Expression Analysis Of Endometrial Immune Factors During Implantation Window In Patients With Repeated Implantation Failure

Research purposeRepeated implantation failure(RIF)generally refers to women with infertility who have failed to obtain a clinical pregnancy after multiple cycles of IVF/ICSI or in vitro fertilization.RIF is an important limiting factor for improving the clinical pregnancy rate after in vitro fertilization-embryo transfer(IVF-ET).The pathogenesis of RIF is complex and unclear,among which the relationship between endometrial immune factors and embryo implantation has attracted much attention in recent years.A large number of studies have shown that the effects of immune cells and cytokines in the endometrium on embryo adhesion,trophoblast invasion,vascular remodeling,and immune tolerance are important factors determining embryo implantation and development.Current studies suggest that the balance of T lymphocyte subsets Th1/Th2 cells in peripheral blood and various cytokines secreted by them are involved in the formation of maternal immune tolerance during pregnancy.However,there are few studies on the expression of cytokines in the endometrium,especially during the implantation window period,and their relationship with the mechanism of RIF occurrence.This study through research on RIF patients with non-transplant patients’ first transplants successful pregnancy period mid-luteal endometrium and the level of a variety of immune factors in the peripheral blood,including Interleukin 6(IL-6),IL-10,the TNF alpha,and the nf-kappa B,analysis of the endometrium and whether the level of cytokines in the peripheral blood has consistency,and analyze its correlation with clinical indicators,to reveal the mechanism of RIF from the aspect of endometrial immunity.MethodsThe study was conducted in the Reproductive Medicine Center of Shandong University Qilu Hospital from December 2020 to November 2021.A total of 16 participants who had received at least three previous embryo transfers were recruited RIF patients who had received at least 4 high-quality embryos in total but failed to obtain clinical pregnancy,and 7 control patients who received IVF/ICSI assisted first embryo transfer and obtained clinical pregnancy.Ovulation monitoring was performed in both groups during the non-embryo transfer cycle.Endometrial sampling and peripheral blood sampling were performed on the 7th day after ovulation.The peripheral blood samples were examined by flow high-throughput multifactor assay.Endometrial tissue samples were divided into two parts:one part was immunohistochemistry to detect the expression of IL-6,IL-10,TNF-α,and NF-κB;the other part was real-time fluorescence quantitative PCR to detect the expression of IL-6,IL-10,and NF-κB.Statistical analysis was performed using IBM SPSS Statistics 25.0 software.Results1.Immunohistochemistry showed that the expression of IL-6,IL-10,TNF-α,and NF-κB in the endometrium of the RIF group was lower than that of the control group on day 7 after ovulation(P<0.05).2.Rt-qPCR showed that the expression of IL-6 in the endometrium of the RIF group was lower than that of the control group on day 7 after ovulation,but the difference was not statistically significant(P>0.05),and the expression of IL-10 was significantly lower than that of the control group,the difference was statistically significant(P<0.05).There was no significant difference in the level of NF-κB in the endometrium of the two groups.3.Compared with the control group,the level of IL-6 in the peripheral blood of the RIF group was significantly increased and the level of IL-10 was significantly decreased,with statistical significance(P<0.05).There was no significant difference in TNF-α level between the two groups.4.Correlation analysis results showed that il-6 in peripheral blood of patients in the RIF group was positively correlated with age(R=0.507,P=0.045),IL-10 was negatively correlated with AMH(R=-0.571,P=0.021)and TSH(R=-0.802,P=0.000)there was no significant correlation between other immune factors and clinical indicators.5.Endometrial IL-6 in the RIF group was negatively correlated with basal FSH(r=-0.539,P=0.031),IL-10 was positively correlated with BMI(R=0.597,P=0.015),TNF-αwas negatively correlated with basal FSH(R=-0.528,P=0.035).There was no significant correlation between other immune factors and clinical indicators.In the control group,endometrial IL-10 was positively correlated with infertility years(r=0.762,P=0.047),while other immune factors had no significant correlation with clinical indicators.6.There was no significant correlation between the levels of IL-6,IL-10,and TNF-α in the endometrium and the levels of IL-6,IL-10,and TNF-α in peripheral blood in the RIF group(P>0.05).Conclusions1.Through a small sample study,we found that low expression levels of IL-6,IL-10,TNF-α,and NF-κB in the endometrium during the implantation window may be one of the adverse factors for embryo implantation.2.Through a small sample study,this study found that increased IL-6 levels and decreased IL-10 levels in peripheral blood during the implantation window may be one of the adverse factors for embryo implantation.3.There was no significant correlation between the expression levels of IL-6,IL-10,and TNF-α in the endometrium and corresponding cytokines in peripheral blood of patients with failed embryo repeated implantation at the implantation window stage.