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Comprehensive transcriptomic analysis reveals stress physiology behind differential gene expression in vitrified–warmed and fresh mouse blastocysts

ABSTRACT: Blastocyst vitrification has improved embryo transfer methods, leading to higher implantation success rates and better pregnancy outcomes in subsequent frozen embryo transfer cycles. In this study, we used mouse blastocysts as a model to simulate the transcriptional changes caused by vitrifying human blastocysts and to further investigate their effects. Utilizing a human vitrification protocol, we implanted both vitrified and fresh embryos into mice and observed a higher implantation success rate for the vitrified embryos. Transcriptomic analysis revealed that vitrified-warmed blastocysts exhibited differential gene expression primarily associated with thermogenesis, chemical carcinogenesis-reactive oxygen species, oxidative phosphorylation, immune response, and MAPK-related signaling pathways. To validate the next-generation sequencing results, we performed reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), which confirmed increased expression of genes such as Cdk6 and Nfat2, and decreased expression of genes such as Dkk3 and Mapk10. Among these pathways, immune responses and MAPK-related signaling pathways indicate that embryos need to communicate with the external environment, likely through microRNAs. We used gene-microRNA interaction predictions to anticipate potential embryonic microRNA expressions and conducted an analysis of embryonic microRNA expression. Twelve microRNAs exhibited expression patterns consistent with the predicted results, potentially affecting uterine epithelial cell adhesion, trophectoderm development, invasive capacity, or immune responses. Our findings suggest that vitrification induces transcriptomic changes in mouse blastocysts, and even small changes in gene expression can increase implantation success.

ORGANISM(S): Mus musculus

PROVIDER: GSE268758 | GEO | 2024/08/07

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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Comprehensive transcriptomic analysis reveals stress physiology behind differential gene expression in vitrified–warmed and fresh mouse blastocysts

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