Studies On LHR, INSR, AR Genes' Methylation States In Both PCOS Animal Models And PCOS Patients

ObjectivePolycystic ovary syndrome(PCOS) is one of the most common endocrine disorders in women of reproductive age,occurring in 4-12%of these women, occupying 30-60%of anovulatory infertile patients.PCOS is heterogeneous in its clinical and biochemical presentation,and has significant racial diversify.Besides,it is also associated with an increased risk of diabetes mellitus,cardiovascular diseases and endometrial cancer in later life.The diagnosis of PCOS in young women may therefore have major implication for long term health.But the precise etiology, diagnose standards and appropriate individual treatment of PCOS are still unclear. Currently,most of investigators focus on the PCOS,especially its etiology.Although the etiology of PCOS remains uncertain,both genetic and environmental influences have been strongly implicated based on the familial clustering and heterogeneous characters of PCOS cases.Therefore,it has been accepted that PCOS may result from abnormal genomic variants under the influence of environmental factors.However,the numerous investigations intended to identity the specific gene(s) involved have yet to yield any valid conclusion.Additionally,it also remains an open question as to what underlying mechanism mediates the influences of environmental factors on PCOS occurrence. Epigenetics is an investigation focus on the relationship between genotypes and phenotypes.It is defined as the molecular phenomenon that regulates gene expression without alterations to the DNA sequence.Epigenetic modifications are essential for mammalian development and epigenetic states can be modified by environmental factors,which contribute to the development of numerous diseases.Besides its role in mediating the disease phenotype,epigenetic alterations also lead to transgenerational transmission of specific genetic traits or molecular events,which has recently been identified.So,it has been accepted that abnormal epigenetic modification is involved in the development and inheritance of numerous diseases.In 2005,the article of LaVoie inferred that abnormal epigenetic modifications may be also involved in the pathophysiology of ovarian endocrine.Thus,the epigenetic abnormality hypothesis has established a novel mechanism for PCOS development and inheritance.The study of the relationship between epigenetics and the etiology of PCOS is seemed to be necessary and urgent.As high LH level,hyperinsulinemia and hyperandrogenism are well know as three typical pathophysiologic abnormalities and DNA methylation is the most common epigenetic modification style,we decided to detect the DNA methylation patterns of LHR,INSR and AR genes in PCOS patients and PCOS animal models and hope to breakthrough the retardant of the investigation on genes of PCOS getting some new thoughts.PART ONE:The study of LHR,INSR,AR genes' methylation states in DHEA induced SD rat modelAbout 50%of PCOS patients have the symptom of high adrenal androgen level. Moreover,the onset of this disease is around the period of puberty menarche and its pathophysiologic appearances are just the same as the physiologic changing of adrenarche.So,some scholars thought that PCOS was the stretch of adrenarche.The typical physiologic change of adrenarche is hypersecretion of dehydroepiandrosterone and dehydroepiandrosterone sulfate.After this theory,young female SD rats were injected with DHEA daily S.C.to establish an ideal PCOS model.Subsequently we detected the DNA methylation status of LHR,INSR and AR genes in this model.Methods23-day-old female Sprague-Dawley(SD) rats were randomly divided into two groups.The rats in the experimental group were injected with DHEA daily S.C.for up to 20 days while the rats in the control group injected with oil at the same time. Ovarian morphologic changes,sex hormone levels,fasting blood insulin and glucose were detected.The LHR,INSR and AR genes' DNA methylation patterns were checked by methylation specific PCR in modeling group and control group.Results1) The ovarian weight in experimental group was lower than that in control group (P=0.000).Considering about the influence of body weight,there was no significant difference in ovarian weight between the two groups.2) The ovaries in experimental group showed multiple follicular cysts,and the number of theca cells and interstitial cells increased while the number of granulose cells decreased.Kinds of developing follicles and corpus lutea were almost absent.3) The serum estrodiol,testosterone,FSH,fasting insulin and fast glucose concentration were significantly higher in experimental group than those in control group(P＜0.05),so as the HOMA index(P=0.000).There were no obvious differences in the serum LH level(P=0.000) and LH/FSH ratio(P= 0.000) between the two groups.4) No methylation of LHR and AR genes were detected in modeling or control group. The methylation frequency of INSR gene(73.3%) was significantly higher in modeling group than that in control group(P=0.000). Summary1) The rat model induced by DHEA was proved to be an ideal PCOS animal model with similar morphological and hormonal changes to those of PCOS patients.2) The outcomes of no difference in LHR,AR genes' methylation states and significant difference in INSR genes' methylation states between modeling and control group indicated that the transcription of LHR and AR genes were not influenced when the transcription of INSR gene was inhibited,which suggested hyperinsulinemia and insulin resistance maybe the exact key point of PCOS induces by adrenal androgen.3) Either blood insulin level and HOMA index or DNA methylation rate of INSR gene were significant higher in modeling group compared with control group, which indicated that abnormal methylation modification of INSR gene is a novel mechanism for insulin resistance occurring in PCOS.At present,investigators thought signal pathway defects after combining to insulin receptors is the mechanism behind the origin of insulin resistance of PCOS.But we found that the function of insulin receptor itself was changed because of the gene methylation, which is a good supplementary of the mechanism about the origin of insulin resistance in PCOS.PART TWO:The study of LHR,INSR,AR genes' methylation states in levonorgestrel with hCG induced SD rat modelMost researchers regard HPO axis dysfunction especially the hypersecretion of luteinization hormone as the key point of the origin of PCOS.Moreover,LH relates to almost all the typical biochemical abnormality in patients with PCOS,such