Methylation level of CpG island promoter sites of CYP19A1, AMH and AMHR2 in granulosa cells of patients with PCOS
ZHU Jin-hu
YANG Xiu-wei
HONG Xiao-shan
MAO Ting
WAN Ying-xiang
Abstract：Objective To verify the methylation of differential gene promoters in granulosa cells and investigate the correlation between gene methylation and gene expression .Methods The observation group was composed of 30 pa-tients with the Uygurs PCOS, control group of 30 with tubal or male factor infertility .General clinical parameters and basal hormone level of FSH, LH, E2, T and PRL were compared between the two groups .On the day of oocyte retrieval , all follicular fluid (FF) and particle separation granulosa cells were collected .The E2, A and AMH level in FF was assayed by commercially available enzyme -linked immunosorbent assy (ELISA) method.Methylation status of CYP19A1, AMH and AMHR2 were detected by methylation specific PCR (MSP) in PCOS and control granulosa cells .The mRNA and pro-tein expression of these genes were measured by Real -time RT PCR and Western Blots .To further study the role of DNA methylation in PCOS, we also investigate the correlatioin between promoter methylation and mRNA expression of these genes.Results The basal hormone levels of LH , T, LH/FSH was significantly higher in the Uygurs PCOS than those in controls.The E2/T was significantly lower in the Uygurs PCOS than that in control , which indirectly reflecting the activity of aromatase, a product of CYP19A1 gene.The other parameters (age, BMI, FSH, E2, PRL) were not significant differ-ent between PCOS patients and controls .The E2 level in FF was similar in the Uygurs PCOS patients and controls .A and AMH levels were significantly higher and E2/T was significantly lower in FF in Uygurs PCOS patients than that in con-trols.The MSP results suggested that DNA methylation level of CYP 19A1 was significantly higher, and the methylation levels of AMH and AMHR2 were significantly lower in the Uygurs PCOS patients compared with controls .These results of MSP were in line with the microarray results and confirmed the reality and reliance of the chip .The mRNA and protein ex-pression levels of CYP19A1 were significantly lower and the mRNA and protein expression levels of AMH and AMHR 2 were significantly higher in the Uygurs PCOS patients compared with controls .The mRNA expression of CYP19A1, AMH and AMHR2 decreased gradually as the promoter methylation level of these gene increased .Furthermore, the expression of CYP19A1 was negatively correlated with AMH expression .Conclusion The altered promoter CpG islands methylation in CYP19A1, AMH and AMHR2 cause aberrant expression of these genes in the Uygurs PCOS granulosa cells .AMH sup-pression of CYP19A1 activity may represent a mechanism by which AMH modulates normal antral follicular maturation and oocyte quality.Despite an apparent normal development under ovarian stimulation , PCOS dominant follicles retain some of their basal abnormalities possibly impacting on oocyte quality .
Keywords：in vitro fertilization-embryo transfergranulosa cellsPCOS
Publication Date：2018-01-01
Pages：6( 1171-1176 )