Effects Of BPA And TCS On Female Reproduction And Mechanism Involved

Environmental endocrine disruptors（EEDs）are a large class of compounds that can change the function of human normal endocrine system,including detergents,plasticizers,polychlorinated biphenyls,drugs,pesticides and plastics,as well as by-products of pollutants and degradation products in the production process.Its universality and reproductive toxicity had increasingly been attracting peoples’attention.Bisphenol A（BPA）and triclosan（TCS）are the two common EEDs.BPA is a2,2-dipropane synthesized from acetone and phenol,which is mainly used to produce epoxy resin and polycarbonate plastics.TCS（5-chloro-2-phenol）is a broad-spectrum antibacterial agent,which is widely used in family health care and personal care products.BPA and TCS can be detected in various human body fluids and tissues,including urine,serum,follicular fluid,saliva,liver,fat and placenta tissues.BPA and TCS are completely removed from urine within 24 hours.The chemical structure of BPA and TCS is similar to that of 17β-estradiol,which can show weak estrogen activity by binding to a variety of estrogen receptors.However,there is still controversy about the results of BPA epidemiological studies on human reproduction.In 2018,our research group reported for the first time that BPA might inhibit the expression of ENa Cαthrough ER/SGK1/ENa Cαpathway through mouse model of early pregnancy exposure to BPA,affect the angiogenesis in the process of mouse decidualization,thus reducing the success rate of embryo implantation. Studies have shown that TCS may interrupt the implantation of mouse blastocysts and alter the estrogen synthesis of placenta,leading to adverse pregnancy outcomes.Due to the lack of effective human epidemiological investigation,the relationship between TCS and female reproduction is still unclear.In vitro fertilization and embryo transfer（IVF-ET）is one of the best in vivo models to evaluate the potential impact of EEDs on the early stage of human reproduction.A recent study in t a cohort of 256 women undergoing 375 IVF fresh cycles,with only 7.1%of the participants having tubal factor infertility,indicated that exposure to BPA did not adversely affect the IVF outcomes in terms of the ovarian stimulation,implantation,clinical pregnancy and live birth rates.In this study,the etiology of the population was diverse,including male factors,unexplained infertility and female factors such as ovarian diseases,uterine diseases,endometriosis and ovarian reserve dysfunction.However,the above infertility factors all affect the outcome of IVF.The effects of BPAAND tcs on early female reproductive outcomes were investigated.Therefore,in the first part of this study,female patients with tubal infertility undergoing conventional IVF were used as the research object.The concentrations of BPA and TCS in urine on the day of oocytes retrieval were measured.The multivariate generalized linear mixed model was used to analyze BPA or TCS between and ovarain stimulation and clinical outcomes,separately.Gap junction（GJ）plays an important role in oocyte development and maturation.GJA1（Gap Junction Protein Alpha 1）as a transmembrane protein is an important component of gap junction.Gap junctions can exist between the innermost layer of cumulus cells and oocytes,as well as between adjacent granular cells.It has been reported that oocyte growth retardation and follicular growth are suppressed in ovaries of GJAl-/-cultured in vitro.The GJA1 has been identified as a marker gene for assessing oocyte quality.Therefore,in the second part of the study,the follicular puncture solution on the day of oocyte retreival was collected and human granulose cells were isolated and cultured.Cell models of BPA10ug/ml treatment group and control group were established respectively.GJA1 was selected by m RNA microarray analysis.The expression of GJA1 in human granulosa cells and KGN cells was analyzed by RT-PCR and Western blot.To explore the effect of BPA exposure on GJA1 expression,immunohistochemistry and cellular immunofluorescence assays were used to detect the expression of GJA1 protein in mouse ovary and human granular cells,respectively.To analyzed the effect of BPA on hormone synthesis,the expression of progesterone in the culture supernatant of human granulosa cells and kgn cells was detected by ELISA.In KGN cells,the expression of P450Scc,3β-HSD and Star at m RNA level and protein level was analyzed by downregulating GJA1 gene via siRNA,and exploring whether GJA1 gene is involved in the regulation of key enzymes in sex hormone synthesis.Then,a Cp G rich sequence in the promoter region of GJA1 was sequenced by pyrosequencing.To explore whether methylation is involved in the regulation of GJA1 