Study On The Expression And Mechanism Of Notch1 And VEGFA In The Endometrium For Recurrent Implantation Failure Patients

The Implantation failure in assisted reproductive technology(ART)is closely related to multiple factors such as embryo quality,endometrial receptivity or immune factors.To date,Recurrent Implantation Failure(RIF)has been an important cause for the failure of in vitro fertilization-embryo transfer(IVF-ET).Studies confirm that even after excluding uterine factors in the implantation of high-quality blastocysts,RIF is still at a rate of up to 10% during IVF-ET.Due to observing the dynamic process in vivo is difficult,there are limited research data on the interaction between human blastocyst and endometrium and the mechanism of embryo implantation,leading to the slow progress of effective treatment in RIF,therefore which is still hot and difficult issue in reproductive medicine.In recent years,it has been reported that Notch signaling pathway plays an important role in embryogenesis,organ development and angiogenesis.Studies have shown that there is the regulation of DLL4 / Notch1 signaling pathway in endometrial cells,embryos and placenta.Notch1 regulates endometrial decidualization and regulates embryo implantation by regulating cell differentiation,proliferation and apoptosis through cell receptor-ligand interaction,which is an important regulatory pathway in endometrial receptivity.As we all known,VEGFA is the vascular endothelial growth factor family,is currently the most potent and most potent in the endometrium.However,In the endometrium of patients with repeated implantation failure,the studies of Notch1 and VEGFA detection and correlation analysis have not yet been clearly reported.Therefore,this study focused on Notch1 and VEGFA,to detect the expression of Notch1(and related genes)and VEGFA in endometrium with recurrent implantation failure,and to analyze the relationship between that in cells,exploring its role in the occurrence and development of RIF.In order to provide a theoretical basis for clinical research and pathogenesis of assisted reproductive technology(ART).ObjectiveTo investigate the expression of Notch1 and VEGFA in the endometrium of LH + 7 days' women with RIF and compared with the normal endometrium of control group,analyzing the relationship between Notch1 and VEGFA by using cell lines in order to explore its role in the occurrence and development of RIF.Methods1.selected normal endometrium at different period in menstrual,and tissues were blocked using wax,by immunohistochemical to analysis the expression of Notch1 in female endometrial and compared with dynamic differences in menstrual.36 patients were selected from March 2017 to December 2017 in the reproductive center from the First Affiliated Hospital of Zhengzhou University,which were conformed inferility and treated with IVE-ET.All patients' endometrium were collected in LH+7 Day,which was the seventh day after injecting with hCG.According to the patient's transplant history,they were divided into recurrent implantation failure,named RIF group(excluding embryonic chromosome abnormalities),and normal female,named control group,using immunohistochemistry to detect the expression of Notch1 and VEGFA in the endometrium.2.RT-PCR technique was used to detect the mRNA expression differences of Notch1,HES1,HEY1,RBPJ,DLL4,VEGFA and HIF-1? in the two groups;Western blot was used to detect the difference in protein expression of Notch1 and VEGFA between the two groups.3.Using human endometrial stromal cells,according to whether treating with decidualization and whether blocking Notch1 by DAPT were divided into A?B?C and D groups: control group,decidualization group,plus Notch pathway inhibitor control group,plus Notch pathway inhibitor decidualization group.The expression of Notch1,HES1,HEY1,RBPJ and VEGFA and HIF in Notch pathway were compared by RT-PCR.Using human endometrial cancer epithelial cell line(Ishikawa),by adding pathway inhibitor(DAPT),to detect the expression of Notch1 and related genes and VEGFA and to explore the mutual regulation between them.Results1.Immunohistochemical results showed that Notch1 and VEGFA protein were mainly expressed in the cytoplasm of endometrial glandular epithelial cells,of which Notch1 expression in early and middle secretory phase was significantly higher than the proliferative phase,VEGFA was also highly expressed in secretory phase.The expression of Notch1 and VEGFA in the endometrium of recurrent implantation failure(RIF)group was lower than that in control group.2.The WB results showed that the expression of Notch1 and VEGFA in the RIF group was lower than that in Control group.RCR showed that the expressions of Notch1,DLL4,HES1 and RBPJ were down-regulated in RIF group,and the expression of VEGFA in RIF group was lower than that in control group(P <0.05).HIF-1? was highly expressed in RIF group.3.The expression of Notch1,HES1,HEY1,DLL4,VEGFA and HIF-1? in decidualization stromal cells were significantly higher than control group.After adding Notch pathway inhibitor,its expression were all lower than that in the decidualization group.After adding Notch pathway inhibitor into Ishikawa cells,Notch1 and related genes were significantly decreased in Ishikawa cells,HIF-1? and VEGFA were further down-regulated.Conclusion1.The decreased expression of Notch1 and VEGFA in the endometrium in the preimplantation stage may be an important reason for the recurrent implantation failure.2.In the endometrium where repeated implantation failures have occurred,the reduction of Notch1 may inhibit VEGFA activity,breaking the equilibrium between Dll4-Notch1 and VEGFA,leading to abnormal decidualization of the implantation phase and inappropriate angiogenesis affects the final implantation of the embryo,resulting in repeated implant failures.