BioProject

Cryopreservation is an important technique for preserving fertility potential through ovarian tissue banking. More...

Cryopreservation is an important technique for preserving fertility potential through ovarian tissue banking. Slow freezing has traditionally been the standard protocol used for ovarian tissue cryopreservation. However, vitrification, as an emerging method, is considered to be an alternative to slow freezing, thus generating some debate in the field. This study utilizes high-resolution spatial transcriptomics to comprehensively profile and compare the molecular impacts of two cryopreservation methods - slow freezing and vitrification - on human ovarian tissues at an unprecedented resolution. Over 135,000 spots were profiled from Fresh and cryopreserved ovarian tissue slices, identifying 8 major cell types and 25 subtypes through marker expression. Detailed spatial localization patterns revealed heterogeneous stromal cell subpopulations and ovarian structures. Transcriptional profiling showed that both cryopreservation methods significantly repressed gene expression, indicative of tissue damage. However, slow freezing induced a broader pro-survival inflammatory and tissue remodeling response compared to vitrification. Gene set enrichment analysis identified elevated ribosomal processes, inflammatory signaling pathways like TNF-?/NF-?B and IL-6/STAT3, as well as extracellular matrix (ECM) organization and collagen synthesis as dominant response themes in slow frozen tissue, which were observed to a lesser degree in vitrified tissue. Key response drivers included MYC, TIMP1, and DCN, with stromal cells located in the cortical regions showing the strongest pathway enrichment. Ribosomal gene expression displayed spatial gradients and supported stromal responses after freezing. We finally demonstrated signaling pathway differences between the two cryopreservation methods, noting TGF-? as a highly possible pathway responsible for coordinating with certain endothelial subgroups to enhance ECM deposition. In summary, this study provides a comprehensive atlas of cell types and their spatial structures in human ovary. It reveals that slow freezing elicits a strong but balanced inflammatory and tissue regenerative state compared to the vitrification. It also offers insights on cryoinjury to ovarian stroma, 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. Less...