Molecular Mechanism Of Hyperandrogenism And PCOS Pathogenesis

Backgroud and ObjectivePolycystic ovary syndrome(PCOS)is a highly prevalent endocrine/metabolic disorder in women of reproductive age,which is characterized by heterogeneous clinical features of menstrual irregularities,polycystic ovary,hyperandrogenism and insulin resistance.PCOS may increase the risk for infertility,type 2 diabetes mellitus,cardiovascular disease and endometrial cancer,emphasizing the need for early diagnosis of the syndrome.The pathogenesis of PCOS remains elusive,but there are indications for a strong genetic basis.Hyperandrogenemia has been considered to be one of the major characteristics of PCOS,thus androgen production and/or action related genes were regarded as candidate genes based on knowledge of the underlying pathophysiology of the syndrome.It is reported that AR(CAG)n and SHBG(TAAAA)n polymorphisms were inversely correlated with their transcriptional activity.Our aim was to investigate whether polymorphic variants of AR and SHBG genes were associated with PCOS.Furthermore,increasing evidence from clinical and experimental animal research supports the developmental programming of PCOS by androgen excess.It is suggested that epigenetic change in gene expression may be the underlying mechanism in the fetal programming of the syndrome.The best-characterized epigenetic modification of DNA is the methylation of cytosine residues within CpG dinucleotides in gene promoters region.In this study,our objective was to evaluate the methylation status of genes involved in androgen production and/or action(AR,CYP11A1 and CYP17A1)and to understand their roles in the development of PCOS.Methods and resultsA case-control study including polycystic ovary syndrome(PCOS)patients and healthy controls was conducted.Fluorescently labeled DNA fragments containing AR(CAG)n and SHBG(TAAAA)n were obtained by PCR and genotyped via capillary electrophoresis.We found that CAG biallelic mean distributions also showed statistical difference between the two groups.Especially,a higher frequency of short AR(CAG)n alleles was found in PCOS patiens compared with that of controls.Besides,carriers of the longer allele genotypes had lower SHBG levels than those with shorter alleles in both PCOS patients and controls.However,no statistically significant associations were found between the SHBG(TAAAA)n allele and genotype with PCOS riskPCOS rat model induced by prenatal administration of 3 mg testosterone from Embryonic Day 16 to 19 showed polycystic ovaries,irregular oestrous cycles and endocrine disorders in adulthood.The methylation status of CpG sites in the promoter regions of the AR,CYP11A1 and CYP17A1 genes were measured by pyrosequencing We identified three hypomethylated sites in AR and one hypomethylated site in CYP11A1 in peripheral blood cells of PCOS rats.In ovarian tissue,five CpG sites of AR and one single CpG site in CYP11A1 were significantly hypomethylated in PCOS rats.Furthermore,transcription of AR,Cyp11Al and Cyp17A1 genes in ovarian samples were measured using quantitative Q-PCR.We found that AR expression was significantly elevated in PNA rats.However,the mRNA levels of CYP11A1 and CYP17A1 genes were not significantly altered in PNA ratsAnother PCOS model was induced by daily administration of DHEA s.c.to 3-week old female rats,and the control groups were injected daily with oil.The daily treatments lasted for up to 35 days consecutively.Serum steroid hormone levels were measured by enzyme-linked immunosorbent assay.Samples were stained with hematoxylin and eosin for histological morphology,and Sirius Red and immunohistochemistry for revealing collagens.The expression of fibrosis-related genes was analyzed both at mRNA(real-time RT-PCR)and protein(western blot)levels.DHEA-induced rats with PCOS exhibited significantly higher levels of fibrosis(collagen Ⅳ)in ovarian tissue.In ovarian tissue of PCOS rats,the expression of profibrogenic cytokines(TGF-β,CTGF,Fibronectin)was increased,while collagenase(MMP2)was decreased.ConclusionsFirst of all,shorter AR(CAG)n alleles enhanced the susceptibility to PCOS,either by upregulating AR activity or by causing hyperandrogenism.Secondly,polymorphism in the regulatory sequence of the SHBG gene is associated with circulating SHBG levels,but there is insufficient evidence to demonstrate a conclusive association between the SHBG(TAAAA)n polymorphism with the risk of PCOS.Thirdly,prenatal androgen exposure may lead to methylation pattern changes and the development of PCOS,but further studies are required to reveal their causal relationships.Fourthly,Ovarian hyperfibrosis may occur in patients with PCOS and result in anovulatory disorder.