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Identification of cryosensitive niches and a targetable FOS/AP‑1 program in the human ovarian cortex by single‑cell
Fanghao Guo1,2, Di Sun3,4, Haixia Ding3
1Center for Reproductive Medicine & Fertility Preservation Program, School of Medicine, International Peace Maternity and Child Health Hospital, Shanghai Jiao Tong University, Shanghai, 200030, China. fhguo@sibs.ac.cn.
Background:
The ovary is a vital and dynamic reproductive organ. Ovarian tissue cryopreservation (OTC) plays a vital role in preserving female fertility. However, the cellular subtypes most susceptible to cryoinjury and the molecular mechanisms underlying cryopreservation-associated damage remain poorly understood. This study aimed to identify cell populations vulnerable to freezing-thawing and to elucidate the key transcriptomic alterations and signaling pathways associated with ovarian cryoinjury at the single-cell and spatial levels.
Methods:
Ovarian cortical tissues from patients undergoing three gender reassignment surgery (GRS) were divided into fresh and vitrification-rapid warming groups. Following collagenase IV digestion, 10x Genomics single-cell RNA-seq was used for dissociated ovarian cell suspensions (27,185 fresh and 25,480 frozen-thawed cells). Eight major cell clusters were identified. Additionally, 110 oocytes (66 fresh, 44 vitrification-rapid warming) were isolated and analyzed using the Smart-seq2 platform. Spatial transcriptomics was performed via BGI Stereo-seq. Molecular validation was performed via β-galactosidase staining, immunofluorescence, and qRT-PCR.
Results:
Cryopreservation significantly altered the activity of pathways related to focal adhesion, oxidative stress, and apoptosis, particularly in stromal and perivascular cells. The number of FOS-positive perivascular cells was notably increased after vitrification-rapid warming, whereas the number of PTGDS-positive stromal cells decreased. Oocyte analysis revealed that cryopreservation primarily disrupted pathways involved in the cell cycle and meiosis, although the damage was not irreversible, supporting the relative safety of long-term cryostorage. Spatial transcriptomics and functional validation further confirmed the rapid and robust activation of the FOS/AP-1 pathway after vitrification-rapid warming, particularly in perivascular and granulosa cells. Treatment with T-5224 (a FOS/AP-1 inhibitor) significantly rescued the morphology and function of cultured frozen-thawed ovaries.
Conclusions:
Stromal and perivascular cells are the main cell types that are sensitive to ovarian cryopreservation. The FOS/AP-1 pathway is markedly activated after, suggesting the exacerbation of metabolic impairment. In oocytes within the ovarian cortex, the cell cycle and meiosis-related physiological processes were the primary processes affected.
Insights
Ovarian cryopreservation damages stromal and perivascular cells by activating the FOS/AP-1 pathway. Inhibiting this pathway rescued frozen-thawed ovarian function, offering insights into fertility preservation.
Area of Science:
- Reproductive biology and cryobiology
- Single-cell and spatial transcriptomics
- Molecular mechanisms of cryoinjury
Background:
- Ovarian tissue cryopreservation (OTC) is crucial for preserving female fertility.
- Cellular targets and molecular pathways of cryoinjury in ovarian tissue are not well understood.
- Identifying vulnerable cell types and mechanisms is essential for improving OTC protocols.
Purpose of the Study:
- To identify ovarian cell populations susceptible to cryopreservation-induced damage.
- To elucidate molecular alterations and signaling pathways affected by freezing-thawing.
- To investigate the role of the FOS/AP-1 pathway in ovarian cryoinjury.
Main Methods:
- Ovarian cortical tissues were subjected to vitrification-rapid warming.
- Single-cell RNA sequencing (scRNA-seq) analyzed 52,665 cells (fresh and frozen-thawed).
- Smart-seq2 analyzed 110 oocytes; spatial transcriptomics (Stereo-seq) and molecular validation were performed.
Main Results:
- Cryopreservation significantly impacted focal adhesion, oxidative stress, and apoptosis pathways in stromal and perivascular cells.
- Vitrification-rapid warming increased FOS-positive perivascular cells and decreased PTGDS-positive stromal cells.
- The FOS/AP-1 pathway was robustly activated in perivascular and granulosa cells; its inhibition partially restored ovarian function.
Conclusions:
- Stromal and perivascular cells are highly sensitive to ovarian cryopreservation.
- FOS/AP-1 pathway activation is a key mechanism contributing to cryoinjury and metabolic impairment.
- Oocyte cryopreservation primarily affects cell cycle and meiosis, with potential for recovery.
