Vaginal microbiota alterations under supraphysiological estradiol state during in vitro fertilization-embryo transfer (IVF-ET) and the association with reproductive outcomes

Abstract

Background: The process of in vitro fertilization-embryo transfer (IVF-ET) induces a maternal supraphysiological estradiol environment during embryo implantation and early development. Estrogen is crucial in modulating the colonization of microbiota within the vaginal epithelium. However, the impact of supraphysiological estradiol levels on the vaginal microbiome and the relationship with pregnancy outcomes remains unclear.

Results: The study aimed to characterize the vaginal microbiota under supraphysiological hormonal conditions. A total of 67 patients undergoing fresh embryo transfer were divided into three groups based on their peak estradiol levels: high-estradiol (HE) group (E₂ > 11,000 pmol/L), median-estradiol (ME) group (E₂ 5,000-11,000 pmol/L), and low-estradiol (LE) group (E₂ < 5,000 pmol/L). Twenty-five patients undergoing frozen-thawed embryo transfer were categorized into natural cycle (NC) group and hormone replacement cycle (HRT) group according to endometrial preparation protocols. Using 16S rRNA sequencing, we found that the vaginal microbiome exhibited variations with changes in peak estradiol levels. The elevated estradiol levels during ovarian stimulation or exogenous estrogen supplementation, significantly reduced alpha diversity, altered beta diversity within the vaginal microbiome, and shifted the vaginal community state types (CSTs) in Chinese infertile women toward Lactobacillus-dominant profiles, resembling those observed in most Asian women previously. However, the reproductive outcomes were not improved by these variations. The Streptococcus_anginosus and Akkermansia abundance correlated with estradiol levels positively, whereas Escherichia-Shigella showed a negative correlation. The abundance of Streptococcus, Atopobium, and Bifidobacterium on the day of embryo transfer may serve as predictors for adverse pregnancy outcomes, as determined by calculating the area under the curve (AUC) values.

Conclusions: Supraphysiological estradiol levels induced by IVF-ET significantly alter vaginal microbiota and shift the CSTs in Chinese infertile women toward patterns of most Asian women. The Lactobacillus dominance under supraphysiological estradiol conditions does not help improve assisted reproductive outcomes. The abundance of Streptococcus, Atopobium, and Bifidobacterium on the day of embryo transfer may serve as predictors for adverse pregnancy outcomes. Among them, Streptococcus correlates positively with peak estradiol levels and may act as a microbial mediator impairing reproductive success under hyperestrogenic conditions. However, further larger-scale researches are needed to identify and elucidate the potential mechanisms.

Keywords: In vitro fertilization-embryo transfer (IVF-ET); Pregnancy outcomes; Supraphysiological estradiol (E2) level; Vaginal microbiome.

Fig. 1

The diversity and taxonomy of vaginal microbiota among groups with different peak estradiol levels. (NC: natural cycle group; HRT: hormone replacement cycle group; LE: low-estradiol group; ME: median-estradiol group; HE: high-estradiol group). (A) Comparison of Shannon diversity indexes of vaginal microbiota. The p value was determined by the Kruskal-Wallis test. ^*p < 0.05, ^**p < 0.01. (B) Principal coordinates analysis (PCoA) of vaginal microbial community structure based on jaccard distance (Adonis p = 0.030). (C) Distribution of vaginal bacterial composition at the phylum level. (D) Distribution of vaginal bacterial composition at the genus level. The top 20 bacterial species are displayed. The horizontal axis represents different groups. The vertical axis represents the relative abundance of vaginal microbiota

Fig. 2

The distribution of microbial community state types (CSTs) between Asian women and five study groups. (NC: natural cycle group; HRT: hormone replacement cycle group; LE: low-estradiol group; ME: median-estradiol group; HE: high-estradiol group) No significant difference was detected among the five groups (p = 0.290) *The distribution map of CSTs in Asian women was replotted based on data from Ravel J et al. [18]

Fig. 3

Differentially abundant species across the groups with different peak estradiol levels. Histogram of LEfSe analysis for vaginal microbiome across above groups. Linear discriminant analysis (LDA) scores greater than 3 are presented (NC: natural cycle group; HRT: hormone replacement cycle group; LE: low-estradiol group; ME: median-estradiol group; HE: high-estradiol group)

Fig. 4

Differentially abundant species across the groups with different pregnancy outcomes. Histogram of LEfSe analysis for vaginal microbiome across above groups. Linear discriminant analysis (LDA) scores greater than 2 are presented (UP: un-pregnancy group; BP: biochemical pregnancy group; AB: abortion group)

Fig. 5

ROC curves for the abundance of Streptococcus, Atopobium and Bifidobacterium in predicting pregnancy outcomes. (A) ROC curve for the abundance of Streptococcus in predicting non-ongoing pregnancy. (B) ROC curve for the abundance of Streptococcus in predicting non-pregnancy. (C) ROC curve for the abundance of Atopobium in predicting early abortion. (D) ROC curve for the abundance of Bifidobacterium abundance in predicting biochemical pregnancy

Fig. 6

Schematic view of our important findings. Although supraphysiological estradiol levels—whether induced by controlled ovarian stimulation or exogenous estrogen supplementation during IVF—may drive a more pronounced shift toward Lactobacillus-dominant vaginal microbiota, this change does not translate into improved pregnancy outcomes. Notably, the abundance of Streptococcus positively correlates with peak estradiol levels and has been linked to adverse pregnancy outcomes, suggesting that Streptococcus may act as a microbial mediator impairing reproductive success under hyper-estrogenic conditions. (IVF: in vitro fertilization; COS: controlled ovarian stimulation; HRT: hormone replacement)