Relationship Between Arsenic And Diminished Ovarian Reserve(DOR) And Its Effect On The Steroidogenesis Of Human Ovarian Granulosa Cells

ObjectiveTo explore the relationship between follicular fluid metal elements and the diminished ovarian reserve,and further explore the effect of arsenic exposure on ovarian steroidogenesis and its mechanism through cell experiments and animal experiments.MethodsPart I Research on the Correlation between Follicular Fluid Metal Elements and DORA hospital-based case-control study was used to collect the basic clinical data and follicular fluid of DOR patients and control groups who underwent assisted reproductive technology in the Maternal and Child Health Hospital of our school from January 2019 to December 2020.The 21 metals elements in follicular fluid were detected by ICP-MS;the general clustering trends and outliers were identified by principal component analysis after data standardization;orthogonal partial least squares discriminant analysis was used to verify the PCA model and identify each elements with different concentrations between groups;the correlation between metal elements was tested by Person correlation;univariate logistic and multivariate logistic regression analysis were performed to assess the associations between metal elements and DOR;the correlations between follicular fluid concentration of metal elements and serum hormone levels was performed by multiple linear regression analysis.PartⅡThe effect of arsenic exposure on steroidogenesis in human ovarian granulosa cellsIsolate the ovarian granulosa cells of non-ovarian infertility patients who underwent assisted reproductive technology in the Maternal and Child Health Hospital of our school from January 2019 to December 2020,and treated them with 0,0.5,1,2and 4μM sodium arsenite.After 24 h,CCK8 was used to detect cell proliferation activity;ELISA was used to detect the levels of progesterone and estradiol in the culture medium;RT-PCR and Western blot were used to detect the m RNA and protein expression levels of genes related to steroid hormone synthesis in human ovarian granulosa cells.Part III The effect of arsenic exposure from weaning to sexual maturity on the synthesis of ovarian steroid hormones in rats and its mechanismForty clean first-weaned female Wistar rats,21 days old,weighing（49.8±4.7）g.Randomly divided into 4 groups according to body weight,each group of 10 animals,exposed to 0,2,10,and 50 ppm of sodium arsenite through water to sexual maturity.At the age of 35 days,a vaginal smear was performed to detect the estrous cycle of the rats.After the intervention,the ovaries were weighed and their organ coefficients were calculated;the histological changes of the ovaries were observed by HE staining and the follicle composition ratios at all levels were calculated;the serum E₂and Pg levels were determined by ELISA;the genes related to ovarian steroid hormone synthesis were determined by RT-PCR and Western blot.And DNMTs m RNA level were detected by RT-PCR;BSP was used to detect the methylation level of the promoter region of SF-1gene.ResultsPart I Research on the Correlation between Follicular Fluid Metal Elements and DORThe case group is older,and the serum FSH level is higher than that of the control group（P<0.05）,but the levels of T and AMH are lower than the control group（P<0.05）;compared with the control group,the metal elements As,Mo,Sr and Ca in the follicular fluid of DOR group are increased,while the content of Cu,Zn,Ti,Cr,Mg and Fe are decreased（P<0.05）;the results of Pearson correlation analysis showed that there is a linear correlation between those metal elements;PCA and OPLS-DA analysis results show that the profile of metal elements in the follicular fluid of the case group has undergone significant changes.The metal elements that VIP>1 are:Sr,Mg,Zn,Cu,As and Mo;univariate logistic and multivariate logistic regression analysis show that As,Sr and Ca in follicular fluid are related to the risk of DOR.PartⅡThe effect of arsenic exposure on steroidogenesis in human ovarian granulosa cellsAfter 24 hours of exposure to arsenic in human ovarian granulosa cells,the E₂level of each dose group decreased significantly,but the P₄level did not change significantly;compared with the control group,HSD3β1 in the 2μM and 4μM groups were significantly down-regulated（P<0.05）;2μM and 4μM arsenic arsenic exposure significantly reduced human ovarian particles Cell St AR and CYP11A1 protein expression（P<0.05）.Part III The effect of arsenic exposure from weaning to sexual maturity on the synthesis of ovarian steroid hormones in rats and its mechanismAfter exposure to arsenic from weaning to sexual maturity,the ovarian organ coefficient in the high-dose group decreased significantly（P<0.05）,and the uterine organ coefficient in the middle-dose group also increased significantly（P<0.05）;Arsenic exposure has no significant effect on the vaginal opening time of rats（P>0.05）,but it will significantly change the estrus cycle of rats,which is specifically expressed as prolonging the preestrus of the middle and high-dose groups（P<0.05）and shortening the middle-dose group Estrus interval（P<0.05）,and significantly prolonged the total length of the estrus cycle in the high-dose group（P<0.05）;The follicle composition ratio of each dose group changed significantly（χ²=115.03）.Compared with the control group,the composition ratio of primordial follicles in the high-dose group was significantly reduced（2.85%vs.7.79%）.In addition,antral and atretic follicles in each dose group showed a dose-dependent decrease（11.11%vs.19.20%,9.11%vs.19.20%and 5.07%vs.19.20）and increase（56.41%vs.43.60%,57.35%vs.57.35%and 66.09%vs.43.60%）,the differences were statistically significant（P<0.001）Medium and high-dose arsenic exposure can significantly increase the serum E₂level in rats,and there is a significant dose-response relationship between each dose(P_trend<0.001);compared with the control group,the expression of St AR and HSD3β1 in the medium and high-dose groups Significantly down-regulated（P<0.001）,the CPY19A1 m RNA level of each dose group was significantly lower than that of the control group（P<0.001）;the m RNA expression level of SF-1 in each dose group was compared with the control Compared with the control group,the expression level of St AR protein in each dose group was significantly increased（P<0.05）,and showed a significant dose relationship;medium and high dose groups The expression of CYP17A1,HSD3β1 and SF-1 protein increased significantly（P<0.05）;Compared with the control group,the m RNA expression level of DNMT3a in the low-dose and high-dose groups was significantly increased（P<0.05）.In addition,the m RNA expression of DNMT3b in the high-dose group also increased significantly（P<0.05）,but DNMT1 did not change significantly in each dose group（P>0.05）;the total DNA methyl group of the SF-1 promoter region in each dose group The DNA methylation levels were 65.71%,60.38%,68.00%and 72.38%respectively.After theχ²test analysis,it was found that the DNA methylation rate of the low and high-dose groups was significantly different from that of the control group（χ²=1.918,P=0.001）,which was mainly manifested as the high-dose methylation level at the+17bp site Significantly increased（P<0.05）,and the methylation level at the-184bp site at low doses was significantly decreased.Conclusion1.The serum hormone levels of patients with DOR have significantly changed,and there are significant differences between their follicular fluid metal profiles and normal women.These significantly different metals may be related to the onset of DOR and the disorder of serum hormones;2.Arsenic exposure can affect the expression of key enzymes in steroid hormone synthesis,which in turn affects the synthesis of steroid hormones in human ovarian granulosa cells;3.SF-1 is an important target for arsenic exposure to cause steroid hormone synthesis disorders;arsenic can significantly increase the DNA methylation level in the promoter region of SF-1 to reduce the m RNA expression of SF-1,but SF-1 may exist The post-transcriptional regulatory mechanism makes its protein expression increase significantly,thereby promoting the secretion of ovarian E₂,and ultimately leading to the premature loss of the follicular pool;the low and medium dose groups may affect the expression of SF-1 through other ways.