Currently, there is a high demand for efficient pig embryo cryopreservation procedures in the porcine industry as well as for genetic diversity preservation and research purposes. To date, vitrification (VIT) is the most efficient method for pig embryo cryopreservation. Despite a high number of embryos survives in vitro after vitrification/warming procedures, the in vivo embryo survival rates after embryo transfer are variable among laboratories. So far, most studies have focused on cryoprotective agents and devices, while the VIT effects on porcine embryonic gene expression remained unclear. The few studies performed were based on vitrified/warmed embryos that were cultured in vitro (IVC) to allow them to re–expand. Thus, the specific alterations of VIT, IVC, and the cumulative effect of both remained unknown. To unveil the VIT-specific embryonic alterations, gene expression in VIT versus (vs.) IVC embryos was analyzed. Additionally, changes derived from both VIT and IVC vs. control embryos (CO) were analyzed to confirm the VIT embryonic alterations. Three groups of in vivo embryos at the blastocyst stage were analyzed by RNA–sequencing: (1) VIT embryos (vitrified/warmed and cultured in vitro), (2) IVC embryos and (3) CO embryos. RNA–sequencing revealed three clearly different mRNA profiles for VIT, IVC and CO embryos. Comparative analysis of mRNA profiles between VIT and IVC identified 321, differentially expressed genes (DEG) (FDR < 0.006). In VIT vs. CO and IVC vs. CO, 1901 and 1519 DEG were found, respectively, with an overlap of 1045 genes. VIT-specific functional alterations were associated to response to osmotic stress, response to hormones, and developmental growth. While alterations in response to hypoxia and mitophagy were related to the sum of VIT and IVC effects. Our findings revealed new insights into the VIT procedure-specific alterations of embryonic gene expression by first comparing differences in VIT vs. IVC embryos and second by an integrative transcriptome analysis including in vivo control embryos. The identified VIT alterations might reflect the transcriptional signature of the embryo cryodamage but also the embryo healing process overcoming the VIT impacts. Selected validated genes were pointed as potential biomarkers that may help to improve vitrification.

中文翻译：

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揭示玻璃化如何影响猪胚泡：转录组学研究的线索

目前，猪行业对高效的猪胚胎冷冻保存程序以及遗传多样性保存和研究目的有很高的需求。迄今为止，玻璃化（VIT）是猪胚胎冷冻保存最有效的方法。尽管大量胚胎在玻璃化/加热程序后在体外存活，但胚胎移植后的体内胚胎存活率在实验室之间存在差异。到目前为止，大多数研究都集中在冷冻保护剂和装置上，而 VIT 对猪胚胎基因表达的影响仍不清楚。进行的少数研究是基于体外培养 (IVC) 的玻璃化/加热胚胎，以使它们能够重新扩张。因此，VIT、IVC 的具体变化以及两者的累积效应仍然未知。为了揭示 VIT 特异性胚胎改变，分析了 VIT 与（vs.）IVC 胚胎中的基因表达。此外，对 VIT 和 IVC 与对照胚胎 (CO) 的变化进行了分析，以确认 VIT 胚胎的变化。通过 RNA 测序分析了三组处于囊胚阶段的体内胚胎：（1）VIT 胚胎（玻璃化/加热和体外培养），（2）IVC 胚胎和（3）CO 胚胎。RNA 测序揭示了 VIT、IVC 和 CO 胚胎的三种明显不同的 mRNA 谱。VIT 和 IVC 之间 mRNA 谱的比较分析确定了 321 个差异表达基因 (DEG) (FDR < 0.006)。在 VIT vs. CO 和 IVC vs. CO 中，分别发现 1901 和 1519°，有 1045 个基因重叠。VIT 特异性功能改变与对渗透压的反应、对激素的反应、和发育成长。而对缺氧和线粒体自噬反应的改变与 VIT 和 IVC 效应的总和有关。我们的研究结果通过首先比较 VIT 与 IVC 胚胎的差异，其次通过包括体内对照胚胎在内的综合转录组分析，揭示了对胚胎基因表达的 VIT 程序特异性改变的新见解。已确定的 VIT 改变可能反映了胚胎冷冻损伤的转录特征，但也反映了克服 VIT 影响的胚胎愈合过程。选定的经过验证的基因被指出为可能有助于改善玻璃化的潜在生物标志物。我们的研究结果通过首先比较 VIT 与 IVC 胚胎的差异，其次通过包括体内对照胚胎在内的综合转录组分析，揭示了对胚胎基因表达的 VIT 程序特异性改变的新见解。已确定的 VIT 改变可能反映了胚胎冷冻损伤的转录特征，但也反映了克服 VIT 影响的胚胎愈合过程。选定的经过验证的基因被指出为可能有助于改善玻璃化的潜在生物标志物。我们的研究结果通过首先比较 VIT 与 IVC 胚胎的差异，其次通过包括体内对照胚胎在内的综合转录组分析，揭示了对胚胎基因表达的 VIT 程序特异性改变的新见解。已确定的 VIT 改变可能反映了胚胎冷冻损伤的转录特征，但也反映了克服 VIT 影响的胚胎愈合过程。选定的经过验证的基因被指出为可能有助于改善玻璃化的潜在生物标志物。