expression induced by BPA,the methylation status of BPA treated group and control group were detected respectively.Through overexpression of mi R-206,we explored the functional role of mi R-206 in KGN cells.Finally,a double luciferase reporter gene assay was carried out to prove whether mi R-206 could change the expression of GJA1 by targeting the GJA1 3’UTR region,thus providing scientific evidence for the potential toxic effects of BPA on oocyte development.Part I Associations between concentrations of BPA and TCS in urines and the outcomes of IVF-ET in women with tubal factorinfertility.Objective:To observe the distribution of BPA and TCS in female urines on the day of oocyte yield.To understand the potential toxic effects of BPA and TCS on human early reproduction.Materials and methods:1. The participants were female partners from couples seeking IVF-ET treatments at theCentre of Reproductive Medicine in the Women’s Hospital School of Medicine at Zhejiang University from September 2013 to October 2016.The urine was collected before the operation of oocytes yield.2. Urine samples were measured using solid-phase extraction and liquid chromatography coupled with tandem mass spectrometry.The results were further standardized by using the concentration of Creatinine（Cr）in urine.Cr was determined by the basic bitter taste algorithm and quantified by colorimetry.In order to correct the concentration of BPA and TCS in urine,Cr corrected BPA/TCS value（1*10-4 mg/g Cr）was used.3.The associations between the urinary BPA concentration and demographic and baseline reproductive characteristics were evaluated using the Kruskal-Wallis tests for continuous variables and the chi-squared tests for categorical variables.4.The concentrations of BPA and TCS in urine were ranked from low to high,and were divided into group according to the quartile method.Q1 was considered as the control group.The other groups（Q2,Q3 and Q4）were compared with Q1 respectively.Multivariate generalized linear mixed model was used to analyze the effects of BPA and TCS on ovulation stimulation outcomes including GN dosage,intimal thickness,dominant follicle number,E2 peak and oocytes,fertilization rate,cleavage rate and excellent embryo rate.5.A generalized linear mixture model was used to analyze the correlation between BPA/TCSconcentrations and the clinical outcomes of the fresh cycles and the first frozen cycles in terms of biochemical pregnancy,clinical pregnancy,implantation rate,abortion rate and live birth rate.The covariates were age,BMI,basal FSH,basal E2 and AFC.6.The correlation between BPA and TCS compound exposure and IVF outcome was analyzed by generalized linear mixed model with interaction items.Results:1.The detection rate of BPA was 83.4%（293/351）,with the concentration ranging from below the LOD to 49.282μg/g Cr.The median（Q1–Q3）value was 0.720（0.246–5.616）μg/g Cr.2. Women with urinary BPA concentrations in the highest quartile exhibited lower numbers of oocytes than that in the women with urinary BPA concentrations in thelowest quartile.However,the overall trend of the number of the total oocytes was not significant in the adjusted models.3. Urinary BPA concentration was not associated with the E₂peak level,endometrial wall thickness,the number of dominant follicles,or the total dose of gonadotropins.Similarly,the urinary BPA concentrations were not associated with the fertilisation rate,cleavage rate,and quality of the embryos4. In fresh cycles:the implantation in the BPA high group was significantly lower than that in the control group in the adjusted and adjusted models.The overall trend p value of clinical pregnancy rate and implantation was lower than 0.05.However,no significant dose-response associations were found between the urinary BPA concentrations and biochemical pregnancy,abortion,and live birth as determined by the models adjusted for age,BMI,basic FSH level,basic E₂level,and AFC.5. In frozen cycles:there was no significant difference in biochemical pregnancy,clinical pregnancy,implantation and live birth rate between the BPA high concentration group and the control group.6. The detection rate of TCS was 94.7%（286/302）.The distribution of TCS was from limit of detection（LOD）to 34.95ng/ml（49.282μg/g Cr）.The median（Q1-Q3）was0.894（0.316-3.048）μg/g Cr.7. In adjusted and unadjusted analysis models,urinary TCS concentrations had no correlations with the E₂peak levels,endometrial thickness,the number of dominant follicles,the dose of gonadotropin on HCG day,fertilization rate,cleavage rate and embryo quality.8. In adjusted and unadjusted analysis models,there was no significant dose-response correlation between urinary TCS concentrations and biochemical pregnancy,clinical pregnancy,implantation,abortion and live