Abstract Study question How does embryo vitrification regulates the transcriptome of preimplantation embryos through epigenetic regulation and how are the effects transmitted to the female offsprings? Summary answer Embryo vitrification changed the DNA methylation level of preimplantation embryo and disturbed glycolipid metabolism of female offsprings in mice. What is known already Vitrification is a type of embryo cryopreservation increasingly frequently used clinically. The effect of embryo freezing on the health of offspring has been widely concerned. Some clinical studies have shown that offspring obtained from frozen embryo transplantation has higher birth weight and may be at higher risk of certain birth defects, increased risk of type 1 diabetes and cancer. But not all studies agree. The offspring safety of vitrification needs further follow-up research. Mouse model is a helpful tool for us to carry up follow-up study and explore mechanism. Study design, size, duration After superovulation and mating of ICR mice, we obtained 8-Cell embryos. In Vitrify Group, the 8-Cell embryos underwent vitrification and thawing and culture in vitro till blastocysts. In InVitro Group, the 8-Cell embryos were directly cultured in vitro till blastocysts. We transplanted these blastocysts into surrogate mice to get offspring of InVitro and Vitrify. Along with offspring of natural conception (NC), we observed their growth and development and glycolipid metabolism until 18 weeks old. Participants/materials, setting, methods Single embryo RNA-seq and Whole Genome Bisulfite Sequencing (WGBS) were performed to get transcriptome and DNA methylation atlas. Immunofluorescence of blastocysts was performed to test level of 5mC/5hmC and Tet2. Glucose and insulin tolerance test (GTT/ITT), serum lipid level test were performed on offspring to examine the glycolipid metabolism status. Liver transmission electron microscope photographs were take to observe ultramicrostructure. Liver RNA-seq and WGBS were performed to verify the variation on pathways concerning glycolipid metabolism. Main results and the role of chance Pseudo-time analysis from RNA-seq of 16-Cell, morula and blastocyst showed a relative developmental time lag in Vitrify. Differentially expressed genes (DEGs) of vitrify group at the blastocyst stage are mainly enriched in lipid metabolism, insulin signal, TOR signal regulation, gamete production related DNA methylation pathways. GSEA analysis revealed down-regulation of insulin response pathway genes. WGBS showed a trend of genome-wide methylation increase in vitrify group. Immunofluorescence confirmed this observation as the fluorescence intensity ratio of 5mC/5hmC was increased. RNA-seq, qPCR and western-blot showed a lower expression level of Tet2. These results suggested that the demethylation was insufficient in vitrify group. Blastocyst differentially methylated genes (DMRs) from WGBS were enriched in insulin response pathways. In offspring, birth weight of Vitrify offspring is higher than InVitro. GTT and ITT showed an elevated level of AUC in Vitrify 10-week-female, indicating an impaired glucose tolerance. And the fasting serum cholesterol level was elevated in Vitrify 10-week-female, indicating an impaired lipid metabolism. Under transmission electron microscope, we saw swollen mitochondria in liver of Vitrify 10-week-female. We extracted RNA from 10-week-female for RNA-seq, DEGs were enriched in cholesterol biosynthesis pathway. We extracted DNA from 10-week-female for WGBS, DEGs were enriched in cAMP and AMPK signaling pathway. Limitations, reasons for caution We have not confirm the precise molecular site affected by vitrification, clarify the mechanism and conduct the rescue test yet, which we are currently working on. This study is just the first step in this direction, and more efforts are needed. Wider implications of the findings Although more clinical trials and basic science studies are required, our findings provide valuable information for preventive and clinical decisions, as ‘freeze-all’ strategy springs up in many centers. Embryo vitrification needs more cautious consideration. Optimizing embryo vitrification protocols are needed according to the mechanism to minimize unwanted effects. Trial registration number 2021YFC2700601
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