Vaginal and endometrial microbiome dysbiosis associated with adverse embryo transfer outcomes

Abstract

Background: Assisted reproductive technology (ART) is the most effective method to treat infertility and the pathogenesis of implantation failure after in vitro fertilization-embryo transfer (IVF-ET) is a challenging filed in infertility. Microbes in the female reproductive tract are considered to be associated with gynecological and obstetric diseases. However, its effects on embryo implantation failure are unsured.

Purpose: This study aimed to investigate reproductive tract dysbiosis, identify different bacteria in reproductive tract as potential biomarkers of embryo implantation failure and demonstrate the pathogenesis through metabolites analysis.

Methods: We compared the data from 16S rRNA gene and metagenome in reproductive tracts through QIIME2 and HUMAnN2 by the times of embryo implantation failure on 239 infertile patients and 17 healthy women.

Results: Our study revealed a strong positive correlation between Lactobacillus abundance and embryo implantation success (IS) after IVF-ET. The microbial community composition and structure in reproductive tract showed substantially difference between the embryo implantation failure (IF) and healthy control. Moreover, we established a diagnostic model through receiver operating characteristic (ROC) with 0.913 area under curve (AUC) in IS and multiple implantation failures (MIF), verified its effectiveness with an AUC = 0.784 demonstrating microbial community alterations could efficiently discriminate MIF patients. Metagenome functional analyses of vaginal samples from another independent infertile patients after IVF-ET revealed the L-lysine synthesis pathway enriched in IF patients, along with ascended vaginal pH and decreased Lactobacillus abundance.

Conclusions: This study clarifies several independent relationships of bacteria in vagina and endometrial fluid on embryo implantation failure and undoubtedly broadens the understanding about female reproductive health.

Keywords: Lactobacillus; Diagnostic model; Implantation failure; In vitro fertilization-embryo transfer; Infertility; Microbiome; Reproductive tract.

Fig. 1

Study design and flow diagram. a Samples were collected from CL, CU, CV, and EF sites during embryo transfer; control group was set in the air of lab. b In total, 594 samples were collected from 154 participants, including 17 HC (17 CL, 17 CU, 17 CV, and 17 EF samples), 54 infertile women with IS (53 CL, 54 CU, 54 CV, and 50 EF samples), and 83 infertile women with IF (83 CL, 83 CU, 82 CV, and 67 EF samples). All samples were subjected to 16S rRNA sequencing to characterize the microbiome and construct diagnostic models. HC, healthy control; IS, implantation success; IF, implantation failure; CL, the lower third of the vagina; CU, the posterior fornix; CV, cervical mucus drawn from the cervical canal; EF, endometrial fluid

Fig. 2

Endometrial and vaginal microbiomes in healthy and infertile women. a The numbers of samples in each group in this study. b Comparisons of microbial alpha diversity (determined by Faith’s PD index) in CL and EF samples based on OTU profiles among the three groups. HC, healthy control; IS, implantation success; IF, implantation failure. * P < 0.05, ** P < 0.01, *** P < 0.001. P-values were calculated by Wilcoxon rank-sum test. c PCoA of Bray–Curtis distances in CL and EF samples. d Bacterial community dissimilarities for each group of pairwise comparisons in CL and EF samples. e Relative abundances of vaginal bacterial genera in HC, IS, and IF groups. Bars on the left show the mean relative abundances of each group. f Abundances of Lactobacillus in HC, IS, and IF groups. P = 0.027 across groups; χ² test. g LEfSe was performed to identify genera in endometrial fluid that significantly differed among HC, IS, and IF groups; genera are ranked according to effect size

Fig. 3

Microbial differences associated with number of failures. a The numbers of samples in each group after regrouping. b PC1 based on OTU distribution in CL microbial communities. IS: implantation success; SIF: single implantation failure; MIF: multiple implantation failures. c PCoA of EF microbial communities. P = 0.032, IS vs. MIF; P = 0.199, SIF vs. MIF; P = 0.869, IS vs. SIF; PERMANOVA. Each circle shows a 70% confidence interval for the indicated group. d Z-scores in the heatmap according to relative abundances of 18 genera in CL (across groups). Genera with P < 0.05 are marked with red stars, P < 0.1 with pink stars, and P ≥ 0.1 with white stars. e ROC analysis of performance of the IS–MIF diagnostic model. f Validation of IS–MIF diagnostic model by another fifty-nine samples

Fig. 4

Metagenomic pathway variations in the vaginal microbiome and their correlations with clinical factors. a PCoA based on metabolic pathway distribution. P = 0.049, IS vs. MIF; P = 0.350, SIF vs. MIF; P = 0.406, IS vs. SIF. Each circle shows a 60% confidence interval for the indicated group. b Heatmap comparing relative abundances of metabolic pathways among IS, SIF, and MIF groups. Red, more abundant; blue, less abundant. c Serum progesterone concentrations in patients undergoing IVF-ET. d Line chart of L-lysine biosynthesis II/III/VI. Points and short lines represent the means and 95% confidence intervals. e Vaginal pH values in patients undergoing IVF-ET. f Abundances of Lactobacillus in IS, SIF, and MIF groups. P = 0.091 across groups; χ² test

Fig. 5

Schematic of hypotheses concerning mechanisms by which the L-lysine–BAs–Lactobacillus axis regulates the vaginal microbiome