Abstract

Introduction Trophoblast dysfunction and abnormal cell death are associated with unexplained spontaneous miscarriage (USM), while the underlying mechanisms remain unclear. This research aims to elucidate the pathogenic functions of the RNA-binding protein KH-type splicing regulatory protein (KHSRP) in USM. Methods We first identified reduced KHSRP expression in USM villi by performing integrative analysis of our and public RNA-seq data, followed by validation in clinical samples via RT-qPCR, western blotting, and immunohistochemistry. Functional studies in HTR-8/SVneo and JEG-3 cells with siRNA knockdown and plasmid overexpression assessed proliferation, apoptosis, cell-cycle progression, and DNA damage through CCK-8, EdU, flow cytometry, and immunofluorescence. Downstream targets of KHSRP were identified by integrating siKHSRP RNA-seq, villi RNA-seq, and RIP-seq data, and subsequently validated at both the transcript and protein levels. In vivo , placental Khsrp knockdown was achieved via intrauterine injection of self-complementary adeno-associated virus 8 carrying a short hairpin RNA (shRNA) targeting the Khsrp gene (scAAV-sh Khsrp ). Results KHSRP was downregulated in USM villi. In trophoblast cells, KHSRP loss impaired proliferation, caused G2/M arrest, and triggered DNA damage accumulation and apoptosis in a p53-independent manner. Mechanistically, KHSRP bound and destabilized tissue factor pathway inhibitors 2 (TFPI2) mRNA. KHSRP deficiency upregulated TFPI2, promoting DNA damage accumulation and apoptosis. In mice, intrauterine scAAV-sh Khsrp injection induced embryo resorption, placental structural abnormalities, and increased TFPI2 expression. Discussion KHSRP is downregulated in USM villi, and its loss elevates TFPI2 by impairing mRNA decay, leading to DNA-damage accumulation and trophoblast apoptosis. Thus, KHSRP and TFPI2 may serve as potential therapeutic targets for USM.