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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE285362 | GEO | 2026/04/15

REPOSITORIES: GEO

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Project description:The Portuguese oyster Crassostrea angulata, a bivalve of significant economic and ecological importance, has faced a decline in both production and natural populations due to pathologies, climate change, and anthropogenic factors. To safeguard its genetic diversity and improve reproductive management, cryopreservation emerges as a valuable strategy. However, the cryopreservation methodologies lead to some damage in structures and functions of the cells and tissues that can affect post-thaw quality. Transcriptomics may help to understand the molecular consequences related to cryopreservation steps and therefore to identify different freezability biomarkers. This study investigates the molecular damage induced by cryopreservation in C. angulata D-larvae, focusing on two critical steps: exposure to cryoprotectant solution and the freezing/thawing process. Expression analysis revealed 3 differentially expressed genes between larvae exposed to cryoprotectant solution and fresh larvae and 511 differentially expressed genes in cryopreserved larvae against fresh larvae. The most significantly enriched gene ontology terms were “carbohydrate metabolic process”, “integral component of membrane” and “chitin binding” for biological processes, cellular components and molecular functions, respectively. Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified the “neuroactive ligand receptor interaction”, “endocytosis” and “spliceosome” as the most enriched pathways. RNA sequencing results were validate by quantitative RT-PCR, once both techniques presented the same gene expression tendency and a group of 11 genes were considered important molecular biomarkers to be used in further studies for the evaluation of cryodamage. The current work provided valuable insights into the molecular repercussions of cryopreservation on D-larvae of Crassostrea angulata, revealing that the freezing process had a more pronounced impact on larval quality compared to any potential cryoprotectant-induced toxicity. Additionally, was identify 11 genes serving as biomarkers of freezability for D-larvae quality assessment. This research contributes to the development of more effective cryopreservation protocols and detection methods for cryodamage in this species.

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

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