Therapeutic targeting of the Tryptophan-Kynurenine Axis for HTR-8/SVneo trophoblast proliferation and migration in unexplained recurrent spontaneous abortion

INTRODUCTION: Recurrent spontaneous abortion (RSA) is associated with maternal-fetal interface dysfunction, particularly abnormal trophoblast invasion and proliferation. However, our understanding of the cause of RSA remains limited. METHODS: Plasma Trp and Kyn levels were measured in two groups using enzyme-linked immunosorbent assay. Immunofluorescence and western blot analyses were employed to evaluate the expression of IDO1, VEGFA, and proteins associated with epithelial-mesenchymal transition (EMT) in villous and decidual tissues from patients with RSA. The effects of Tryptophan (Trp) and IDO1-driven Trp-Kynurenine (Kyn) metabolism on trophoblast proliferation, migration, EMT, and angiogenesis were investigated in the HTR-8/SVneo cell line using wound healing, transwell migration, quantitative real-time PCR (RT-qPCR), Western blotting, and tube formation assays. RNA sequencing (RNA-seq) identified differentially expressed genes in cells treated with 500 μM exogenous L-Trp. RESULTS: RSA patients exhibited elevated plasma Trp levels and significantly reduced Kyn levels, indicating decreased IDO1 activity (as assessed by the Kyn/Trp ratio) compared to controls. IDO1, EMT-related proteins, and VEGFA were downregulated in RSA patient tissues. In vitro, L-Trp enhanced trophoblast migration, invasion, EMT, and microvasculature formation via IDO1 activation. The reduced functional capabilities induced by the IDO1 antagonist 1-MT (500 μM) were rescued by Kyn (300 μM). RNA-seq revealed that L-Trp upregulation modulates trophoblast gene expression and functional pathways associated with amino acid metabolism, angiogenesis, and vasculature development. DISCUSSION: Our study reveals a novel molecular mechanism by which Trp metabolism regulates HTR-8 cell function, suggesting that modulating IDO1 activity may represent a therapeutic strategy to improve trophoblast function and pregnancy outcomes in RSA.