Decidualization deficiency is a hallmark pathology of unexplained recurrent spontaneous abortion (URSA), but the undefined molecular drivers hinder the development of effective therapies. Hyperoside, a bioactive flavonoid from Hypericum perforatum, exhibits therapeutic potential against URSA, yet its underlying mechanism of action remains unknown. In this study, we employed an integrated multi-omics approach coupled with a multi-dimensional validation framework that spanned URSA patient decidual tissues, in vivo mouse models, and in vitro telomerase-immortalized human endometrial stromal cell (T-hESC) decidualization system, to systematically investigate hyperoside's mechanism in URSA, with a focus on R-loop-driven endometrial stromal cell senescence. We found that hyperoside dose-dependently reduced embryo resorption and rescued decidualization deficiency by preventing stromal cell senescence. Mechanistically, hyperoside effectively alleviated aberrant intracellular R-loop accumulation, thereby suppressing excessive activation of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway which contributes to the initiation of the cellular senescence program. Further target identification and validation experiments confirmed that DExH-box helicase 9 (DHX9) was the functional molecular target of hyperoside, with the Thr419 residue serving as the critical binding site. Functional validation revealed that DHX9 knockdown or introduction of the T419A point mutation markedly attenuated the anti-senescence and pro-decidualization effects of hyperoside. In vivo experiments further confirmed that uterine-specific knockdown of DHX9 reduced hyperoside's protective effects against R-loop accumulation, cGAS-STING pathway activation, and embryo loss. Collectively, these findings demonstrate that hyperoside alleviates stromal cell senescence and decidualization deficiency in URSA through DHX9-dependent resolution of R-loops and subsequent suppression of cGAS-STING-associated senescence signaling. More broadly, this work identifies R-loop-mediated genomic stress as a previously underappreciated contributor to URSA-associated decidual dysfunction and provides a mechanistic basis for the protective effects of hyperoside through DHX9-dependent R-loop homeostasis.
