| Background:Studies have shown that the uterine cavity contains a lower abundance of bacterial communities,similar to the vagina,which also has a predominant composition of Lactobacillus.Currently,there is a lack of clarity regarding the potential impact of different uterine flora on embryo implantation as well as pregnancy maintenance and their mechanisms.In previous studies,pregnancy success after embryo transfer was significantly lower in germ-free mice reared under aseptic conditions than in mice reared under conventional conditions,suggesting a potential role for intrauterine bacteria in the process of embryo implantation in mice.So,do bacteria colonizing the human uterine cavity play a similar role in embryo implantation?Given that this potentially critical issue is largely unexplained,the potential impact of human intrauterine flora on embryo implantation and the mechanisms of their influence deserve further exploration.Objective:Part I:Analysis of the compositional characteristics of the flora in reproductive tract samples at the endometrial receptive stage in patients who achieved different pregnancy outcomes after embryo transfer and the differences between groups(including species and metabolic pathways)to provide clues for further studies on the potential association between reproductive tract flora characteristics and pregnancy outcomes after embryo transfer.Part II:To assess the correlation between the uterine flora and the degree of endometrial gene expression/immune cell infiltration during the receptive period.To provide a basis for further research into the biological pathways by which the uterine flora may influence embryo implantation.Part III:Screening of differential genera from uterine cavity samples and a preliminary exploration of their regulatory mechanisms during embryo implantation.Methods:Part I:64 patients receiving human assisted reproductive technology(human assisted reproductive technology)treatment were grouped according to their pregnancy outcomes(live birth and non-pregnancy)after embryo transfer,and the composition characteristics of the microflora in their uterine secretion(CS),endometrial fluid(EF)and endometrial biopsy,EB)samples derived from receptive period,and differences between groups(including species and metabolic pathways)were analyzed using bacterial 16S ribosomal RNA sequencing method.Part II:RNA sequencing was conducted for the same EB samples,the differences in gene expression were analyzed,and the function of differentially expressed genes were annotated.CIBERSORT and Immu Cell AI network tools were used to calculate the infiltration degree of22 kinds of immune cells and 18 kinds of T cell subgroups in EB.Correlational analysis were performed using DEGs/differential immune cells and differential microbial taxa in EB and EF samples from the same patient,so as to provide clues for further research on the biological pathway through which reproductive tract microflora that may affect embryo implantation.Part III:The mechanism experiment is designed according to the data of different microorganisms in reproductive tract samples between groups,and the correlation between different microorganisms and different immune cell infiltration obtained from the precious two parts.Immunofluorescence analysis was used to analyze the differences in the expression levels of corresponding immune cells in EB between the two groups of patients,and the correlation between the above indexes and the relative abundance of differential bacteria between groups were analyzed,and Western Blot was used to analyze the differences in the expression levels of related proteins for immune cells.Then,RT-q PCR was used to analyze the differences in the expression levels of related genes for the immune cells,to verify the results of the previous two parts.Then,conditional culture,sorting and passage of key bacteria in uterine fluid were performed.16S r RNA full-length sequencing was performed to identify their species composition,verify the activity and culturability of functional species in reproductive tract,and preliminarily determine the specific composition of key bacteria in uterine fluid.Finally,the selective expanded culture solution was co-cultured with immune cells in vitro to further verify the possible molecular mechanism of the key bacteria genera colonizated in reproductive tract affecting embryo implantation.Research results:Part I:The bacterial composition of EF and EB is relatively similar,while the CS differs significantly from the other two samples,indicating that compared with the CS samples,the uterine microflora had its unique composition,and the EF and EB samples were not contaminated by CS.The results obtained can represent the real situation of uterine microbiota.The genera with significant differences in CS between the two groups are:Methylobacterium-Methylorubrum,Cutibacterium,Corynebacterium,Pelomonas,Pseudomonas,Sphingosine and Veillonella;in EF which are:Lactobacillus,Gemella,Neisseria,Gastraeromonas,Gracilibacteria,Moraxella,Massilia,Enhydrobacterium and Corynebacterium;and in EB are Lactobacillus and Chryseobacterium.At the genus level,the relative abundance of Lactobacillus showed significant differences in both upper reproductive tract samples(EF and EB samples)between the two groups of patients.The metabolic functions of the microbiome were also analyzed with PICRUST2 based on the Metacyc database.Using the STAMP software,it was found that pathways related to lipid metabolism such as fatty acid elongation(ko00062),and energy metabolism such as thermogenesis(ko04714)have high activity in the EF of non-pregnant patients,while functions in the EF of live birth patients are mainly enriched in pathways such as carbohydrate metabolism,glucose and mannose metabolism(ko00051),lipid metabolism such as secondary bile acid biosynthesis(ko00121),and drug metabolism(ko00983).In the EB sample,the main pathways for differential microbial function enrichment in the non-pregnant group include immune related pathways such as antigen processing and presentation(ko04612),Th17 cell differentiation(ko04659),and lipid metabolism related pathways such as adipocyte factor signaling(ko04920)and pyruvate metabolism(ko00620).However,the function of microbiota in live birth group are mainly enriched in the energy metabolism related pathways such as