Integrative Multi-Omics Analysis Unveils Biomarkers Linking the Gut Microbiota, Blood Metabolites, and Recurrent Pregnancy Loss

Weiming Hao,1 Ruigao Song,1 Huimin Lv,1 Chunying Song2 1Reproductive Medicine Center, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Third Hospital of Shanxi Medical University, Tongji Shanxi Hospital, Taiyuan, People’s Republic of China; 2Human Sperm Bank, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Third Hospital of Shanxi Medical University, Tongji Shanxi Hospital, Taiyuan, People’s Republic of ChinaCorrespondence: Weiming Hao, Reproductive Medicine Center, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Third Hospital of Shanxi Medical University, Tongji Shanxi Hospital, Taiyuan, People’s Republic of China, Email haoweiming@sxbqeh.com.cnPurpose: Emerging evidence suggests that dysbiosis of gut microbiota and metabolic disturbance can adversely affect reproductive health. This study seeks to shed light on the connection between gut microbiota, blood metabolites, and recurrent pregnancy loss (RPL), and identify potential biomarkers linking them.Patients and Methods: The associations of gut microbiota and blood metabolites with RPL were explored through Mendelian randomization (MR) and mediation analyses. Differential expression analysis combined with three machine learning algorithms was then used to identify biomarkers that link the gut microbiota-blood metabolite network in RPL. Additionally, a nomogram was constructed to evaluate their predictive performance for RPL. On this basis, immune infiltration analysis and single-cell RNA sequencing (scRNA-seq) were further conducted to gauge the immune characteristics of RPL.Results: A total of 28 gut microbiota and 82 blood metabolites/metabolite ratios showed significant potential associations with RPL. Among them, mediation analysis revealed that 3-amino-2-piperidone amplified the hazardous effect of Photobacterium abundance on RPL (mediation proportion = 14.4%, β = 0.017, P = 0.0478), whereas CAG-495 attenuated the protective effect of cysteine-glutathione disulfide levels on RPL (mediation proportion = 15.5%, β = 0.003, P = 0.0497). ASH1L, G6PD, SETDB1, and LAP3 were identified as biomarkers linking the gut microbiota-blood metabolites network in RPL. The nomogram constructed based on these biomarkers exhibited excellent ability to discriminate RPL, with an area under the curve (AUC) value of 0.972. Finally, scRNA-seq demonstrated an increasing proportion of decidual macrophages and enhanced cell-cell communication in the RPL group.Conclusion: Significant potential links were observed between the gut microbiota, blood metabolites, and RPL. Integrative multi-omics analysis further identified key biomarkers linking gut microbiota, blood metabolites, and RPL, and highlighted the role of the gut microbiota-metabolites-immune axis in the pathogenesis of RPL.Keywords: gut microbiota, blood metabolites, recurrent pregnancy loss, multi-omics analysis, biomarkers, immune