Association Of AMHRâ…¡, INSR Gene DNA Methylation And Epigenetics In PCOS

Polycystic ovary syndrome is a common endocrine disorder in adolescents and reproductive age women. The classic features include menstrual irregularity, biochemical or clinical hyper androgens, and ultrasound appearance of polycystic ovaries. The etiology of PCOS is still unclear, but environmental and genetic factors may contribute to the pathogenesis of PCOS. It is reported to be a heterogenic common genetic disorder, and features such as hyperandrogenemia, abnormal insulin resistance/secretion in relatives of PCOS women are heritable. However, only a limited number of studies have aimed to extensively profile DNA methylation expression and function within a PCOS study population,and the results are at times, contradictory. However, it follows that a resilient motivation should arise to further research the actions and regulations of DNA methylation to clarify their role in PCOS.Anti-mullerian hormone(AMH) is a local regulator secreted by ovarian granulose cells,which was found to be of great importance in the growth and development of follicle. The abnormal expression and regulation of ovarian local regulation factor may cause path physiological changes and it varied. AMH levels reflect the number of small anural follicles in ovaries and expression changes of AMH and its receptor(mainly the type II receptor) are suspected to be involved in the pathogenesis of polycystic ovary syndrome(PCOS). Reports about the local role in PCOS ovarian and endometrial pathology of AMHR II were limited. In recent years, genes involved in insulin signaling pathway have been suggested as candidate genes for PCOS and significant associations between the syndrome and genetic variants in insulin receptor(INSR) have been found. Previous epidemiologic studies have also shown significant associations of INSR gene variants with insulin resistance and obesity.Furthermore, a significant association between INSR gene variants and insulin resistance hasbeen observed. Epigenetic is the study of heritable changes in gene expression that is not caused by DNA sequence alterations, but are mitotically and transgenerationally heritable.Inappropriate epigenetic reprogramming has been identified as contributing to common diseases with fetal origins such as type 2 diabetes, and prostate cancer, suggesting it may also contribute to PCOS, given that PCOS is a common disease with both reproductive and metabolic abnormalities. Furthermore, DNA methylation, the principal mechanism of epigenetics, has been reported to play a role in cancer, aging, and complex chronic diseases.To investigate the role of epigenetics in PCOS, we analyzed the clinical informations and described the clinical features of PCOS subjects compared with the control ones. Therefore,we conducted a pilot epigenetic study of DNA methylation in PCOS comparing the methylation percentage of AMHRII and INSR between PCOS and matched controls, in order to find out the possible association of AMHRII, INSR gene DNA methylation and epigenetics in PCOS. However, further designs to determine the causality was still needed.Objective:1. Describe the biochemical and clinical features of PCOS.2. Using immunohistochemical method, study localization, distribution and expression of MHRII/INSR in ovary and endometrium and then discover the correlation of AMHRII/INSR and histopathological changes, ovarian follicular dysplasia at same time.3. By bisulfate genomic sequence, study AMHR II/INSR and its role in gene methylation in Ovarian and endometrial pathology of PCOS subjects, and then discover the epigenetics in PCOS.Methods:1. Collected and analyzed the clinical informations of PCOS subjects and the control ones going to see a doctor in Guangzhou Family Planning Specialty Hospital during March.2014 to December.2014.2. Applied ELISA to test the level of serum luteinizing hormone(LH), follicle stimulating hormone(FSH), Estradiol2(E2), Progesterone(P), testosterone(T), Fasting Insulin(FINS) andfasting plasma glucose( FPG). The value of FSH/LH and HOMA-IR index were also calculated.3. The ovarian and endometrium tissue underwent paraffin imbedding, microtome section, HE staining and then were observed patho-histological changes.4. AMHRII and INSR were marked respectively by immunohistochemistry method. Observed the orientation and distribution of AMHRII as well as INSR in the ovary and endometrium tissue.5. Tested AMHRII /INSR gene methylation qualitative of the blood in the subjects by bisulfite genomic sequence(BSP), and calculated the methylation rate.6. Statistical analysis was