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 ELISA. 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 recurrent spontaneous abortion (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 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 sequencing 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.