Transcriptomic differences in bovine blastocysts following vitrification and slow freezing at morula stage [EmbryocryoVit]. Bos taurus

This study aimed to compare the transcriptome of vitrified and slow frozen embryos with the untreated control. Bovine embryos (compact morulae) were vitrified or slow frozen and post-warm embryos were cultured to expanded blastocyst stage. The vitrified- and slow frozen-derived were subjected to microarray analysis and compared with untreated control embryos for differential gene expression. Morula to blastocyst conversion rate was higher (P<0.05) in control (72%) and vitrified (77%) embryos compared to slow frozen (34%) embryos. Total 20 genes were upregulated and 44 genes were downregulated in the vitrified embryos (fold change ≥ +-2, P<0.05). In slow frozen embryos, 102 genes were upregulated and 63 genes were downregulated (fold change ≥ +-1.5, P<0.05) in comparison with untreated embryos. Vitrified embryos exhibited significant changes in gene expression mainly involving embryo implantation (PTGS2, CALB1), lipid peroxidation and ROS generation (HSD3B1, AKR1B1, APOA1) and cell differentiation (KRT19, CLDN23). The slow frozen embryos, however, showed significant changes in the expression of genes related to cell signaling (SPP1), cell structure and differentiation (DCLK2, JAM2 and VIM), and lipid metabolism (PLA2R1 and SMPD3). In silico comparison between vitrified and slow-frozen (reference) embryos revealed similar changes in gene expression as between vitrified and untreated embryos. In conclusion, the vitrified bovine embryos demonstrated better post-warming embryo development than slow-frozen bovine embryos but their gene expression related to lipid metabolism, steroidogenesis, cell differentiation and placentation changed significantly. Slow freezing killed more embryos than vitrification, and the survived embryos did not express significant change in their gene expression. Overall design: Vitrified embryos intensities compared with fresh bovine embryos (control)

本研究旨在比较玻璃化冷冻（vitrified）胚胎、慢速冷冻胚胎与未处理对照组胚胎的转录组（transcriptome）。本研究以牛致密桑椹胚（compact morulae）为实验材料，分别实施玻璃化冷冻与慢速冷冻处理，解冻后将胚胎培养至扩张囊胚阶段。随后对解冻获得的玻璃化冷冻来源胚胎与慢速冷冻来源胚胎开展微阵列分析（microarray analysis），并与未处理对照组胚胎对比差异基因表达情况。
对照组（72%）与玻璃化冷冻组（77%）的桑椹胚向囊胚转化率显著高于慢速冷冻组（34%，P<0.05）。与未处理对照组相比，玻璃化冷冻胚胎中共鉴定出20个上调基因、44个下调基因（倍数变化≥±2，P<0.05）；慢速冷冻胚胎则存在102个上调基因、63个下调基因（倍数变化≥±1.5，P<0.05）。
玻璃化冷冻胚胎的差异表达基因主要涉及胚胎着床相关基因（PTGS2、CALB1）、脂质过氧化与活性氧（ROS，Reactive Oxygen Species）生成相关基因（HSD3B1、AKR1B1、APOA1）以及细胞分化相关基因（KRT19、CLDN23）。而慢速冷冻胚胎的差异表达基因则主要与细胞信号转导（SPP1）、细胞结构与分化（DCLK2、JAM2及VIM）以及脂质代谢（PLA2R1与SMPD3）相关。
以慢速冷冻胚胎为参照的生物信息学（in silico）比较分析显示，玻璃化冷冻与慢速冷冻胚胎间的基因表达变化模式，与玻璃化冷冻胚胎与未处理对照组胚胎间的变化模式相似。
综上，牛玻璃化冷冻胚胎的解冻后发育能力优于慢速冷冻胚胎，但其与脂质代谢、类固醇生成、细胞分化及胎盘形成相关的基因表达发生了显著改变。慢速冷冻对胚胎的致死率高于玻璃化冷冻，且存活胚胎的基因表达未出现显著变化。
总体实验设计：以新鲜牛胚胎为对照组，比较玻璃化冷冻胚胎的微阵列信号强度。