Research Of Efficacy And Safety Of MTOR Signaling Pathway Activator MHY1485 On In Vitro Activation Of Human Ovarian Tissue

Background and ObjectivePremature ovarian insufficiency(POI)refers to women with menstrual disturbance and amenorrhea accompanied by persistent lower estrogen level and higher gonadotropin level before the age of 40 and its clinical manifestations are usually infertility.It is very difficult for some POI patients in childbearing age to obtain pregnancy with conventional assisted reproductive technology,while egg donation as an alternative method of preserving fertility is hard to implement due to our country's ethics and other restrictions.Studies have shown that there are still a certain number of primordial follicles which show no response to gonadotropin in the ovary of POI patients.So how to activate these residual primordial follicles to promote their growth,maturity,and even ovulation? It will be a new breakthrough in female fertility preservation and successful pregnancy in POI patients.In vitro activation(IVA)is a new method of female assisted reproductive therapy,which is the focus of current research.Researchers activate the primordial follicles with short time in vitro,transplant the activated ovarian tissue back to the body and then use ovulation methods and IVF-ET technology to obtain pregnancy.In recent years,it has been found that PI3K-AKT pathway,Hippo pathway,etc.,which are related to the development and mature of follicle,and researchers have successfully obtained the birth of offspring mice and human healthy babies by utilizing the corresponding activating means of these signaling pathways.Scientists recently found that mTOR signaling pathway activator MHY1485 can activate mouse primordial follicle development.Whether the mTOR signaling pathway have the same effectiveness to activate the primordial follicles of human ovarian or not? Even though it is effective,its biosafety has not yet been strongly substantiated.The purpose of this study is to establish a standardized mouse model of human ovarian tissue activation-xenograft and then use the model to verify the effectiveness and safety of the mTOR signaling pathway activator MHY1485 on human ovarian primordial follicle activation.This study will explore for the first time the effect of in vitro ovarian tissue activation on the DNA methylation level of ovarian tissue.Then it will conduct a preliminary assessment of the operation's safety and provide a powerful experimental evidence for IVA technique in the clinical application of fertility preservation in POI patients.MethodsIn our research,ovary tissues of patients with excision surgery of non-ovarian factors were collected from the Department of Obstetrics and Gynecology in Changzheng Hospital and were cultured in vitro after fragmentation.Different concentrations of activator MYH1485 were used in vitro culture of ovarian tissue after a short period of time and then we used Western blot to detect mTOR signaling pathway protein and their phosphorylated protein levels.HE staining method was used to compare human ovarian tissue morphology and follicular changes before and after in vitro activation.The ovarian tissues of control group and activation group were respectively transplanted into the kidney capsule of ovariectomized mice and a normalized human ovarian tissue xenograft model was established.After some time,the growth of the grafts were observed and grafts were recovered.The ovarian tissue weight was measured,the development of ovarian follicles in each group was observed and compared by HE staining method and the changes of hormone levels in human ovarian tissue before and after ovariectomy and transplantation were detected by ELISA.Our researches use the human high-throughput methylation chip Infinium Methylation EPIC BeadChip(850k chip)technology to analyze of the activation group,control group and normal group human ovarian tissue genome methylation,and then assess preliminarily the safety of human ovarian tissue in vitro culture and activation technology from the epigenetic aspects.Results1.The activator MYH1485 of mTOR signaling pathway can increase the phosphorylation of downstream mTOR and rps6 protein levels without affecting total mTOR and rpS6 levels,and phosphorylated protein levels increase with increasing concentration(1?M?3?M?10?M).The differences were statistically significant.Treatment with 20?M MYH1485 cannot further increase the downstream p-mTOR and p-rps6 protein levels any more in comparison to 10?M MYH1485.2.After short-term in vitro culture of ovarian tissue,there are no significant difference in tissue morphology between the activation and control group.3.After the castrated surgery of SCID mice,the serum level of AMH drop while FSH increase.After one month of transplantation of human Ovarian tissue to the kidney capsule of SCID mice,the serum level AMH slightly increase while FSH decrease.The differences were statistically significant.4.One month after transplantation,ovarian tissue survived in mice and no obvious tissue necrosis was found.Compared with the control group ovarian,activation group ovarian showed larger growing follicles.The results of weighing of ovary transplantation group showed that the weight of ovary in activation group was significantly higher than that of control group,the difference was statistically significant.5.The results of human ovarian tissue DNA methylation chip show that:(1)There are no significant difference in overall DNA methylation level between three groups of human ovarian tissue.(2)There are 9 methylation-variable sites between control group and normal group including 5 methylation-variable genes,52 methylation-variable sites between activation group and normal group including 43 methylation-variable genes,24 methylation-variable sites between control group and activation group including 18 methylation-variable genes.Conclusions1.The activator MHY1485 can effectively activate the mTOR signaling pathway,activate the primordial follicles and promote follicular growth and development.2.There are no obvious ovarian tissue necrosis and morphological changes in the in vitro culture and activation of human ovarian tissue.3.Human ovarian tissue can survive in the animal model and have hormone secretion.4.The in vitro culture and activation process of ovarian tissue did not cause abnormal DNA methylation,which confirmed the safety of human ovarian tissue in vitro activation technology preliminarily from the epigenetic aspects.5.The method of in vitro activation of ovarian tissue and transplantation of ovarian tissue can provide new assisted reproductive technology for POI patients.