birth.9. Compound exposure of BPA and TCS has no correlation with ovarian stimulations and clinical outcomes in IVF patients.Conclusion:1.BPA exposure can affect the oocyte acquisition,embryo implantation and pregnancy of IVF patients.2.TCS exposure has no definite effect on the ovarian stimulations and IVF clinical outcomes.3.BPA and TCS combined exposure had no synergistic or antagonistic effect on IVF outcomes.Part II Alteration of GJA1 expression and relatedmechenisms involved in the effect of BPA on ovariangranular cellsObjective:To explore gene expression changes of key enzyme of hormone synthesis and connexins in human granulosa cells and KGN cells after BPA exposure.To investigate the molecular regulation mechanisms involved in BPA-induced alteration.Materials and methods:1.The follicular fluid of IVF patients on the day of oocyte retrieval was collected and separated with lymphocyte separation fluid.After 48 h of DMEM-F12 medium culture,the medium was changed.After 48 h of BPA10ug/ml medium and 0.05% DMSO medium culture,the cells were collected and total RNA was extracted.MRNA microarray analysis（biological repetition of three independent samples）was carried out first,and the sequencing results were analyzed by bioinformatics.The expression of GJA1 gene in human granulosa cells and KGN cells exposed to five different concentrations of BPA in termsof0.001ug/ml,0.01ug/ml,0.1ug/ml,1ug / ml and 10 ug /ml)for 48 hours was verified by RT-PCR and Western blot.2.Flow cytometry and Western blot were used to analyze the percentage of apoptosis and protein expression of apoptosis related genes in BPA（10ug/ml）treated human granulosa cells.3.Immunofluorescence and Immunohistochemistry were used to detect the protein expression of GJA1 in human granulosa cells and ovaries in mice.4.The expression of P450 Scc,Star and 3β-HSD was verified by RT-PCR and western-blot following GJA1 knocked down using siRNA.5.RT-PCR and Western blot were used to analyze the effects of different BPA concentration treatments on gene expression of P450 Scc,Star and 3β-HSD in human granulosa cells and KGN cells.6.Methylation of GJA1 promoter in BPA-induced KGN cells was evaluated using pyrosequencing analysis.7.In BPA exposure group,the mi RNAs expression was detecteded by RT-PCR.The expression of GJA1 in m RNA and protein level was observed by over-expression of mi R-206.Meanwhile,double luciferase reporter gene experiment was used to verify the binding target of mi R-206 and GJA1.8.In the cell of 293 T,double luciferase reporter gene assay was used to verify the binding target of mi R-206 and gene GJA1.Results:1.Flow cytometry showed that there was no significant difference in the percentage of apoptosis between BPA group and control group.Protein level: there was no significant difference in the expression of Caspase-3,Bcl-2 and Bax between BPA group and control group by using Western blot.2.RT-PCR and Western blot were used to confirm the decreased expression of GJA1 gene in BPA treated group of human granulosa cells and KGN cells,respectively.3.Immunofluorescence of human granulosa cells and immunohistochemistry of ovaries of mice exposed to BPA showed that the expression of GJA1 protein decreased significantly.4.The expression level of progesterone was decreased with dose-dependent in BPA exposure in the culture supernatant of human granulosa cells and KGN cells by using ELISA.5.The expression of GJA1 gene was down regulated by siRNA in KGN.The expression of P450 scc and star was down regulated at m RNA and protein levels,and the difference was statistically significant.6.We sequenced a Cp G island rich sequence in the promoter region of GJA1 by using pyrosequencing method.The results showed that there was no difference between the average methylation level of BPA treatment group and control group.However,in the comparison of the methylation level of each Cp G site,the methylation level of the fourth Cp G island was significantly higher than that of the control group.7.Overexpression of mi R-206 can reduce the expression of GJA1 gene in KGN at m RNA and protein levels,and the difference is statistically significant.8.Double luciferase reporter gene assay showed that mi R-206 significantly reduced the relative luciferase activity of wild type 293 T cells,but had no significant effect on mutant 293 T cells.Conclusion:1.No dose-response relationship between BPA exposure and apoptosis of human granulosa cells.2.BPA exposure decreased the progesterone level in human granulosa cells and KGN cells.GJA1 regulated the expression of P450 Scc and Star in genes of steroid hormone key enzyme.3.Methylation of promoter mediated the expression of GJA1 induced by BPA.4.Mi R-206 targeted GJA1 and regulated its BPA-induced abnormal expression.