| BackgroundEmbryo implantation is one of the key steps in the establishment of normal pregnancy.It refers to the process in which the implantation-competent blastocyst interacts and establishes a tight connection with the receptive uterus.The success rate of in vitro fertilization(IVF)depends largely on embryo implantation.Aneuploid embryos lacking developmental capacity may be the main reason for implantation failure,while around one-third of the normal euploid blastocysts are also unable to be implanted successfully,which suggests that endometrial receptivity plays an important role in embryo implantation.It is reported that about 10%of patients have recurrent implantation failure(RIF)with unknown causes after several IVF treatments,which is a major clinical problem to be solved in the field of assisted reproduction technology(ART).There is currently no consistent definition of recurrent implantation failure in clinical practice,which mostly refers to the situation where female infertility patients under the age of 40 have not achieved clinical pregnancy after at least 3 fresh or freeze-thaw embryo transfer cycles and transferring a total of at least 4 high-quality embryos.The etiology of recurrent implantation failure is complex,involving gamete or embryo,uterus,hydrosalpinx,immunity,thrombosis and so on.Notch signaling pathway is a highly conserved signaling pathway in phylogeny,which plays an important role in embryogenesis,cell development,epithelial-mesenchymal transition(EMT),angiogenesis and other life processes.Changes in certain molecules or downstream events in the Notch signaling pathway can lead to the occurence of pathological conditions.It has been confirmed that Notch1 is expressed in endometrial cells,embryos,and placenta,and is involved in the regulation of endometrial decidualization,which in turn affects the process of embryo implantation.Autophagy is a highly conservative mechanism of lysosomal-mediated self-digestion of eukaryotic cells and protein degradation.It works by circulating amino acids,reducing the number of damaged proteins and regulating protein levels to give response to extracellular signals.It is reported that it plays an indispensable role in the endometrial-related physiological and pathophysiological processes and embryo implantation.However,the role of Notch1 and autophagy in the endometrium with RIF is still unclear.ObjectiveTo explore the effects of Notch1 pathway on autophagy and decidualization of endometrial cells,and the expression of Notch1 pathway and autophagy in the endometrium with recurrent implantation failure to provide a theoretical basis for further research on the molecular mechanism of RIF.Methods1.Immunohistochemical method was used to analyze the localization and dynamic.expression difference of autophagy marker LC3B in the defferent stages of endometrium.Western Blot method was used to detect the different expression levels of LC3B and P62 protein in endometrial tissues at the proliferative phase and mid-secretory period and decidual tissues.2.The third-generation human endometrial stromal cells and Ishikawa cell lines were cultured in vitro.They were divided into control group,decidualization group,DAPT control group and DAPT decidualization group according to whether the cells were treated with decidualization and whether Notch pathway inhibitor(DAPT)was added.The expression levels of LC3B and P62 in each group were detected by Western Blot method.Besides,the mRNA expression levels of Notch1 pathway-related and decidual marker molecules were compared by RT-qPCR method.3.The expression levels of Notchl and LC3B in endometrial tissues of normal and RIF groups during the window of implantation were measured by Immunohistochemistry.The expression levels of LC3B and P62 in the two groups were detected by Western Blot method.Besides,the RT-qPCR method was used to detect the mRNA expression levels of Notchl pathway-related molecules in the two groups of endometrial tissues.Results1.In endometrial tissues,LC3B protein was mainly localized in endometrial epithelial cells,and its expression level was dynamic during the menstrual cycle.The protein expression level of LC3B in the mid-secretory period was significantly higher than that in the proliferative stage(P<0.05),while the protein expression level of P62 showed the opposite trend(P<0.05).2.In the third generation of endometrial stromal cells,the expression level of LC3B protein in the decidualization group was higher than that in the control group(P<0.05).Compared with the decidualization group,the expression of LC3B protein in the decidualization group with DAPT showed a downward trend(P>0.05).Besides,the mRNA expression levels of Notch1 signaling pathway-related molecule,Hesl and decidualization marker molecules IGFBP1 and PRL also decreased(P<0.05).In the Ishikawa cell line,the expression level of LC3B protein after decidualization treatment was higher than that in the control group(P<0.05),and the expression level of LC3B protein in the decidualization group with DAPT decreased compared with the decidualization group(P>0.05).3.The expression level of Notch1 protein in RIF group was lower than that in the normal group(P<0.05),but the expression level of LC3B protein in RIF group was higher than that in the normal group(P<0.05),the expression level of P62 protein in RIF group was lower than that in the normal group(P<0.05).The mRNA expression of Notchl and Hesl in RIF group decreased(P<0.05).Conclusion1.During the window of implantation in the normal endometrium,the Notchl pathway was up-regulated and the level of autophagy increased.2.During the window of implantation in the endometrium with RIF,the Notchl pathway was down-regulated and the level of autophagy increased.3.Notchl pathway-mediated abnormal autophagy may be one of the important reasons for RIF.In addition to the Notchl pathway,other factors are likely to be involved in the regulation of endometrial autophagy and decidualization with RIF,and further research is needed. |
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