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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE267323 | GEO | 2024/05/18

REPOSITORIES: GEO

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Project description:Studies on embryo cryopreservation efficiency had been focused mainly on technical and embryo factors. While structural damages can be easily evaluated, physiological damages only can be estimated by analyzing their in vitro and in vivo development to later stages. In order to determine how cryopreservation process affect embryo pre-implantory development, a transcriptional microarray study has been performed comparing gene expression of 6 days old rabbit embryos, previously vitrified or frozen and transferred into recipients rabbit females, to their in vivo counterparts. For each experimental group, control, vitrified and frozen late blastocysts, total RNA was extracted from 3 pools of approximately 10 embryos and labeled with Cy3 or Cy5 dyes. Then, six competitive hybridizations were carried out including two dye-swaps to compensate dye-bias. A specifically microarray designed to study rabbit gene expression profiling, the Rabbit 44K oligonucleotide array (Agilent Technologies), was used in this study. Identification of differentially expressed transcripts from 6 day old blastocysts was achieved using the Limma algorithm, and functional annotation was performed by Blast2GO software. Compared to 6 day old in vivo derived embryos, viable vitrified embryos only present 3 differentially expressed genes, in contrast to frozen viable embryos with 24 genes upregulated and 46 genes downregulated. These results reveal that effects of cryopreservation still remain in late blastocyst pre-implantatory gene expression, and potential damage and alterations produced by vitrification and slow-freezing procedures are differentially resolved after 3 days of in vivo development. Transcriptional microarray study that compares gene expression of viable 6 day old rabbit embryos, previosly vitrified or frozen and tranferred into recipient rabbit females, to their in vivo counterparts. Experiment 1: Control embryos vs. Vitrified embryos and Experiment 2: Control embryos vs. Frozen embryos. Biological replicates used: 3 replicates for control embryos, 3 replicates for vitrified embryos and 3 replicates for frozen embryos.

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

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