Endometrial Stromal Cell-Derived Trimethylamine N-oxide Sustains Decidualization via 14-3-3η/FOXO1 Signaling to Prevent Recurrent Spontaneous Abortion-Metabolomics raw data of human decida and plasma samples & raw data of LiP-SMap

Recurrent spontaneous abortion (RSA), often linked to defective endometrial stromal cell (ESC) decidualization, lacks effective therapeutic strategies targeting metabolic drivers. Here, we identify in situ synthesis of trimethylamine N-oxide (TMAO) in human decidua as a critical safeguard against RSA. Metabolomic profiling revealed markedly reduced TMAO levels in decidual tissues of RSA patients compared to healthy controls. Mechanistically, cAMP-PKA-CREB1 signaling upregulated flavin-containing monooxygenase 3 (FMO3) expression in ESCs during pregnancy, driving local TMAO accumulation to millimolar concentrations. TMAO directly bound the C-terminal domain of 14-3-3η, enhancing its interaction with 3-phosphoinositide-dependent protein kinase 1 (PDK1) to relieve PDK1-mediated suppression of FOXO1. This promoted FOXO1 nuclear translocation, activating decidualization markers (PRL, IGFBP1). Through mouse models employing dietary choline restriction, pseudopregnancy induction, or FMO3 inhibition via shRNA, pharmacological and CRISPR conditional knockout methods, we demonstrated that endometrial TMAO deficiency impairs decidualization and increases pregnancy loss. Notably, TMAO supplementation effectively reversed these pathological outcomes. Strikingly, TMAO restored decidualization capacity in 15% of patient-derived ESCs with inherent dysfunction, correlating with FOXO1 activation. Our findings unveil endometrial in situ TMAO synthesis as a metabolic checkpoint for decidualization and propose TMAO as a therapeutic candidate for RSA rooted in decidual metabolic insufficiency.