The Effect And Mechanism Of Bushen Huoxue Recipe On The Endometrial Receptivity In Mice With Implantation Dysfunction Caused By Controlled Ovarian Hyperstimulation

Background and ObjectiveInfertility has been a public health issue worldwide.Approximately 9% of couples around the world suffer from infertility.With the development of medicine and its increasing success rate,assisted reproductive technology(ART)has been applied in the clinic extensively.The relatively low implantation rate and clinical pregnancy rate are the bottlenecks during the development of in vitro fertilization(IVF).Controlled ovarian hyperstimulation(COH)is a critical process of IVF.During COH,exogenous gonadotropins are commonly used to stimulate the maturation of multiple follicles,which may result in supraphysiological levels of estrogen and progesterone from the ovary.The steroid hormone secretion disorder is detrimental to the development of endometrium during the window of implantation,which leads to the un-synchronization between endometrium receptivity and embryo development,resulting in the implantation failure and early pregnancy loss.Traditional Chinese medicine(TCM)has a long history in the treatment of infertility and other gynecological diseases.According to the theory of TCM,kidney governs reproduction and is the basis of innateness,and women are blood-based and Qi-based.As deficiency of kidney and imbalance of Chong Ren,Qi and blood are the common causes of infertility,tonifying the kidney and activating blood circulation is a common treatment for gynecological diseases.Bushen Huoxue Recipe(BSHXR),the foundational formula for regulating menstruation and fertility treatment,is frequently used in the assisted treatment of IVF patients with repeated implantation failure.Its efficacy has been proved in clinical practice after several years of application.However,the effect and underlying mechanisms of Bushen Huoxue Recipe on embryo implantation dysfunction resulting from COH remains unclear.In this study,we established the implantation dysfunction mouse model through COH and explored the effect of BSHXR on the implantation in mice undergoing COH and also the potential mechanisms.MethodsFirst of all,to establish a mouse model of implantation dysfunction after COH,different doses of pregnant mare serum gonadotropin(PMSG)and human chorionic gonadotropin(h CG)were injected intraperitoneally in mice to induce ovarian stimulation: PMSG 5 IU + h CG 5 IU,PMSG 10 IU + h CG 10 IU,PMSG 30 IU / 100 g + h CG 30 IU / 100 g,PMSG 40 IU / 100 g + h CG 40 IU / 100 g and PMSG 40 IU / 100 g + h CG 100 IU / 100 g.The dosage of PMSG and h CG was determined by comparing the pregnancy rate and the average number of implantation sites.Then,the mouse model of implantation dysfunction after COH was established and was used for the intervention of BSHXR.Female Kunming mice were randomly divided into 6 groups: control group,spontaneous pregnancy with a medium dose of BSHXR(control + BSHXR-M),model group,a low dose of BSHXR(BSHXR-L),medium dose of BSHXR(BSHXR-M)and a high dose of BSHXR(BSHXR-H).The mice in the model,BSHXR-L,BSHXR-M,and BSHXR-H groups were injected intraperitoneally with PMSG(40 IU / 100 g)and h CG(100 IU / 100 g)for superovulation.The mice in the control group and control + BSHXR-M group were intraperitoneally injected with the same amount of normal saline at the same time points.After h CG injection(the control group with saline),the female mice in estrous were caged with the fertile male mice.On the next morning,female mice were examined for vaginal plugs.The day when plug observed was defined as day 1 of pregnancy(D 1).From D 1 on,the mice in the BSHXR groups were intragastrically administrated daily with the corresponding doses of Bushen Huoxue Recipe,with BSHXR-L(5.7 g/kg/d),control + BSHXR-M and BSHXR-M(11.4 g/kg/d),and BSHXR-H(22.8 g/kg/d).The control and model group were given an equal volume of distilled water by gavage at the same time points.All pregnant mice were given by gavage once a day until the day of execution.Some mice were killed on D 8 to determine the pregnancy rate and the number of implantation sites.The serum levels of estrogen and progesterone on D 4 were measured by radioimmunoassay.The protein expression of epithelial cadherin(E-cadherin),integrin β3(ITGB3),Homeobox A10(Hoxa 10),estrogen receptor α(ERα),leukemia inhibitory factor(LIF),heparin-binding EGF-like growth factor(HB-EGF),progesterone receptor(PR)and Indian hedgehog(Ihh)in the endometrium were evaluated by immunohistochemistry and Western blot.ResultsAfter the comparison among mice treated with different doses of PMSG and h CG,intraperitoneal injection of 40 IU / 100 g PMSG and h CG 100 IU / 100 g based on the bodyweight of mice significantly reduced the pregnancy rate and the number of implantation sites on D 8.The implantation dysfunction mouse model was constructed by using this combination.The medium dose of BSHXR did not negatively affect the pregnancy outcomes in mice with spontaneous pregnancy.The low,medium and high doses of BSHXR significantly increased the pregnancy rate and the number of implantation sites,and reduced the fetal loss caused by COH in early pregnancy.COH downregulated the protein expression of E-cadherin,ITGB3,and Hoxa 10 in the endometrium on D 4,which diminished the endometrial receptivity.COH negatively affected the maternal-embryo crosstalk during the window of implantation.While BSHXR up-regulated the protein expression of E-cadherin and ITGB3 in the endometrium,thus improving the endometrial receptivity and enhancing the embryo implantation.Ovarian hyperstimulation caused the disorder of estrogen and progesterone secretion.High levels of serum sex hormones resulted in the down-regulation of ERα and PR protein expression in the endometrium,which further reduced the expression of LIF,HB-EGF,and Ihh protein.As a result,epithelial-stromal molecular dialogues in the endometrium during the implantation window were interrupted after COH.BSHXR could regulate the secretion of ovarian sex hormones,up-regulated the protein expression of ERα and PR in the endometrium,and enhanced the activation of downstream of ER and PR signaling,ensuring the normal dialogue between endometrial epithelial and stromal cells.BSHXR could promote embryo implantation and subsequent decidualization,and improve the pregnancy outcome in mice after COH.ConclusionIn this study,we found that BSHXR could attenuate the embryo implantation dysfunction caused by COH by improving endometrial receptivity and strengthening the dialogue between uterine and embryo during the window of implantation.Furthermore,BSHXR could control the excessive secretion of estrogen and progesterone in mice undergoing COH,promote the function of estrogen and progesterone receptor signaling,and enhance the interaction between endometrial epithelium and stroma,which is critical to embryo implantation and endometrium decidualization.Additionally,BSHXR did not negatively affect the embryo implantation during natural pregnancy.