performed using SPSS 21.0 software(Chicago, IL, USA). To analysis AMHR II, INSR gene DNA methylation level relationship with the clinical features,and expounds PCOS and epigenetic mechanisms.Results:1.The basic characteristics of PCOS patients with clinical data various clinical manifestation were found. The patients with PCOS met criteria for clinically requirements. In the collected75 cases of patients with PCOS, Menstruation normal group were only in 18 cases(24%).PCO group were in 54 cases(72%), HI or HA ware in 53case(70.7%), IR ware in 48(64%).To compare between amenorrhea groups and abnormal menstruation groups, abnormal menstruation groups was lower aged, Two sets of data showed statistically significant differences(P<0.05). To compare with IR and non-IR, IRgroups, IR groups abnormal menstruation phenomenon is more obvious, Two sets of data showed statistically significant differences(P<0.05).2. HE pathological tissue morphology analysis Visible PCOS patients with ovarian multiple expansion of vesicle, granular cell layers decreased, the stormily hyperplasia, mature corpus lustrum;Normal groups see different development stages of the follicles, granular cell layer thicker, visible mature corpus luteum 。 A group of patients with PCOS endometrial thickening, glandular epithelial cell hyperplasia, distorted glands and stroma hyperplasia obviously;No obvious hyperplasia group normal endometrium.3. Immunohistochemical analysis of AMHRII、INSR There were all expressed in PCOS group and a control group of ovarian and endometrial withAMHRII, INSR. AMHRII expression mainly located in cell membrane, INSR expression mainly located in cytoplasm. INSR were detected in ovarian granulosa cell and ovarian stromal cells to express of PCOS group. AMHRII expressed in granulosa cells more apparent,and ovarian stroma in see. Control group AMHRII, INSR mainly expressed in immature follicular granulosa cells, stroma in see.INSR are expressed more positive in PCOS group than in Control group. AMHRII expression in ovarian slightly higher than the control group,INSR expressed in ovarian PCOS and control group no significant difference, AMHRII in PCOS and the control group no difference between the expression of endometrium.4. Bisulfite sequencing method of quantitative analysis of DNA methylation4.1AMHR2 gene DNA methylation.60 cases sample AMHR2 gene detected four sites of DNA methylation, 40 patients with PCOS group, 20 cases of normal group, the Pos1 and Pos2 there was no significant difference between the two groups, P3, P4 have significant differences between the two groups.The 40 cases of PCOS group including : ①oligomenorrhea② HA or HI ③ side or both sides PCO, Meet two or three clinical behavior.Is divided into four groups, they are no correlation between groups(P > 0.05). Different clinical features in PCOS group AMHRII gene methylation level and insulin resistance relations have significant differences(R = 0.532, p = 0.532)4.2 INSR gene DNA methylation. 60 cases sample INSR gene detected four sites of DNA methylation, 40 patients with PCOS group, 20 cases of normal group, the Pos1 and Pos2 there was no significant difference between the two groups, P3, P4 have significant differences between the two groups. The 40 cases of PCOS group including : ①oligomenorrhea② HA or HI ③ side or both sides PCO, Meet two or three clinical behavior. Is divided into four groups, they are no correlation between groups(P > 0.05). Different clinical features in PCOS group INSR gene methylation level and insulin resistance relations have significant differences(R=0.281,p=0.03)Conclusion1. The basic characteristics of PCOS patients with clinical data Various clinical manifestation were found.the patients with PCOS met criteria for clinically requirements and express heterogeneous; oligomenorrhea relevance to the low age, the age person with menstrual dilute rate is high; IR is more associated with oligomenorrhea phenomenon.2. INSR’expression in the endometrium was significantly positive in PCOS group, and a little higher was found of AMHRII’expression, Do not rule out AMHRII participate in endometrial lesions, INSR may be correlated with endometrial hyperplasia.3. INSR’expression did not change significantly in the follicles and interstitial of PCOS patients, But in interstitial vascular epithelial express clearly, Speculating that may have correlation with ovarian stromal hyperplasia.4. AMHRII, INSR gene DNA methylation correlated with clinical characteristics of PCOS,With no correlation between different clinical features in PCOS groups. IR is associated with AMHRII, INSR gene of DNA methylation.