The Relationship Between Copper Exposure And PCOS And Its Effects On The Steroidogenesis Of Human Ovarian Granulosa Cells

Objective To investigate the correlation between copper exposure and polycystic ovary syndrome and to further investigate the effect of copper exposure on steroidogenesis in human ovarian granulosa cells and its mechanism.Methods 1.Basic clinical data and follicular fluid of PCOS patients and the control group who underwent intracytoplasmic sperm injection from March 2017 to March 2018 in the assisted reproductive technology laboratory of the Provincial Maternal and Child Health Hospital affiliated to our university were collected.ICP-MS was used to detect 22 elements in follicular fluid.PCA and PLSDA were used to analyze the elemental spectra changes of follicular fluid.Logistic regression was used to analyze the relationship between the copper concentration of follicular fluid and the rate of high-quality embryo.Linear regression analysis of the relationship between copper concentration in follicular fluid and the number of oocytes obtained and AMH-mediated effects.Correlation analysis was used to analyze the relationship between follicular copper concentration and age,AMH and steroid levels in follicular fluid.The levels of testosterone and progesterone in follicular fluid were measured by ELISA.2.The ovarian granulosa cells were isolated from patients with non-ovarian infertility who underwent intracytoplasmic sperm injection from April 2018 to January 2019 in the assisted reproductive technology laboratory of the Provincial Maternal and Child Health Hospital affiliated to our university.After 0,1,2,5,10,20 and 40μg/m L copper exposure for 24 h,the cell proliferation activity was detected by CCK8 method,and the levels of progesterone,estradiol and testosterone in the culture medium were detected by ELISA method.Real-time PCR was used to determine the expression of steroidogenesis-related genes St AR,HSD3B1,CYP11A1,HSD17B1,CYP19A1,FSHR and sf-1 in human ovarian granulosa cells.The expression levels of FSHR and sf-1 were detected by western-blot and immunofluorescence.3.(1)Human ovarian granulosa cells cultured in vitro after 48 h were divided into six groups: control group,20μg/m L Cu group,FSH group(agonist: 50 m IU/m L),FSH+20μg/m L Cu group,BI-10 group(inhibitor: 30 m M),BI-10+20μg/m L Cu group.The expression levels of FSHR were detected by real-time PCR,western-blot and immunofluorescence after exposure for 24 h,and the levels of progesterone and estradiol in cell culture medium were detected by ELISA.(2)Human ovarian granulosa cells were cultured in vitro for 48 h,and then exposed to copper at 0,2,20 and 40μg/m L for 24 h.Combined bisulfite sequencing PCR(BSP)was used to detect the methylation level in the promoter region of SF-1 gene.Further,the expression levels of methyltransferases DNMT1,DNMT3 A and DNMT3 B were detected by real-time PCR.Results 1.(1)There were no statistically signifcant differences in BMI,E2,PRL,endometrial thickness,fertilization rate,cleavage rate,the amount of follicular fluid retrieved or high-quality embryo rate between PCOS and the patients with fallopian tube obstruction and the control group(P>0.05).Compared with the controls,LH,T,AMH levels and retrieved mature oocytes in the PCOS group increased significantly(P>0.05);while FSH decreased significantly(P<0.05).(2)PCA analysis and PLS-DA analysis showed that there was a significant difference between PCOS group and the other two groups.After calculating the variable importance plot value,the 6 most significantly altered trace elements were identified,namely,Cu,Mg,Ca,Ti,As,and Sr.Among these changed trace elements,Cu had the largest VIP score.(3)One-way ANOVA showed that the copper concentration(1064.53±243.75)μg/L in the follicular fluid of PCOS group was significantly higher than that of the other two groups(920.91±305.89,877.98±239.73)μg/L(P <0.05).(4)Correlation analysis showed that the copper concentration of follicular fluid was positively correlated with the number of oocytes obtained(Β=1.785,P=0.001).It was negatively correlated with the rate of high-quality embryos(Β=-6.360,P=0.05).Moreover,It was negatively correlated with age(r=-0.168,P=0.002)and positively correlated with serum AMH level(r=0.228,P< 0.001),follicular fluid T level(r=0.250,P=0.022)and P4 level(r=0.275,P=0.010).2.(1)Compared with the control group,40μg/m L of copper resulted in decreased cell proliferation(P<0.05).(2)Compared with the control group,the level of P4 in human ovarian granulosa cell culture medium was decreased in 40μg/m L Cu group(P<0.05),and the E2 level in human ovarian granulosa cell culture medium was increased in 1,2μg/m L Cu group(P<0.05).In the 20,40μg/m L Cu group,the E2 level in human ovarian granulosa cell culture medium was decreased(P<0.05),and the level of T in human ovarian granulosa cell culture medium was decreased in 1,2μg/m L Cu group(P<0.05).(3)Compared with the control group,m RNA expression levels of St AR,CYP11A1,HSD3B1 and CYP19A1 were down-regulated in the 20,40μg/m L Cu group(P<0.05);CYP19A1 m RNA expression was up-regulated in 1,2 and 5μg/m L Cu group(P<0.05);the m RNA expression level of HSD17B1 was up-regulated in the 2,5,10,20 and 40μg/m L Cu groups(P<0.05).(4)Compared with the control group,the m RNA and protein expression of FSHR were up-regulated in the 2μg/m L Cu group(P<0.05),while the expression of FSHR was down-regulated in the 20μg/m L and 40μg/m L Cu groups(P<0.05).The results of immunofluorescence assay were consistent with the results of PCR and Western blot.(5)Compared with the control group,the expression of SF-1 in 2μg/m L Cu group was up-regulated(P<0.05),while the expression of SF-1 in 20μg/m L and 40μg/m L Cu group was down-regulated(P<0.05).3.(1)Compared with the 20μg/m L group,the expression of FSHR was further down-regulated after adding inhibitor(BI-10)(P<0.05).The expression of FSHR was up-regulated after the addition of FSH(P<0.05).(2)Compared with the 20μg/m L Cu group,the m RNA expression level of CYP19A1 and E2 level were further decreased after the addition of inhibitor(BI-10)(P<0.05).After FSH was added,the m RNA expression level of CYP19A1 and E2 level increased.(P<0.05).(3)The total methylation rates of the SF-1 promoter region in the control group,2,20 and 40μg/m L Cu group were 27.14%,7.62%,17.62% and 0.71%,respectively.Compared with the control group,the total methylation rate of the 2,20 and 40μg/m L Cu groups decreased(χ2=146.50,P<0.001).(4)Compared with the control group,the m RNA expression of DNMT1 and DNMT3 A were down-regulated in the 2,20 and 40μg/m L Cu groups(P<0.05),while the m RNA expression level of DNMT3 B was not statistically significant between the various dose groups(P<0.05).Conclusion 1.The elemental profile of PCOS follicular fluid changed significantly,and the copper concentration of follicular fluid increased significantly and was associated with follicular developmental abnormalities.2.Copper can affect the steroidogenesis in human ovarian granulosa cells by affecting the expression of genes involved in steroidogenesis.3.FSHR is an important target of steroidogenesis abnormalities in human ovarian granulosa cells induced by copper exposure.Copper can lead to changes in the methylation pattern of SF-1 promoter region in human ovarian granulosa cells,thus affecting steroidogenesis,which is related to copper changing methyltransferase.