comparison with the corresponding controls.Bioinformatics analysis predicted that synthesis of lipid and steroid was activated in GCs from PCOS women.The expression pattern of genes related to the synthesis of steroid and lipid metabolism was in accordance with the RNA-Seq.An approximate 25%reduction was observed in global DNA methylation of GCs from PCOS women(4.44±0.65%)compared with the controls(6.07±0.72%,P<0.05).The promoters of several genes related to the synthesis of steroid and lipid metabolism were hypomethylated,which showed different degrees of negative correlations with the expression levels.Conclusion:BP A disrupts the gene expression patterns and activate the synthesis of steroid.DNA methylation aberrations in genome-wide scale or gene-specific manner identified in our study may be an inducer,which could at least partially explain the gene expression changes and consequent hyperandrogenism in PCOS.Our results suggest epigenetic mechanisms may participate in the pathogenesis of PCOS.