Objective This study aims to analyze the relationship between reproductive tract microecological changes, metabolic differences, and pregnancy outcomes at different time points in the frozen-thawed embryo transfer cycle while patients are undergoing hormone replacement therapy, which will be a breakthrough point for improving outcomes.

Methods A total of 20 women undergoing frozen-thawed single blastocyst transfer for the first time at the Reproductive Medicine Center of Fujian Maternal and Child Health Hospital between July 2022 and January 2023 were recruited for this study. Their vaginal and cervical secretions were collected for 16S rRNA sequencing and non-targeted metabolomics analysis on days 2-5 of menstruation, day 7 after estrogen replacement therapy started, the day when progesterone was added, and the day of transplantation. The subjects were divided into different groups according to their clinical pregnancy status and the sequencing results were analyzed using bioinformatics methods.

Results 1) The alpha-diversity index of the vaginal and cervical microbiota was higher on days 2-5 of menstruation (P<0.01), but did not differ significantly on day 7 after oral estrogen replacement therapy started, the day of progesterone administration, and the day of transplantation (P≥0.1). 2) Both the pregnant group and the non-pregnant group showed a variety of microorganisms and metabolites with significant differences in the lower reproductive tract at different time points. 3) Microbial analysis at different time points showed that there were significant differences in vaginal flora, including Peptoniphilus, Enterocloster, Finegoldia, Klebsiella, Anaerobutyricum, Agathobaculum, Sporanaerobacter, Bilophila, Prevotella, and Anaerococcus in the pregnant group (P<0.05). 4) Metabolite analysis at different time points showed that there were significant differences in 3-hydroxybenzoic acid, linatine, (R)-amphetamine, hydroxychloroquine, and L-altarate in the vaginal secretions of the pregnant group (P<0.05), and that there were significant differences in isocitric acid, quassin, citrinin, and 12(R)-HETE in the cervical secretions (P<0.05). 5) Metabolite analysis at different time points showed that, in the non-pregnant group, there were significant differences in linatine, decanoyl-L-carnitine, aspartame, sphingosine, and hydroxychloroquine in the vaginal secretions (P<0.05), and the isocitric acid, quassin, ctrinin, and 12(R)-HETE in the cervical secretions (P<0.05). 6) Combined microbiome and metabolomics analysis showed that certain metabolites were significantly associated with microbial communities, especially Klebsiella.

Conclusions Significant differences in the microbiota genera and metabolites at different time points were found during the frozen-embryo transfer cycle of hormone replacement therapy, which may be used as potential biomarkers to predict pregnancy outcomes of embryo transfer.