USP9X as a Candidate Mediator of Prenatal Aspirin‐Induced Ovarian Reserve Reduction in Offspring Mice

This study suggests that prenatal aspirin exposure is associated with reduced ovarian reserve in offspring, associated with HDAC1‐linked epigenetic downregulation of Usp9x as a candidate mechanism. These preclinical findings provide new insights into fetal‐origin ovarian disorders and contribute to the evidence base concerning aspirin's gestational safety. ABSTRACT Aspirin, widely used during pregnancy to prevent complications, may adversely affect fetal development. This study investigates prenatal aspirin exposure (PAE) on ovarian reserve in female offspring. Pregnant mice received aspirin (5, 10, and 20 mg/kg·d) from gestational days 918. Morphological and functional analyses of ovaries across prenatal to postnatal stages revealed PAE (20 mg/kg·d) reduced primordial and antral follicle counts, increased follicular atresia, and impaired ovulation in adulthood. Transcriptomics identified sustained downregulation of Usp9x as a potential mediator, which correlated with suppression of the HIF1α/NOBOX signaling axis. Mechanistically, our data suggest that aspirin exposure is associated with increased HDAC1 activity and enrichment of HDAC1 at the Usp9x promoter, accompanied by a reduction of H3K27ac marks, thereby potentially epigenetically silencing Usp9x expression. This enhanced HIF1α ubiquitination and degradation, ultimately attenuating NOBOX‐mediated transcriptional regulation of downstream follicular genes. Using in vitro fetal ovarian cultures and NIH3T3 cells, we confirmed aspirin's disruption of primordial follicle assembly via the USP9X‐HIF1α‐NOBOX pathway. Crucially, HDAC1 knockdown or pharmacological inhibition rescued USP9X expression and restored follicular development. Our findings indicate that PAE reduces ovarian reserve through HDAC1‐linked epigenetic silencing of Usp9x, exacerbating HIF1α/NOBOX pathway dysfunction, thereby informing aspirin's gestational safety and future interventions for fetal‐origin ovarian disorders. This study suggests that prenatal aspirin exposure is associated with reduced ovarian reserve in offspring, associated with HDAC1-linked epigenetic downregulation of Usp9x as a candidate mechanism. These preclinical findings provide new insights into fetal-origin ovarian disorders and contribute to the evidence base concerning aspirin's gestational safety. ABSTRACT Aspirin, widely used during pregnancy to prevent complications, may adversely affect fetal development. This study investigates prenatal aspirin exposure (PAE) on ovarian reserve in female offspring. Pregnant mice received aspirin (5, 10, and 20 mg/kg·d) from gestational days 918. Morphological and functional analyses of ovaries across prenatal to postnatal stages revealed PAE (20 mg/kg·d) reduced primordial and antral follicle counts, increased follicular atresia, and impaired ovulation in adulthood. Transcriptomics identified sustained downregulation of Usp9x as a potential mediator, which correlated with suppression of the HIF1α/NOBOX signaling axis. Mechanistically, our data suggest that aspirin exposure is associated with increased HDAC1 activity and enrichment of HDAC1 at the Usp9x promoter, accompanied by a reduction of H3K27ac marks, thereby potentially epigenetically silencing Usp9x expression. This enhanced HIF1α ubiquitination and degradation, ultimately attenuating NOBOX-mediated transcriptional regulation of downstream follicular genes. Using in vitro fetal ovarian cultures and NIH3T3 cells, we confirmed aspirin's disruption of primordial follicle assembly via the USP9X-HIF1α-NOBOX pathway. Crucially, HDAC1 knockdown or pharmacological inhibition rescued USP9X expression and restored follicular development. Our findings indicate that PAE reduces ovarian reserve through HDAC1-linked epigenetic silencing of Usp9x, exacerbating HIF1α/NOBOX pathway dysfunction, thereby informing aspirin's gestational safety and future interventions for fetal-origin ovarian disorders. Advanced Science, EarlyView.