Identification of Cryosensitive Niches and a Targetable FOS/AP‑1 Program in the Human Ovarian Cortex by Single‑Cell and Spatial Transcriptomics

Ovary is a vital and dynamic reproductive organ. Ovarian tissue cryopreservation (OTC) plays vital role in female fertility preservation, especially for adolescent female cancer patients. Yet, the sensitive cell populations and cryoinjury molecular mechanisms underlying cryopreservation remain elusive. This study employs single-cell RNA sequencing and spatial transcriptomics to dissect the impacts of temperature stress and cryoprotectant toxicity on the human ovarian cortex. The spatiotemporal molecular characteristics of eight ovarian cell types following vitrification-thawing had been comprehensively characterized. Granulosa, perivascular and stromal cells are identified as most sensitive cell types to OTC procedure. Further analysis using Smart-seq2 on oocytes highlights alterations in “cell cycle” and “DNA methylation” pathways. Notably, the FOS/AP-1 pathway emerges as a crucial response factor to stress and toxicity during cryopreservation. Inhibition of this pathway with T-5224 mitigates vascular damage and reduces apoptosis in vitrification-thawed ovaries. These findings provide insight into the spatiotemporal dynamics during ovarian vitrification and thawing, aiding in prioritizing therapeutic strategies for pre- and post-cryopreservation interventions.