High-Resolution Spatial Transcriptomics Reveals Stroma Damage in Human Ovarian Tissue Response to Cryopreservation

Ovarian tissue cryopreservation is an important technique for preserving fertility potential, albeit the associated tissue damage may severely impact post-thaw tissue viability. We collected human ovarian tissues from multiple samples and performed Stereo-Seq high-resolution spatial transcriptomics to comprehensively profile and compare the molecular impacts of two cryopreservation methods - slow freezing and vitrification. We identified 8 major spatial clusters and revealed their functional heterogeneity by subclustering. We then detailed cryopreservation response at both the global and subcluster levels to illustrate overall and niche specific effects. Compared to fresh samples, we observed a decrease in major metabolic pathways in frozen samples with both techniques, whereas vitrified samples have severer decrease than slow-frozen samples. The affected metabolic pathways included those related to proteins, such as ribosomal processes and proteasomal degradation; lipids, specifically sterol and cholesterol metabolism; and overall energy production, which encompassed cellular respiration and mitochondrial processes. On the other hand, slow freezing elicited a strong but balanced inflammatory and tissue remodeling state compared to vitrification. We also reported upregulated cell-cell signaling related to angiogenesis, cellular adhesion and extracellular matrix remodeling in slow-frozen tissue. These pathways were responsible for enhancing tissue repair by coordinating with certain stromal and endothelial subclusters. In summary, our study offered insights on ovarian cell response to cryopreservation, which may guide optimization of ovarian tissue cryopreservation protocols for clinical applications. Overall design: Human ovarian tissues were donated from one patient aged 35 years who underwent oophorectomy due to cervical cancer. We conducted Stereo-seq profilling of transcriptomic data on three tissue pieces stored under different conditions. According to the Stereo-Seq protocol, one piece of fresh group tissues was fixed in O.C.T. compound (Tissue Tek) for subsequent sequencing, tissues of the two cryopreservation groups were processed after freezing and thawing.