| Polycystic ovary syndrome (PCOS) is the most common endocrine disorder. It affects an estimated 4-12% of women of reproductive age. PCOS is characterized by hyperandrogenism, chronic anovulation and/or polycystic ovaries. Besides, it is also associated with an increased risk of diabetes mellitus, cardiovascular diseases and endometrial cancer in later life. PCOS patients have a endometrial cancer risk 4 times higher than young women without PCOS.It was reported up to 60% of PCOS women who show compensatory hyperinsulinemia exhibit insulin resistance in worldwide. More than 50% of PCOS patients are insulin resistant and obesity PCOS women have a higher risk than lean PCOS women. The hyperinsulinemia present in a high percentage of PCOS women, may affect the function of several tissues including the endometrium. Siegel et al. examined an SNP at the tyrosine kinase domain of INSR and found an association in lean patients with PCOS. This SNP could be a susceptible variant for PCOS, or a result of linkage disequilibrium with another INSR polymorphism. The insulin receptor affinity and number in PCOS patients is associated with insulin resistance. However, little information is available about the regulatory role exerted by insulin receptor in the endometria from PCOS women. Many researchers had tried to explore whether the mutations of INSR could explain insulin resistance in PCOS. Insulin resistance is presumably mediated through downstream changes in the insulin receptor-mediated signal transduction cascade. In the signaling pathway, the binding of insulin to its receptor provokes auto-phosphorylation in tyrosine residues and phosphorylation on IRS, which is necessary for insulin signal transduction. This is an important issue because glucose has been proposed as the main energy source of the endometrial cell and high levels of energy are needed to fulfill endometrial functions. Metformin administration resulted in normal endometria as if PCOS patients suffer complex endometrial hyperplasia which failed to provera treatment. These findings could be considered a rough indicator of the relationship between endometrial hyperplasia and the insulin resistance phenotype of PCOS.Although clustering of cases in families strongly support the role of genetic factors in the development of PCOS, heterogeneity of phenotypic features in different families and even within the same family underscores the importance of the environmental contribution. Modifications of molecular structure of insulin, their receptors and of the enzymes involved in insulin action and secretion have been under continuous and intense investigation with variable results. Polycystic ovary syndrome has been proven to be a familial condition. Although the role of genetic factors in PCOS is strongly supported, the genes that are involved in the etiology of the syndrome have not been fully investigated. As a branch of genetics, epigenetics is defined as the investigation of heritable changes in gene expression that occur without changes DNA sequence. Epigenetic regulation mechanisms include DNA methylation, histone modifications and gene imprinting. Its research objective is gene modification, including DNA methylation. Epigenetic regulation, especially through DNA methylation, is a flexible, yet stable, mechanism for maintaining such a cellular memory. DNA methylation is a postreplicative modification occurred in most prokaryotic and eukaryotic genomes, which has a variety of important biological functions including regulation of gene expression, gene imprinting, preservation of chromosomal integrity, and X-chromosome inactivation. DNA methylation is a crucial epigenetic modification of me genome that plays an important role in the regulation gene expression and genomic imprinting and regulation of many of cellular processes.As a main enzyme for DNA methylation, DNA methyltransferase (Dnmt) is not only associated with DNA methylation, but also links to many important biological activities, including cell proliferation, senescence and cancer development. According to their structure and functions, DNA methyltransferases (Dnmts) are divided into two major families in mammalian cells:maintenance methyltransferase (Dnmtl) and denovo methyltransferases (Dnmt3a, Dnmt3b, and Dnmt3L). These Dnmts are crucial for mammalian growth and development. Dnmts deficiency will lead to embryonic development defects, cancer, and other diseases. DNA methyltransferases (DNMTs)is the key emzymes of DNA methylation.We wonder hyperinsulinemia and insulin resistance would lead to epigenetic change in PCOS patients. Although multiple genetic factors including mutations and polymorphisms to several genes have been associated with PCOS risk, the inheritance mode and the molecular genetic mechanisms underlying PCOS risk are not fully understood. At the same time, increased risk of endometrial cancer in insulin resistant PCOS patients indicator of the relationship between endometrial hyperplasia and the insulin resistance phenotype of PCOS.The aim of this study is to investigate the possible association between insulin resistence and INSR genes methylation in PCOS women. We want to explore the relationship between insulin resistence and methylation of INSR genes on the endometrium of polycystic ovary syndrome. We also want to investigate the possible roles of DNMT1,DNMT3a and DNMT3b playing in the pathogenesis and development of PCOS by detecting the expression of DNMT1,DNMT3a and DNMT3b in PCOS patients with and without insulin resistance and women without PCOS.Part one Relationship between insulin resistance and methylation of INSR genes in endometria of patients with polycystic ovary syndromeObjectivePolycystic ovary syndrome(PCOS) had been proven to be a familial condition. The role of genetic factors, as well