| BackgroundInfertility is defined as the inability of a couple to achieve a successful pregnancy after a year or more of continuous sexual intercourse without contraception.In Vitro Fertilization-Embryo Transfer(IVF-ET)technology is one of the core technologies of assisted reproduction,and the process includes removing eggs from the ovaries of infertile women and culturing them in an in vitro environment,then combining them with optimally processed sperm to form a fertilized egg and undergo embryonic development,and finally selecting well-developed Finally,the embryos are selected and transferred to the uterus of the woman to achieve pregnancy.The Female Reproductive System(FRS)exhibits a relatively complex anatomical structure.Disruption of the microbial community structure in the reproductive tract can lead to compromised mucosal barriers,increased abundance of anaerobic bacteria,and the release of toxic factors,thereby affecting reproductive health.The role of microbiota in the female reproductive process warrants further investigation.Previously,the monitoring indicators of female pregnancy mainly focused on sex hormones,endometrial thickness,and fewer studies have been conducted in the microbiological direction,especially in the field of embryo transfer.Therefore,this study focused on exploring the microbial community structures in the vaginal and uterine cavities of women undergoing embryo transplantation,as well as serum metabolites.It aims to identify potential microbial biomarkers associated with adverse pregnancy outcomes and explore the underlying pathways and mechanisms,so that diagnostic tests can provide evidence-based and reliable predictors for clinical diagnosis and treatment.Objectives1.Section Ⅰ:To investigate the association between vaginal and uterine microbiome community structure and pregnancy outcome after embryo transfer using 16S rDNA sequencing,and to explore the microbial markers that predict transfer failure.2.Section Ⅱ:To investigate the characteristics of the serum metabolome of patients with IVF-ET pregnancy failure and to search for differential metabolite enrichment pathways by using non-targeted metabolomics techniques.3.Section Ⅲ:To explore the correlation between differential microorganisms and clinical features and differential metabolites in clinical samples.4.Section Ⅳ:To validate the association between signature microorganisms and adverse pregnancy outcomes through animal models,assess the changes in cytokine levels and histopathologic structures,and explore the potential mechanisms by which microorganisms affect endometrial tolerance.Methods1.Section Ⅰ:Infertile patients undergoing IVF-ET were enrolled,vaginal secretions and endometrial fluids were collected from women on the day of transplantation,and follow-up pregnancy outcomes were categorized into infertile and live birth groups.Four different groups were ultimately obtained in the microbiological analysis panel:51 samples in the vaginal-sourced nonpregnant group,50 samples in the vaginalsourced live-birth group,34 samples in the uterine-sourced nonpregnant group,and 50 samples in the uterine-sourced live-birth group.Using 16S rDNA amplicon sequencing technology,the V4-V5 variable region was amplified and sequenced,and the uniformity and abundance of microorganisms within the groups within the samples were evaluated by the diversity index.The community diversity of microorganisms between the two groups was compared by Principal Co-ordinates Analysis,and the relative abundance of species within the samples and groups was analyzed.The relative abundance of species within the samples and groups was determined by linear discriminant analysis.Linear discriminant analysis was used to determine the significantly different species between the groups,and Random Forest modeling was used to verify the value of different microbial differentiation.2.Section Ⅱ:Infertile patients who underwent IVF-ET were enrolled,serum samples were collected on the day of transplantation,and 20 cases were divided into the infertile group and 20 cases were divided into the live-birth group according to the pregnancy outcome.Non-target metabolomics was performed using liquid chromatography-tandem mass spectrometry to characterize and quantify the metabolites,and modeled by Principal Component Analysis(PCA)and Partial Least Squares-Discriminant Analysis(PLS-DA)modeling to analyze the differences between groups,using univariate statistical analysis to calculate the significant differences of metabolites,using Receiver Operating Characteristic(ROC)to search for valuable metabolic markers,and enriching related pathways.3.Section Ⅲ:Employing linear correlation analysis to investigate the associations between differential microbial relative abundances in the non-pregnant group and the live birth group,as well as exploring the relationships between differential microbial relative abundances in the live birth group and endometrial thickness and hormone levels.4.Section Ⅳ:Constructing a mouse model,the experimental group was given Prevotella solution to lubricate the uterus once every other day;the control group was given PBS buffer for the same period of time,and after 21 consecutive days of treatment,they were dissected and observed for the number of gestational sacs.Serum samples and endometrial tissues were collected,while the remaining mice were randomly combined with healthy male mice in a cage,and the number of gestational sacs was dissected and observed 14 days after the combined cage.T-test was used to statistically assess the intergroup differences between the Prevotella solution group and the control group regarding whether the number of gestational sacs resulted in a significant reduction in the number of gestational sacs,using the observation of mouse uterine epithelium,vasculature and glands by HE staining,the expression of VEGFα in uterine tissues was explored by RT-PCR and Western Blot,and the immunohistochemistry was used to observe the VEGFα,CD31,CK18,and CK19 expression.Changes in serum progesterone were analyzed by Elisa,and serum inflammatory factors were detected using protein microarray technology.And the transcription level and protein expression of IL-1β,Ccl-2,TNF-α,IFN-y molecules in uterine tissues were probed by RT-PCR and Western Blot;immunofluorescence was used to observe CD3+T cell infiltration in uterine tissues and the expression of IL-1β,IFN-γ,TNF-α,and Ccl-2 in the tissues.Result1.Section Ⅰ:The differential microorganism in the microecological study of the uterine and vaginal communities in the non-pregnant group was Prevotella;whereas the differential microorganism in the live birth group was Lactobacillus.2.Section Ⅱ:A total of 1028 metabolites were identified in the serum of both nonpregnant and live birth groups,with 92 significantly different metabolites.Compared to the live birth group,the non-pregnant group exhibited an enrichment of metabolites primarily in three signaling pathways:steroid hormone biosynthesis,aldosterone synthesis and secretion,and phenylalanine metabolism.3.Section Ⅲ:There was a negative correlation between the relative abundance of Prevotella and Lactobacillus(r=-0.561,P=0.013),as well as a negative correlation between the relative abundance of Prevotella and serum progesterone levels(r=-0.528,P=0.043).4.Section Ⅳ:Following Prevotella treatment,there was a significant reduction in the number of pregnancy sacs(P<0.001),uterine blood vessels(P<0.001),and glands(P=0.013)in mice.Additionally,the levels of VEGF,CD31,CK18,and CK19 significantly decreased in uterine tissue(all P values<0.001).Serum progesterone levels showed a significant decline(P<0.001).The infiltration of CD3+T cells in uterine tissue significantly increased,accompanied by a significant elevation of inflammatory factors such as IL-1β,Ccl-2,TNF-α,and IFN-γ in both serum and uterine tissue(all P values<0.001).ConclusionCompared to the live birth group,the abundance of Prevotella in the female reproductive tract significantly increased in the non-pregnant group.Combining the enrichment of differential metabolites in serum,it was observed that pathways related to steroids,aldosterone,and phenylalanine metabolism were downregulated in the serum of non-pregnant women.These metabolic pathway changes are likely the primary factors contributing to the reduced serum progesterone levels.Furthermore,an investigation into the correlation between reproductive tract Prevotella abundance and serum progesterone revealed a negative relationship.Animal experiments confirmed that Prevotella,by reducing progesterone levels,interfering with uterine vascular development,and inducing local and systemic inflammatory responses,leads to adverse pregnancy outcomes.Therefore,it is crucial to pay attention to the ecological balance of the female reproductive tract microbiota before embryo transplantation and consider early interventions to reverse adverse pregnancy outcomes. |
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