Unknown,Transcriptomics,Genomics,Proteomics

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Direct comparison of effects of slow-freezing and vitrification procedures on rabbit late blastocyst transcriptome

ABSTRACT: Nowadays there is a need to improve cryopreservation protocols in both human assisted reproduction and domestic animal field. We wanted to compare the transcriptomic changes induced by two commonly used cryopreservation procedures (slow freezing and vitrification) on rabbit late blastocyst before the onset of implantation. We recovered rabbit morulae at day 3 of development, freezing or vitrified and transferred them into recipient maternal tracts till day 6 of development. In this way, we wanted to analyse if different cryopreservation techniques produce different effects on gene expression that embryos are unable to get over after three days of in vivo development. After artificially insemination morulae were recovered at day 3 of development, frozen or vitrified, and then transferred to recipient does. To assess gene expression differences between frozen and vitrified the recipients were slaughtered three days after the transfer, at day 6.

ORGANISM(S): Oryctolagus cuniculus

SUBMITTER: M. Desemparats Saenz-de-Juano

PROVIDER: E-GEOD-50979 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

Project description: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.

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Direct comparison of effects of slow-freezing and vitrification procedures on rabbit late blastocyst transcriptome

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