The Molecular Mechanisms Of Endometrial SEMA4D On Trophoblast Invasion In Patients With Recurrent Implantation Failure

BackgroundRecurrent implantation failure(RIF)is a serious challenge in the assisted reproductive technology,which refers to failure to embryo implantation or achieve a clinical pregnancy following transferred multiple good-quality embryos undergoing multiple in vitro fertilization-embryo transplantation(IVF-ET)cycles.Despite intensive basic researches and large clinical trials were performed in last decades,the underlying causes of RIF remain unclear,and satisfactory treatment options are still in urgent need.The etiology of RIF is very complex.Multiple factors may contribute to RIF,mainly including embryonic chromosomal abnormalities and poor endometrial receptivity.Study suggested that embryo with an implantation ability can successfully implant into the receptive endometrium lining through apposition,adhesion,and invasion,which is together regulated by the embryonic trophoblast cells and endometrial cells.Whereas abnormal embryos can also successfully implant into the maternal uterus with a receptive endometrium.Therefore,endometrial receptivity is the most critical factor for successful embryo implantation,which promotes the embryonic trophoblast cells invading into decidua and provides nutrients and oxygen to the fetus.However,the regulatory mechanism of endometrial receptivity on trophoblast behavior in RIF remains largely elusive.MethodsWe collected the mid-secretory endometrial tissues from normal fertile women and patients with RIF in this study.To identify the key factors of endometrial receptivity involved in regulating the ability of trophoblast cell invasion in patients with RIF,we employed a strategy consisting of initial screening with transcriptomic sequencing and combined with bioinformatics analysis.Further validation the protein level of key factors in the mid-secretory endometrial tissues from normal fertile women and patients with RIF using Real time-PCR(RT-PCR)assay,Western blotting,immunohistochemistry,and flow cytometric analysis in a large sample size.According to the validation results,in vitro cellular model was next used to explore the functional effects and molecular mechanisms of key factors on proliferation,invasion,migration,and the levels of MMP-2 and MMP-9 of human trophoblast cell lines HTR-8/SVneo,using CCK-8 cell proliferation,transwell invasion and migration assay,Western blotting,ELISA and si RNA technology.Meanwhile,to indentify whether there was a cross-talk between the key signaling molecules and their downstream signaling molecules,we measured the phosphorylation levels of downstream signaling molecules after treatment with related signaling inhibitors,and further to reveal whether this key signaling pathway is involved in the regulation of trophoblast invasion and migration.Results1.The endometrial transcriptome profiles of patients with RIF were significantly different compared to the normal fertile women.We performed different gene analysis and 2885 differentially expressed genes(DEGs)were detected,in which 1453 DEGs were up-regulated and 1432 DEGs were down-regulated.GO analysis found the downregulated DEGs were significant enriched in endometrial receptivity related processes,such as intracellular signal transduction,extracellular matrix disassembly,positive regulation of cell migration,Wnt signaling pathway and cell adhesion,while upregulated DEGs were enriched in the positive regulation of GTPase activity,DNA replication,cell adhesion and others.We constructed a network between positive regulation of cell migration and cell adhesion GO terms using their enriched genes.Two genes,SEMA4 D and APC,were identified as the common hub genes between the positive regulation of cell migration and cell adhesion GO terms.Real-time PCR data further validated the level of SEMA4 D m RNA was significant decreased in RIF endometrium,while APC m RNA levels were similar between two groups.2.The protein levels of endometrial SEMA4 D in patients with RIF were significantly lower than those in the normal fertile women,while the percentages of SEMA4D(CD100)-expressing cells in peripheral blood were similar between fertile control and RIF group during the mid-luteal phase.3.To explore the functional assay using human trophoblast cell line HTR-8/SVneo,the results of in vitro cellular model confirmed that HTR-8/SVneo cells had a higher level of high-affinity receptor Plexin-B1 after human recombinant SEMA4 D treatment.The results showed that SEMA4 D markedly enhanced invasive and migration ability of HTR-8/SVneo cells,while the proliferation of HTR-8/SVneo cells was not significant difference between control and SEMA4 D treatment group.Additionally,our results showed that the concentration of MMP-2 in the supernatant of HTR-8/SVneo cells was significantly increased in the SEMA4 D treatment group compared to the control group,whereas the concentration of MMP-9 in supernatant of cell culture was similar between the two groups.4.The levels of phosphorylation of Met and its downstream signaling molecules phosphatidylinositol 3-kinase(PI3K)and protein kinase B(Akt)in HTR-8/SVneo cells were significantly increased upon SEMA4 D treatment for 24 h,while the total expression levels of Met,PI3 K and Akt were not different between SEMA4 D treatment group and control group.5.We transfected HTR-8/SVneo cells with si RNA-Met to knockdown the expression of Met(si-Met group),the cells treated with scrambled si RNA served as negative control(NC group)and the cells treated with transfection reagent alone served as mock group.The effectiveness of the si RNA-Met was firstly validated by using Realtime PCR and western blotting,the expressions of Met m RNA and protein were markedly reduced in si-Met group compared with that in the NC control.Our results showed the decreased cell invasion and migration in si-Met group with or without SEMA4 D treatment when compared with the NC group.In line with this,the level of phosphorylation of Met,PI3 K and Akt as well as MMP-2 expression were significant decreased in si-Met group with or without SEMA4 D treatment when compared with the NC group.6.When cells were treated with PI3K/Akt specific inhibitor LY294002 for 48 h,the expression of phosphorylation of PI3 K and Akt were effectively suppressed.Likewise,the cell invasion and migration of HTR-8/SVneo cell and MMP-2 expression were significantly decreased upon addition of LY294002 alone or combined with SEMA4 D.Conclusions1.The unique gene expression profile in the endometrium of patients and may associate with the development of RIF.2.SEMA4 D serves as a key factor of the mid-luteal endometrium that might affect the embryo implantation in the patients with RIF.3.SEMA4 D promoted HTR-8/SVneo cells invasion and migration by increasing the level of MMP-2 via the activation of Met/PI3K/Akt signaling pathway in human trophoblast cells.4.Knockdown of Met or inhibition of the PI3K/Akt pathway both abrogated SEMA4 D mediated effects on trophoblast behavior.