Immunotherapy Regimens And Immune Marker Evaluating For Recurrent Implantation Failure

Background and objective Declining fertility rates and an aging population are problems are problems the words faces,and infertility is one of the main factors leading to these two problems.In recent years,the incidence of infertility has been increasing.Unfortunately,despite advanced technology-assisted treatment,10% of patients still have intractable infertility.Recurrent implantation failure(RIF)has been recognized as a significant obstacle and major challenge to the successful application of assisted reproductive technology(ART),and it also is a foremost cause of adverse pregnancy outcomes.Some progress has been made in the research on the effectiveness of immunotherapy in improving pregnancy outcomes in RIF patients,and some treatment methods have also been used in clinical practice.Nevertheless so far,there is a lack of high-level evidence-based medical research on immunotherapy drugs in this group,and the effectiveness of immunotherapy for RIF patients is still controversial.In this study,bibliometrics,network meta-analysis and cohort study were used to study the current immunotherapy schemes of RIF.Bioinformatics technology and pathological experiments were used to find the target genes to guide immunotherapy.In addition,real-time fluorescent quantitative PCR(q RT-PCR),Western Blot,immunohistochemistry,cell immunofluorescence,cell transfection silencing CD40 expression,adenovirus overexpression gene,in vitro spheroid adhesion experiment and the construction method of animal blastocyst-endometrial embryo implantation model in vitro were applied to clarify the expression of target gene CD40 in RIF patients and explore its possible molecular mechanism.Furthermore,the change in CD40 expression after immunotherapy and its influence on embryo implantation were studied.To provide research evidence for individualized immunotherapy for RIF patients,from screening the optimal immunotherapy regimen to gene-guided immunotherapy.Methods 1)Bibliometric analysis: We searched the RIF-related literature in the Web of Science core database from 2000 to 2020.Bibliometrics was used to analyse the annual publication volume,distribution of countries/institutions,journals,keyword co-occurrence,and co-citation of documents.Moreover,VOSviewer and Cite Space software were used for visual analysis.We further screened the research literature on immunotherapy RIF,used VOSviewer software to analyze the co-occurrence of keywords,and drew a keyword time view to understand the research status and hotspots in this field according to the changes of keywords over time.2)Network meta-analysis: systematically searched the Cochrane Library,Pub Med,Embase and Web of Science databases,and clinical trials registry sites,tracked references to relevant systematic reviews,and screened and included randomized controlled trials of immunomodulatory agents in the treatment of RIF.Stata13.0 software was used to conduct random-effects network meta-analysis,to compare the therapeutic effects of each immunomodulatory agent under different outcome indicators,and to calculate the surface under the cumulative ranking curve(SUCRA).Finally,various interventions were ranked under different pregnancy outcome indicators,and their pros and cons were compared.3)Retrospective cohort study: according to the inclusion and exclusion criteria,RIF patients who received peripheral blood mononuclear cells(PBMCs),platelet-rich plasma(PRP)or granulocyte colony-stimulating factor(G-CSF),and those who did not receive any immunization were collected.One-way analysis of variance was used to compare the baseline information of patients and the situation of transferred embryos,and the chi-square test was used to compare the implantation rate(IR)and clinical pregnancy rate(CPR)of different treatment groups and then evaluate the clinical efficacy of different treatment regimens.4)Screening of immune genes: datasets GSE16532,GSE120103 and GSE92324 from the GEO database were downloaded.Differentially expressed genes(DEGs)were screened by GEO2 R.Downloaded the immune-related genes in the Imm Port database and took the intersection to match the differentially expressed immune-related genes(DEIRGs).GO enrichment analysis and KEGG pathway analysis were performed on DEIRGs.The protein-protein interaction(PPI)network of DEIRGs was constructed using STRING online software and visualized based on Cytoscape software.Core genes and necessary modules in PPI were extracted by Cyto Hubba and MCODE plug-ins.The immune cell infiltration matrix in the tissue was obtained