AMPK signaling pathway(ko04152),c AMP signaling pathway(ko04024)and thyroid hormone signaling pathway(ko04919),lipid metabolism pathways such as ether lipid metabolism(ko00565).Part II:Using DESeq2,153 genes were found to be differentially expressed(log FC≥|1|,P<0.05).The differentially expressed genes(DEGs)were mainly enriched in immune regulation[phosphatidylinositol3-kinase signal(PI3K signal)(GO:001406),phosphatidylinositol mediated signal(GO:0048015),and regulation of cells on transforming growth factorβ(TGFβ)stimulation response(GO:1903844),regulating TGFβReceptor signaling(GO:0017015),TGFβreceptor signaling(GO:0007179),metabolic pathways[regulating lipid metabolism processes(GO:0045833)and adipocyte differentiation regulation(GO:0045600,GO:0045598,GO:0045444)],MAPK activity regulation(GO:0043405),and cell proliferation and migration,and adhesion process[regulating epithelial cell proliferation(GO:0050678),regulating wound healing(GO:0061041),protein serine/threonine kinase(GO:0016049),transmembrane receptor protein serine/threonine kinase signal pathway(GO:007178),cell junction assembly(GO:0034329),cell-cell adhesion(GO:0016339),cell substrate adhesion(GO:0031589),glycosaminoglycan metabolism process(GO:0030203)related GO(Gene Ontology)terms.The KEGG pathway are mainly enriched in the PI3K-Akt signaling pathway,complement system,focal adhesion,extracellular matrix receptor interaction,hematopoietic cell lineage and calcium signaling pathway.The correlation analysis shown that the functions of DEGs related to differential microorganisms are mainly enriched in immune regulation,metabolism,cell proliferation,migration,and adhesion.In particular,the DEGs related to Lactobacilli are functionally rich in immune and metabolic pathways such as antigen binding(GO:0003823),cytokine receptor activation(GO:0004896),ubiquitin protein transferase activity(GO:0004842),protein binding(GO:0044548),transmembrane transport protein activity(GO:0042910,GO:0015174),and ATPase activity(GO:0018455,GO:0004089).The results of immune-infiltration analysis showed that for the non-pregnant group,naive B cells,NK cells and M1 macrophage were increased while follicular helper T cells,plasma cells,memory B cells and Th 1 cells were downregulated compared to live birth group.Lactobacillus in EF is positively correlated with the infiltration of plasma cell,CD4 memory quiescent T cells and follicular helper T cells in EB.While Lactobacillus in EB was negatively correlated with M1macrophages and NKT cells,and positively correlated with central memory T cell,i Treg(adaptive regulatory T cells),activated dendritic cells and Treg 1 in EB.Part III:The results of the previous two parts were verified by further studies based on the differential species of Lactobacillus which coexisting in the two uterine samples,and macrophages with varying infiltration degree between groups and associated with Lactobacillus.Firstly,immunofluorescence analysis shown that,ompared with the live birth group,the non-pregnant group had an increase in the number of CD86+cells(M1 macrophages)(4.06 vs 2.61,P=0.011)and the rate of CD86+cells(1.29%vs 0.60%,P=0.018)in EB.However,there was no statistical difference in the number of CD163+cells(M2 macrophages)(6.17 vs 5.61,P=0.619)and the rate of CD163+cells(1.63%vs 1.28%,P=0.122)between the two groups.The number(r=-0.654,P=0.021)and rate of CD86+cells(r=-0.657,P=0.020)are negatively correlated with the relative abundance of Lactobacillus in EB,and the rate of CD163+cells(r=-0.594,P=0.042)is negatively correlated with the relative abundance of Lactobacillus.Western Blot results shown that compared with the live birth group,the expression of CD86 protein(0.05vs 0.01,P=0.038)in EB of the non-pregnant group was significantly increased,but there was no statistically significant difference in the expression of CD163 protein(0.18 vs 0.20,P=0.766)between the two groups.RT-q PCR shown that compared with live birth group,the expression level of TNF-αm RNA(3.40 vs 0.97,P=0.003)in EB of non-pregnant group was significantly increased,while the expression levels of IL-10 m RNA(0.72 vs 2.64,P=0.014)and TGF-βm RNA(0.68 vs 6.05,P=0.004)were significantly decreased.There was no statistical difference in the expression level of IL-1βm RNA(0.70 vs 2.07,P=0.058)between the two groups.Lactobacillus colonized in utero could be cultured and transited by MRS medium.The 16Sr RNA full-length sequencing suggested that Lactobacillus spp.was mainly composed of Lactobacillus Crispatus and Lactobacillus Jensenii.The co-culture experiment showed that,high concentration of Lactobacillus liquid(10~7CFU/ml,10~8CFU/ml and 10~9CFU/ml)had significant negative effects on macrophage activity.Generally,the optimal concentration of Lactobacillus solution(10~5CFU/ml and 10~6CFU/ml)induced the polarization of macrophages from M1 to M2.Specifically,after Lactobacillus induction,macrophages significantly increased the secretion of M2 polarization markers TGF-β(P<0.05)and IL-10(P<0.05),significantly decreased the secretion of M1polarization marker TNF-α(P<0.05),but significantly increased the secretion of M1 polarization marker IL-1β(P<0.05).Conclusions:Part I:1.The microbiomes in CS samples are significantly different from those in EF and EB samples,while those in EF and EB are more similar.2.Higher relative abundance of Lactobacillus in the intrauterine(EF and EB)are associated with relatively better pregnancy outcomes,but there is no significant correlation between Lactobacillus abundance in CS and pregnancy outcomes.3.Uterine microbiome may affect pregnancy outcome through immune pathways and glycolipid metabolism.Part II:1.There are differences in the degree of immune cell infiltration in EB of patients with different pregnancy outcomes,among which M1macrophages in EB are negatively correlated with pregnancy outcome.2.The intrauterine flora are correlated with the infiltration degree of various immune cells in EB during the receptivity stage,and the relative abundance of Lactobacillus is negatively correlated with the infiltration degree of M1 macrophages.Part III:The intrauterine colonized Lactobacillus can promote the local immune balance of the endometrium by regulating cytokine secretion of macrophages in the receptive endometrium,thus affecting embryo implantation. |
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