Research On Growth Hormone Improving Ovarian Reproductive Function In Mice Model Of Decreased Ovarian Reserve Function And Explore The Mechanism

Research background and purpose:Decreased ovarian reserve（DOR）leads to menstrual disorders,decreased fertility and even infertility.In in vitro fertilization and embryo transfer（IVF-ET）,patients with DOR were suffered poor ovarian response（POR）characterized by fewer oocytes and embryos,and lower clinical pregnancy rate.Research found that growth hormone（GH）can improve clinical pregnancy rate of patients with DOR by improving the quality of oocytes and endometrial receptivity.Due to ethical constraints,it is difficult to research the etiology,pathogenesis and treatment of DOR with female ovarian tissue.DOR animal model is used to solve this problem.The direct evidence that GH improves ovarian reproductive function in patients with DOR are scarece,and the specific mechanism and effective dose of GH is not fully unknown.At present,DOR modeling method is not uniformed,and comparative study of different methods is lacking.Few literature reported that GH improve ovarian reserve in mice with DOR,with not completely mechanismand unconsistent conclusions.Currently,chemotherapy drugs such as cyclophosphamide（CTX）and cisplatin（CDDP）are widely used to establish DOR mice model.Therefore,this study compared CTX and CDDP,and chose the superior model to research the effect of GH on ovarian function by ovarian histopathology and serology.RNA sequencing（RNA-seq）was performed to investigate the mechanism of GH improving ovarian reproductive function.By the research above,therapeutic measures and theoretical basis was provided to improve the outcomes of IVF-ET in patients with DOR.Materials and Methods:DOR mice model was established in female KM mice aged 6～8 week in the same estrous cycle by CTX or CDDP.The mice were randomly grouped（6 mice in each group）:CTX-DOR group,intraperitoneal injection of CTX 50mg/kg,and then 8mg/kg for 14 days;CTX-negative control group,intraperitoneal injection of 0.4 m L normal saline for 14 days;CDDP-DOR group,intraperitoneal injection of CDDP 1.5 mg/kg for7 days;CDDP-negative control group,intraperitoneal injection of 0.4 m L normal saline for 7 days;untreated group,without any intervention.The levels of serum anti-Müllerian hormone（AMH）,follicle-stimulating hormone（FSH）and estradiol（E₂）were measured by enzyme-linked immunosorbent assay（ELISA）.After HE staining,the number of ovarian follicles at all levels and the thickness of ovarian cortex were measured under the microscope.The most typical model of DOR was chose for GH intervention research.The DOR mice model was randomly divided into 4 groups（10 mice in each group）,then intraperitoneal injection of GH for 21 consecutive days:low-dose GH group,GH 0.4mg/kg;medium-dose GH group,GH 0.8 mg/kg;high-dose GH group,GH 1.6 mg/kg;CDDP-DOR group,GH 0 mg/kg.The negative control group（intraperitoneal injection of 0.2m L normal saline from the beginning of modeling to the end of GH intervention,n=10）and the untreated group（no intervention,n=10）were set up.The levels of serum AMH,FSH and E₂were measured by ELISA.After HE staining,the number of follicles at all levels of development were counted,and the thickness of ovarian cortex and medulla were measured under the microscope.RNA-seq was used to detect gene expression and biological function enrichment analysis of differential genes in ovarian at the optimal dose of GH intervention.Result:1.Comparison the effects of CTX and CDDP in establishing DOR mice model1.1 Compared with the untreated group and the two control groups,both CTX-DOR and CDDP-DOR groups showed increased activity and decreased skin gloss,beginning on the 3rd day in CDDP-DOR group and on the 5-6th day in CTX-DOR group.The weight of mice in CDDP-DOR group was significantly lower than that in the untreated group（35.00±2.31 g vs.38.52±1.68 g,P<0.05）.1.2 Compared with the untreated group and the two control groups,the follicles in the CTX-DOR group and the CDDP-DOR group showed cystic expansion or collapse,decreased granulosa cells,increased stroma,and massive inflammatory cell infiltration.Ovarian cortex thinned,medulla thickened,and cortex/medulla ratio decreased,but there was no statistical difference（P>0.05）.1.3 Compared with the untreated group and the