Spatial Transcriptomic Atlas Reveals That Forkhead Box O3‐Mediated Mitochondrial Dynamics Imbalance Drives Premature Ovarian Insufficiency in Mice

Premature ovarian insufficiency (POI) is a major driver of female reproductive aging, but its mechanisms and the spatial and structural patterns of reproductive aging remain poorly understood. This study, therefore, constructed a spatial transcriptomic atlas of POI mouse models to define the spatial and molecular features of granulosa senescence during disease progression. Spatial analysis revealed disrupted follicular structure and distinct granulosa subpopulations exhibiting blocked differentiation and senescence-associated gene signatures. Integrating multiple gene sets identified structural and functional mitochondrial impairment, excess fission, reduced fusion, mitochondrial membrane potential loss, insufficient ATP production, and reactive oxygen species accumulation as central features of granulosa senescence in POI. KEGG pathway enrichment implicated FOXO signaling in regulating mitochondrial dysfunction, and FOXO3 phosphorylation was significantly reduced in POI. In a triptolide-induced KGN cell POI model, pharmacological inhibition of aberrant FOXO3 activation partially restored mitochondrial morphology and function, whereas suppressing FOXO3 phosphorylation in normal KGN cells induced mitochondrial dysfunction. AAV-mediated FOXO3 overexpression in mouse granulosa cells recapitulated the senescent phenotype and mitochondrial dynamic imbalance, activating PINK1/PARKIN-mediated mitophagy signaling. Physiologically aged 10-month-old mouse ovaries showed identical hallmarks-reduced p-FOXO3, upregulated senescence markers, and disrupted mitochondrial dynamics-suggesting a conserved feature of ovarian functional decline. Together, these findings demonstrate that aberrant FOXO3 pathway activation disrupts mitochondrial dynamic homeostasis, driving granulosa senescence and ovarian failure in POI. By integrating spatial transcriptomics with functional and mechanistic analyzes, this study establishes a spatially resolved framework for understanding ovarian aging and identifies FOXO3-regulated mitochondrial pathways as potential diagnostic and therapeutic targets for POI.