as the environmental factors contribution in PCOS was strongly supported. The genes that are involved in the etiology of the PCOS had not been fully investigated. The epigenetic abnormality hypothesis has established a novel mechanism for PCOS development and inheritance. DNA methylation is the most common epigenetic modification style. We wonder hyperinsulinemia and insulin resistance would lead to epigenetic change in PCOS patients. The aim of this study was to explore the relationship between insulin resistance and methylation of INSR genes on the endometria of PCOS.Methods35 patients with PCOS were included in the study. They were divided into two groups according to their HOMA index. The diagnosis of PCOS was made according to Rotterdam Consensus.18 endometrial samples from PCOS patients without insulin resistance (PCOS-NIR group) and 17 endometrial samples from PCOS patients with insulin resistance (PCOS-IR group) were collected. The data of age, serum estridol, testosterone(T), follicle stimulating hormone(FSH) and luteinizing hormone(LH), fasting insulin(FINS) and fasting blood glucose(FBG), HOMA index between PCOS-NIR group and PCOS-IR group were compared. Human endometria were obtained with a Pipelle suction curette from the corpus of the uteri of patients with PCOS. DNA methylation patterns of INSR genes in endometrial cells were detected by methylation specific PCR in both PCOS groups.ResultsThere were significant difference between two groups on BMI, WHR, level of FBG, level of FINS and HOMA index (P<0.05). In the MSP assay,13 samples in PCOS-IR group and 11 samples in PCOS-NIR group showed partially methylated in the promoter region of INSR. No whole methylation banding of INSR genes was detected in endometrial cells of these two PCOS groups. The difference in the methylation status in the two groups was not statistically significant (p=0.328, two-sided test).Summary1. PCOS patients with insulin resistance had higher BMI, WHR, level of FBG, level of FINS and HOMA index than that of PCOS patients without.2. Our study proved high partially methylation in the promoter region of INSR in PCOS patients. It indicated the association between mechanism of PCOS and epigenetics.Part two The messenger RNA expression of INSR genes in endometria with PCOS ObjectiveThe binding of insulin to its receptor provoked auto-phosphorylation which is necessary for insulin signal transduction. This was an important issue because glucose had been proposed as the main energy source of the endometrial cell and high levels of energy are needed to fulfill endometrial functions. Dunaif et al. had reported that in PCOS, the hyperinsulinemia could be triggered by a defect in the expression or activity of molecules located downstream from the insulin receptor. We had tried to explore whether insulin resistance correlate with the methylation of INSR in PCOS in the previous study. The aim of this study was to examine the messenger RNA expression of INSR in endometrial cells.Methods35 patients with PCOS were included in the study. They were divided into two groups according to their HOMA index. The diagnosis of PCOS was made according to Rotterdam Consensus.18 endometrial samples from PCOS patients without insulin resistance (PCOS-NIR group) and 17 endometrial samples from PCOS patients with insulin resistance (PCOS-IR group) were collected. The data of age, serum estridol(E2), testosterone(T), follicle stimulating hormone(FSH) and luteinizing hormone(LH), fasting insulin(FINS) and fasting blood glucose(FBG), HOMA index between PCOS-NIR group and PCOS-IR group were compared. Human endometria were obtained with a Pipelle suction curette from the corpus of the uteri of patients with PCOS. Control endometria were obtained from ten secondary infertile women with regular menstrual cycle at the time of hysteroscopy. Quantitative real-time reverse transcriptase-PCR (RT-PCR) was carried out to quantify the relative messenger RNA(mRNA) levels of INSR in isolated endometrial stromal cells. ResultsThe menstrual cycle of PCOS patients were significant longer than that of controls. The levels of T, LH, LH/FSH in controls were lower than those of PCOS patients, the difference showed statistically significant (P< 0.05). The WHR, weight, BMI, level of FBG, level of FINS and HOMA index of PCOS-IR patients were significantly higher than those of PCOS-NIR patients and controls(P<0.05).The relative mRNA level of INSR gene was calculated with relative quantitation of gene expression. The relative quantitation of INSR mRNA expression was 0.11±0.16,0.16±0.19 and 0.15±0.16 in PCOS-IR group, PCOS-NIR group and control group, respectively. There were no significant differences in INSR mRNA levels between groups from PCOS patients with or without insulin resistance and controls(P>0.05).Summary1. We proved the mRNA expression of INSR gene in endometrial stromal cells in PCOS patients, which was necessary for insulin combination and signal transduction.2. We found that the mRNA expression of INSR gene in PCOS-IR patients was lower than that in PCOS-NIR patients. However, our studies failed to prove any possible correlation between insulin resistance and mRNA expression of INSR genes in PCOS patients.Part three:The expression of deoxyribonucleic acid methyltransferases DNMT1, DNMT3A, and DNMT3B in patients with PCOS ObjectiveDNA methyltransferases (DNMTs) was the key emzymes of DNA methylation. DNMTs were not only associated with DNA methylation, but also linked to many important biological activities, including cell proliferation, senescence and cancer development. These DNMTs were crucial for mammalian growth and development.The aim of this study was to investigate the possible roles of DNMT1,DNMT3a and DNMT3b playing in the pathogenesis and development of PCOS by detecting the expression