Identification of cryosensitive niches and a targetable FOS/AP‑1 program in the human ovarian cortex by single‑cell...

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.