two control groups,the number of primary follicles was decreased and the number of atresia follicles was increased in the CTX-DOR group and CDDP-DOR group.The number of antral follicles and secondary follicles was decreased in the CDDP-DOR group than that in the CDDP-negative group and untreated group（P<0.05）.The number of atresia follicles in the CDDP-DOR group was more than that in the CTX-DOR group（P<0.05）.The number of follicles at all levels in the CDDP-DOR group was changed more significantly than that in the CTX-DOR group without significance（P>0.05）.1.4 Compared with the two control groups and the untreated group,the level of serum FSH was increased,and the level of AMH and E₂were decreased in the CTX-DOR group and the CDDP-DOR group（P<0.05）.2.Research of GH on ovarian reproductive function in CDDP-DOR mice2.1 Compared with the negative control group and the untreated group,the behavioral activity,mental state,fur gloss and feeding ability were no significant difference in the GH groups.The weight in the high-dose GH group was higher than that in the medium-dose GH group and the CDDP-DOR group（P<0.05）.2.2 Compared with the CDDP-DOR group,the GH groups showed alleviated ovarian hyperemia and inflammatory infiltration,thickened cortex,thinned medulla,and increased cortex/medulla ratio,obviously with the GH dose.The thickness and ratio of ovarian cortex and medulla was significantly different between the high-dose GH group and the CDDP-DOR group（P<0.05）,whereas no difference between the high-dose GH group and the untreated group（P>0.05）.2.3 The number of primary and secondary follicles in the GH groups was higher than that in the CDDP-DOR group,while the number of atresia follicles in the medium-dose and high-dose GH groups was lower than that in the CDDP-DOR group（P<0.05）.The number of primitive follicles in the high-dose GH group was higher than that in the low-dose and medium-dose GH groups（P<0.05）,whereas the number of secondary follicles,sinus follicles and atresia follicles was no statistical difference（P>0.05）.The number of secondary follicles in the high-dose GH group increased compared to the untreated group（P<0.05）.2.4 The levels of serum AMH and E₂in GH groups are higher,whereas FSH was lower than those in CDDP-DOR group,with significant difference only in the high-dose GH group（P<0.05）.Among the three GH groups,the degree of hormonal changes was evident with increasing GH dose,but without significance（P>0.05）.3.Research of GH on RNA-seq of ovarian tissue in CDDP-DOR mice3.1 The differential genes between CDDP-DOR group and untreated group include Atp5o,Ndufs6 and other genes involved in mitochondrial metabolism,and Cyp2b10,Cyp19a1,LHB and other genes involved in reproductive endocrine function.The significantly enriched functions are mainly related to ciliary movement,ribosome and protein synthesis,mitochondrial metabolism and redox reaction.The significantly enriched pathways are mainly thermogenic effect,oxidative phosphorylation,etc.3.2 The differential genes between high-dose group and CDDP-DOR group include Igkc,Jchain,Igha and other genes involved in immune,and Mecr,Atp5o,mt-Rnr2,Cyp11a1 and other genes involved in mitochondrial metabolism,FSHR and other genes involved in reproductive function.The significantly enriched functions are mainly related to ciliary movement,protein synthesis,and the activity of oxidoreductase.The significantly enriched pathway is mainly the interaction of extracellular matrix.3.3 The differential genes between the high-dose group and the untreated group include Mrps18b,Me3,mt-Nd2 and other genes involved in mitochondrial metabolism.The significantly enriched function is mainly related to ribosome synthesis.There was no significant enrichment pathway between the high-dose group and the untreated group.Conclusions:Both CTX and CDDP can establish DOR mice models.The CDDP modeling cycle is shorter,and the pathological changes of DOR are more typical compared with CTX model.GH can improve ovarian reproductive function in CDDP-DOR mice in a dose-dependent manner.High-dose GH intervention can restore the follicular development and sex hormone to normal levels.GH might improve ovarian function of DOR mice by regulating the expression of genes involved in mitochondrial metabolism,redox reaction,protein and ribosome synthesis.