Multi-omics analyses link dysregulated immune hemostasis to decreased endometrial MMP protein in recurrent pregnancy loss

This study aims to explore the potential mechanisms contributing to recurrent pregnancy loss (RPL) and identify potential biomarkers. We analyzed endometrial samples from 39 RPL and 22 healthy controls using proteomics, identifying differentially expressed proteins (DEPs) with DEqMS and quantifying immune cell infiltration with CIBERSORT. We also examined a decidual scRNA-seq dataset (CRA002181) to identify macrophage marker genes, performed WGCNA to correlate macrophage infiltration with protein expression, and conducted functional enrichment analysis. Immunofluorescence staining was conducted to quantify the proportions of CD3+, CD8a+, CD68+, and CD206+ cells, and Western blot analysis was performed to determine MMP9 expression in endometrial tissues from RPL and controls. M2 macrophages and CD8+ T cells were enriched in the endometrium of RPL. Proteomic analysis of endometrial tissues from RPL identified 310 DEPs associated with immune response, cell adhesion, and secretory granule processes. From the scRNA-seq dataset, 295 macrophage marker genes were identified. WGCNA revealed that the brown module exhibited the highest correlation with macrophage infiltration. MMP9 emerged as the sole intersecting candidate, showing marked downregulation in RPL. Mendelian randomization analysis showed that genetically predicted higher circulating MMP9 levels decrease RPL risk (OR 0.75, 95% CI 0.57–0.98; p=0.036). Additionally, RPL patients showed significantly higher proportions of CD3+CD8a+ cells (p<0.05) and CD68+CD206+ cells (p=0.265), and decreased MMP9 protein expression (p<0.05), compared with controls. In conclusion, this study indicates that an imbalance in endometrial M2 macrophage and CD8+ T cell immune infiltration is associated with RPL. MMP9 may represent a potential predictive biomarker for RPL.