levels of gene DNMT1, DNMT3a, and DNMT3b in PCOS patients with and without insulin resistance and controls.Methods35 patients with PCOS were included in the study. They were divided into two groups according to their HOMA index. The diagnosis of PCOS was made according to Rotterdam Consensus.18 endometrial samples from PCOS patients without insulin resistance (PCOS-NIR group) and 17 endometrial samples from PCOS patients with insulin resistance (PCOS-IR group) were collected by suction curette from the corpus of the uteri. Control endometria were obtained from ten secondary infertile women with regular menstrual cycle at the time of hysteroscopy. The data of age, serum estridol(E2), testosterone(T), follicle stimulating hormone(FSH) and luteinizing hormone(LH), fasting insulin(FINS) and fasting blood glucose(FBG), HOMA index between PCOS-NIR group, PCOS-IR group and control group were compared. Immunofluorescene staining and western Blot were used to examine the localizations and the expression of DNMT1, DNMT3a, DNMT3b in the PCOS-IR group, the PCOS-NIR group and controls without PCOS. We also compared the expression levels of these DNMTs in the three groups. ResultsDNMT1,DNMT3a, and DNMT3b were located in the nucleus and cytoplasm of stromal cells by immunofluorescene staining. Compared with control subjects or PCOS-NIR patients, the expression levels of DNMT3b by immunofluorescene staining were significantly higher in PCOS-IR patients, the results had statistically significant (P<0.05). In westem blotting, semi-quantitative analysis showed the relative expression of DNMT3b protein were significantly higher in insulin-resistant PCOS patients, the results had statistically significant (P<0.05). But the expression levels of DNMT1 and DNMT3a were similar in these three groups (P>0.05). We also calculated and tested the statistical significance of Pearson's correlation coefficients of expression levels among the 3 genes. The results indicated that the expression levels of these 3 genes are correlated positively. The expression levels of DNM'T3b and serum testosterone, LH, WHR, BMI, FBG, FINS and HOMA index were correlated positively.Summary1. DNMT1, DNMT3a and DNMT3b were located in the nucleus and cytoplasm of stromal cells. The genes DNMT3b were over-expressed in the endometria of PCOS patients with insulin resistance by immunofluorescene staining.2. The relative expression of DNMT3b protein was significantly higher in insulin-resistant PCOS patients than that in non-insulin resistant PCOS patients and women without PCOS by western blotting analysis. The results obtained from western blot were according with those obtained from immunofluorescene staining.3. The expression levels of DNMT3b measured by immunofluorescene staining and western blot were significantly higher in insulin-resistant PCOS patients than those in women without PCOS. Because DNA methyltransferases DNMT3b was a key enzyme for genes DNA methylation. This provided a strong piece of evidence that PCOS ultimately may be an epigenetic disease.4. The expression levels of DNM'T3b and DNMT1 measured by western blot were correlated positively.5. The changes of over expression levels of the DNM'T3b in insulin-resistant PCOS patients would play an important role in the pathophysiology and development of endometrial cancer in them. Because DNM'T3b over expression would lead to endometrial cancer prior to the Genes DNA methylation.Conclusions1. PCOS patients with insulin resistance had higher BMI, WHR, level of FBG, level of FINS and HOMA index than that of patients without PCOS.2. We found that partially methylated in the INSR promoter region of PCOS patients with insulin resistance were higher than PCOS patients without insulin resistancce. The mRNA expression of INSR gene in PCOS-IR patients was lower than that in PCOS-NIR patients. Our study proved high partially methylation in the promoter region of INSR in PCOS patients. It indicated the association between mechanism of PCOS and epigenetics.3. DNMT1, DNMT3a and DNMT3b were located in the nucleus and cytoplasm of stromal cells. The genes DNMT3b were over-expressed in the endometria of PCOS patients with insulin resistance by immunofluorescene staining.4. The relative expressions of DNMT3b protein were significantly higher in insulin-resistant PCOS patients than those in non-insulin resistant PCOS patients and women without PCOS by western blotting analysis. The results obtained from western blot were according with that obtained from immunofluorescene staining.5. The expression levels of DNM'13b measured by immunofluorescene staining and western blot were significantly higher in insulin-resistant PCOS patients than that in women without PCOS. Because DNA methyltransferases DNMT3b is a key enzyme for genes DNA methylation. This provides a strong piece of evidence that PCOS ultimately may be an epigenetic disease.6. The expression levels of DNM'T3b and DNMT1 measured by western blot were correlated positively.7. The changes of over expression levels of the DNM'T3b in insulin-resistant PCOS patients would play an important role in the pathophysiology and development of endometrial cancer in them. Because DNM'T3b over expression would lead to endometrial cancer prior to the genes DNA methylation.It is important to note that to our knowledge this is the first study to demonstrate over expression levels of the DNM'T3b in endometria of insulin-resistant PCOS patients, This provides a strong piece of evidence that PCOS ultimately may be an epigenetic disease and insulin-resistant PCOS patients had higher risk for endometrial cancer. Nevertheless, the experimental design and the data of the present study do not allow us to conclude that over expression levels of the DNM'T3b can affect the INSR genes DNA methylation.
|
PDF Full Text Request