using CIBERSORT software,and Spearman correlation analysis identified the correlation between core genes and immune infiltrating cells.Candidate genes were subjected to GO and KEGG enrichment analysis using Gene set enrichment analysis(GSEA).Receiver operating characteristic curve(ROC)analysis was used to evaluate the diagnostic value of candidate genes for RIF.Finally,clinical endometrial samples were collected,and Western Blot and immunohistochemical methods verified the bioinformatics results.5)Clinical validation and mechanism exploration of target gene CD40: this study collected endometrial tissue samples from 20 RIF patients and 20 non-RIF women,and the expression level of the CD40 gene was detected by q RT-PCR method,and the expression difference between the two groups was compared.The expression of CD40 in Ishikawa cells was knocked down by transfection of sh CD40 to detect the expression of TGF-β/Smad signal pathway-related proteins.The TGF-β signal pathway activator(SRI-011381)was further used to verify whether CD40 regulates the activity of the TGF-β/Smad pathway and thus affects endometrial receptivity.Changes in the expression of receptive markers(HOXA10 and ITGB3)and in vitro spheroid adhesion assays were used to evaluate the receptivity of endometrial epithelium.6)Study on the relationship between target gene CD40 and prognosis after immunotherapy: the endometrial tissue of RIF patients who received G-CSF immunotherapy and those who did not receive immunotherapy were collected,and q RT-PCR and Western Blot detected the expression levels of CD40 gene and protein in RIF endometrial tissue;In the vitro cell model,the changes of CD40 expression after rh G-CSF stimulation were studied by q RT-PCR,Western blot and cellular immunofluorescence methods;At the same time,the effect of CD40 expression or immunotherapy on the degree of embryo implantation was illustrated by using the animal blastocyst-endometrial embryo implantation model in vitro.Finally,immunohistochemistry was used to observe the relationship between CD40 and pregnancy outcomes after immunotherapy in vivo,and the Be Wo spheroid spreading experiment was further used to observe the relationship between CD40 expression and embryo implantation ability after rh G-CSF in vitro.Results 1)The bibliometric analysis finally included 1,764 articles.The analysis results show that the research interest of RIF has continued unabated in recent years,and the number of related literature publications has shown a steady growth trend year by year.The United States publishes the most significant number of articles,followed by China.The journal with the most papers is Fertility and Sterility.Improving pregnancy outcomes through immunotherapy such as PRP and G-CSF is one of the hot topics in current RIF research.The research status and hotspots of immunotherapy for RIF were analyzed,and the results showed that researchers have long been devoted to the study of the immune etiology and mechanism of RIF and topics such as the development of new treatment regimens and the improvement of pregnancy outcomes have become hotspots in this field.2)The network meta-analysis included 21 studies involving 2277 participants and 8 immunotherapies.PBMCs,G-CSF,intralipid,PRP and sirolimus can effectively improve the clinical pregnancy rate of patients,and sirolimus ranks first,followed by PBMCs,PRP,G-CSF and intralipid.Regarding embryo implantation rate,PBMCs and G-CSF were significantly better than placebo.In terms of live birth rate,PBMCs and sirolimus were effective.However,recombinant human leukemia inhibitory factor(r-h LIF)has been shown to reduce the risk of live birth rate.In terms of miscarriage rate,no treatment options can reduce the risk of miscarriage,but PBMCs rank highest for reducing miscarriage rate.3)A retrospective cohort study included 156 RIF patients,including 30 in the PBMCs-treated group,30 in the PRP-treated group,31 in the G-CSF-treated group,and 65 in the control group.The comparison between the four groups showed that,except for the difference in endometrial thickness on the day of embryo transfer,there was no significant difference in other baseline data characteristics(P>0.05).The comparison of pregnancy outcome showed that the IR and CPR of the PBMCs treatment group were 29.8% and 46.7%,respectively;the IR and CPR of the PRP treatment group were 33.9% and 50.0%,respectively;the IR and CPR of the G-CSF treatment group were 30.4% and 45.2%,respectively;IR and CPR in the control group were 16.9% and 23.1%,respectively.The Chi-square test showed that the three immunotherapy regimens had no statistical difference