as hyperandrogenism and insulin resistance.Therefore,we implanted subcutaneous of levonorgestrel silica gel staff to down regulating the secretion function of juvenile SD rats' hypothalamus and pituitary,then the rats were injected with human chorionic gonadotropin daily S.C.to establish an ideal PCOS model.Subsequently we detected the DNA methylation status of LHR,INSR and AR genes in this animal model.Methods24-days-old female Sprague-Dawley(SD) rats were randomly divided into two groups.The rats in the experimental group were given subcutaneous implanting of levonorgestrel silica gel staff(3mm per rat) and begun to inject 1.5 IU hCG twice daily S.C.3 days after for 9 days.The rats in the control group injected with saline at the same time.Ovarian morphologic changes,sex hormone levels,fasting blood insulin and glucose were detected.The LHR,INSR and AR genes' DNA methylation patterns were checked by methylation specific PCR in modeling group and control group.Results1) The ovarian weight(P=0.000) and volume(P=0.000) in experimental group were higher than those in control group.2) The ovaries in experimental group showed multiple follicular cysts,and the number of theca cells and interstitial cells increased while the number of granulose cells decreased.Kinds of developing follicles and corpus lutea were almost absent.3) The serum progesterone,testosterone,luteinizing hormone,fasting insulin and fast glucose concentration were significantly higher in experimental group than those in control group(P＜0.05),so as the LH/FSH ratio(P=0.000) and HOMA index(P=0.000).4) No methylation of LHR and AR genes were detected in modeling or control group. The methylation frequency of INSR gene(76.7%) was significantly higher in modeling group than that in control group(P=0.000). Summary1) The rat model induced by levonorgestrel with hCG was proved to be an ideal PCOS animal model with similar morphological and hormonal changes to those of PCOS patients.2) The outcomes of no difference in LHR,AR genes' methylation states and significant difference in INSR genes' methylation states between modeling and control group indicated that the transcription of LHR and AR genes were not influenced when the transcription of INSR gene was inhibited,which suggested hyperinsulinemia and insulin resistance maybe the exact key point of PCOS induces by LH.3) Either blood insulin level and HOMA index or DNA methylation rate of INSR gene were significant higher in modeling group compared with control group, which indicated again that abnormal methylation modification of INSR gene is a novel mechanism for insulin resistance occurring in PCOS.PART THREE:The study of LHR,INSR,AR genes' methylation states in luteinizing granulose cells of PCOS patientsOnly after the description of scven cases of amenorrhca and bilateral polycystic ovaries by Stein and Leventhal in 1935 was PCOS considered a separate entity that interested clinicians and researchers worldwide.To present,the origin of the typical ovarian morphology—polycystic ovary of this syndrome remains unclear.To deeply understand the polycystic ovary of PCOS,we detect the DNA methylation status of LHR,INSR and AR gencs,hope to find the relationship betwcen epigenctic and PCOS.MethodsThe objects of this research age 25-34 years and BMI 18.5-22.9 had normal basal sex hormone levels,fasting blood glucose and fasting insulin levels without endometriosis.They were all at their first IVF-ET cycle using down regulation long controlled hyperstimulation protocol.The diagnose standard of PCOS referred to Rotterdam Standard in 2003.The LHR,INSR and AR genes' DNA methylation patterns of luteinizing granular cells were checked by methylation specific PCR in PCOS and control group.Patients in PCOS group were oligo-ovulation or anovulation patients with polycystic ovaries and do not have clinical and chemical changes of hyperandrogenism.Patients in control group were normal response infertile women with pelvic or oviduct factors.Results1) 69 infertile patients included in our research consisted of 39 PCOS patients and 30 control women.The PCOS patients' age,BMI,day 3 hormones' level(E₂,T,P, FSH,LH),fasting blood glucose,fasting insulin levels,LH/FSH ratios and HOMA index were all detected without significant differences to control group's.2) No whole methylation of LHR,INSR and AR genes were detected in ovarian luteinizing granular cells of PCOS or control group and there were no differences in partial methylation rates between PCOS and control group(P＞0.05).SummaryIt was a new attempt to research DNA methylation patterns in PCOS patients. According to this research,there were no differences between PCOS and control group of LHR,INSR and AR genes' methylation states,which suggested that the changing of LHR,INSR and AR genes' methylation patterns may not be the true reason which induces the polycystic ovary of PCOS.The farther researches are needed to explore the mechanism behind the development of polycystic ovary of PCOS patients. Conclusions1) The rat models induced by DHEA or by levonorgestrel with hCG were proved to be ideal PCOS animal models with similar morphological and hormonal changes to those of PCOS patients.2) The outcomes of no difference in LHR,AR genes' methylation states and significant difference in INSR genes' methylation states between modeling and control group indicated that the transcription of LHR and AR genes were not influenced when the transcription of INSR gene was inhibited,which suggested hyperinsulinemia and insulin resistance maybe the exact key point of PCOS induces by adrenal androgen or LH.3) Either blood insulin level and HOMA index or DNA methylation rate of INSR gene were significant higher in modeling group compared with control group, which indicated that abnormal methylation modification of INSR gene is a novel mechanism for insulin resistance occurring in PCOS.At present,investigators thought signal pathway defects after combining to insulin receptors is the mechanism behind the origin of insulin resistance of PCOS.But we found that the function of insulin receptor itself was changed because of the gene methylation, which is a good supplementary of the mechanism about the origin of insulin resistance in PCOS.4) There were no differences in LHR,INSR and AR genes' methylation states between PCOS patients and control group,which suggested that changing of LHR, INSR and AR genes' methylation patterns maybe not the true reason result of polycystic ovary of PCOS.More deeply researches are needed to discover the mechanism behind the development of polycystic ovary of PCOS.