[Analysis of Reproductive Tract Microecological Changes During the Frozen-Thawed Embryo Transfer Cycle and Clinical Pregnancy Outcomes]

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. 目的: 分析激素替代冻胚移植周期中不同时间点生殖道微生态变化及代谢差异与妊娠结局的关系。 方法: 选取2022年7月−2023年1月首次行激素替代治疗冻融单囊胚移植患者20例作为研究对象，采集月经周期第2～5天、雌激素替代治疗7 d后、内膜转化日和胚胎移植日的阴道及宫颈分泌物进行16S rRNA测序及非靶代谢组学检测，按照临床妊娠与否进行分组，通过生物信息学方法对测序结果进行分析。 结果: ①阴道及宫颈微生物群α-多样性指数在月经周期第2～5天更高（P<0.01），在口服雌激素替代治疗7 d后、内膜转化日和胚胎移植日差异无统计学意义（P≥0.1）。②妊娠组、非妊娠组阴道及宫颈分泌物在不同时间点存在多种具有显著差异的微生物及代谢物。③不同时间点微生物分析表明妊娠组阴道分泌物Peptoniphilus、Enterocloster、Finegoldia、Klebsiella、Anaerobutyricum、Agathobaculum、Sporanaerobacter、Bilophila、Prevotella和Anaerococcus差异有统计学意义（P<0.05）。④不同时间点代谢物分析表明妊娠组阴道分泌物3-Hydroxybenzoic acid、Linatine、（R）-Amphetamine、Hydroxychloroquine和L-Altarate差异有统计学意义（P<0.05）；宫颈分泌物Isocitric acid、 Quassin、Citrinin和12（R）-HETE差异有统计学意义（P<0.05）。⑤不同时间点代谢物分析表明非妊娠组阴道分泌物Linatine、Decanoyl-L-carnitine、Aspartame、Sphingosine和Hydroxychloroquine差异有统计学意义（P<0.05）；宫颈分泌物Isocitric acid、 Quassin、Citrinin和12（R）-HETE差异有统计学意义（P<0.05）。⑥微生物与代谢组学联合分析表明，某些代谢物与微生物群落具有显著的相关性，尤其是Klebsiella。 结论: 激素替代冻胚移植周期中不同时间点存在多种菌属及代谢的显著差异，有可能会成为预测胚胎移植妊娠结局的潜在生物标志物。

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

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.

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. 目的: 分析激素替代冻胚移植周期中不同时间点生殖道微生态变化及代谢差异与妊娠结局的关系。 方法: 选取2022年7月−2023年1月首次行激素替代治疗冻融单囊胚移植患者20例作为研究对象，采集月经周期第2～5天、雌激素替代治疗7 d后、内膜转化日和胚胎移植日的阴道及宫颈分泌物进行16S rRNA测序及非靶代谢组学检测，按照临床妊娠与否进行分组，通过生物信息学方法对测序结果进行分析。 结果: ①阴道及宫颈微生物群α-多样性指数在月经周期第2～5天更高（P<0.01），在口服雌激素替代治疗7 d后、内膜转化日和胚胎移植日差异无统计学意义（P≥0.1）。②妊娠组、非妊娠组阴道及宫颈分泌物在不同时间点存在多种具有显著差异的微生物及代谢物。③不同时间点微生物分析表明妊娠组阴道分泌物Peptoniphilus、Enterocloster、Finegoldia、Klebsiella、Anaerobutyricum、Agathobaculum、Sporanaerobacter、Bilophila、Prevotella和Anaerococcus差异有统计学意义（P<0.05）。④不同时间点代谢物分析表明妊娠组阴道分泌物3-Hydroxybenzoic acid、Linatine、（R）-Amphetamine、Hydroxychloroquine和L-Altarate差异有统计学意义（P<0.05）；宫颈分泌物Isocitric acid、 Quassin、Citrinin和12（R）-HETE差异有统计学意义（P<0.05）。⑤不同时间点代谢物分析表明非妊娠组阴道分泌物Linatine、Decanoyl-L-carnitine、Aspartame、Sphingosine和Hydroxychloroquine差异有统计学意义（P<0.05）；宫颈分泌物Isocitric acid、 Quassin、Citrinin和12（R）-HETE差异有统计学意义（P<0.05）。⑥微生物与代谢组学联合分析表明，某些代谢物与微生物群落具有显著的相关性，尤其是Klebsiella。 结论: 激素替代冻胚移植周期中不同时间点存在多种菌属及代谢的显著差异，有可能会成为预测胚胎移植妊娠结局的潜在生物标志物。