in improving pregnancy outcome(P>0.05),but compared with the control group,the IR and CPR of RIF patients were significantly improved,and the difference was statistically significant.4)In the GSE16532 dataset,124 immune-related differential genes were screened.After constructing the PPI network,24 hub genes were obtained.The results of immune cell infiltration matrix analysis showed that compared with the control group,M0 macrophages and resting mast cells were significantly increased(P<0.05),while activated NK cells were significantly decreased(P<0.05).The correlation of 24 hub genes with M0 macrophages,resting mast cells or activated NK cells was analyzed.17 genes significantly related to immune microenvironment disturbances were screened out and verified in GSE120103 independent data set.In the GSE92324 dataset,17 genes were analyzed,and three genes,CD40,PRF1 and EDN3,were found to be differentially expressed between RIF and ordinary pregnant women.GSEA analysis found that these three genes were involved in multiple immune biological processes and were enriched in immune pathways.ROC analysis found that these three genes had good diagnostic values for RIF,with AUC of 0.951 for CD40,1.000 for EDN3,and 0.778 for PRF1.The validation results of the clinical trial are consistent with the results of bioinformatics analysis,and it is found that CD40,PRF1 and EDN3 are lowly expressed in RIF patients.5)In this study,20 RIF patients and 20 non-RIF women were included to verify the expression difference of CD40 and explore its possible mechanism in RIF.The results showed that the expression level of CD40 in the endometrial tissue of the RIF group decreased,and its low expression was related to the decrease of spheroid adhesion rate of Ishikawa cells,and its low expression inhibited the activity of TGF-β/Smad signal pathway in the cells.It was further found that SRI-011381 could significantly increase the spheroid attachment rate and the expression of receptive markers in Ishikawa cells after activating TGF-β/Smad signal pathway while knocking down CD40 could inhibit the effect of SRI-011381 in Ishikawa cells.6)A total of 24 patients with RIF were included.The expression levels of CD40 m RNA and protein in the treatment group were significantly higher than that in the control group(P<0.05);After Ishikawa cells were stimulated with rh G-CSF in vitro,the levels of CD40 m RNA and protein increased(P<0.05),and the results of cell immunofluorescence experiments showed that CD40 protein was significantly expressed in Ishikawa cells stimulated by rh G-CSF.In the spreading experiment,after adenovirus-mediated high expression of CD40 in monolayer Ishikawa cells or after stimulation with rh G-CSF,the spreading area of mouse embryos was higher than those of the control group;Among the collected 30 RIF endometrial specimens treated with immunotherapy,patients with high expression of CD40 had better embryo implantation rate than those with low expression(? 2=5.334,P=0.021).However,the two groups had no significant difference in clinical pregnancy rate(? 2=1.833,P=0.176)and live birth rate(? 2=1.632,P=0.201).Be Wo spheroid spreading experiment found that Ishikawa cells with low expression of CD40 limited the expansion area of Be Wo spheroids,and this inhibition could be reversed by rh G-CSF stimulation.Conclusion 1)Immunotherapy is one of the frontier areas of RIF research.Researchers have long been devoted to studying RIF’s immune etiology and mechanism.In addition,developing new treatment regimens for various causes to provide personalized treatment and ultimately improve pregnancy outcomes are hot spots in immunotherapy RIF research.2)Immunotherapy is expected to be one of the crucial measures to improve pregnancy outcomes in RIF patients.However,the optimal immunotherapy regimen is still controversial.This study found that PBMCs,PRP and G-CSF have more advantages in effectively improving pregnancy outcomes in RIF patients,and they may be beneficial treatment options for RIF in the future.3)CD40,EDN3 and PRF1 genes are involved in the progression of RIF,affect the balance of the immune microenvironment,and have good diagnostic performance diagnosing of RIF.Among them,CD40 may affect endometrial receptivity through the TGF-β/Smad signaling pathway and becomes the critical gene leading to repeated implantation failure.Therefore,it is more valuable in guiding the immunotherapy of RIF and may be used as a potential diagnostic and therapeutic target of RIF to guide the personalized immunotherapy of RIF patients.However,the significance of EDN3 and PRF1 on immunotherapy needs further study.