Downregulation of the biological macromolecule Spondin-2 disrupts maternal-fetal immune tolerance and trophoblast invasion, driving unexplained recurrent pregnancy loss

Recurrent pregnancy loss (RPL) affects approximately 1 %–3 % of couples preparing for pregnancy, of which more than 50 % have unknown etiology, referred to as unexplained recurrent pregnancy loss (URPL). This study aimed to investigate the role of Spondin-2 (SPON2) in the pathogenesis of URPL and to elucidate its underlying molecular mechanisms. Using human trophoblast stem cells (TSCs) and extravillous trophoblast differentiation models, combined with single-cell and spatial transcriptomic analyses, in vitro functional assays, and the abortion-prone mouse model, we systematically evaluated the regulatory function of SPON2 in trophoblast biology and maternal–fetal immune homeostasis. We found that SPON2 was highly expressed in human extravillous trophoblasts but was significantly downregulated in the villus tissue of patients with URPL and in the placenta of an abortion-prone mouse model. Mechanistically, SPON2 deficiency triggered excessive accumulation of reactive oxygen species (ROS), which subsequently activated the NF-κB signaling pathway and upregulated OLR1 transcription. This ROS/NF-κB/OLR1 cascade impaired trophoblast migration and invasion and promoted a proinflammatory microenvironment, characterized by elevated secretion of IL-1β, IL-6, and TNF-α as well as enhanced M1 macrophage polarization. Importantly, the in vivo administration of recombinant SPON2 improved pregnancy outcomes in the abortion-prone mouse model by restoring trophoblast function and local immune balance. Collectively, our findings demonstrate that SPON2 modulates trophoblast function and the maternal-fetal immune microenvironment via the ROS/NF-κB/OLR1 axis, highlighting the crucial role of SPON2 in reproductive health and its potential as a therapeutic target for URPL.