Datasheet1_Seasonal variation in the morphokinetics of in-vitro-derived bovine embryos is associated with the blastocyst developmental competence and gene expression.docx

Summer heat stress is a major cause of reduced development of preimplantation embryos. Nevertheless, seasonal effects on embryo morphokinetics have been less studied. We used a non-invasive time-lapse system that allows continuous monitoring of embryos to study the seasonal impact on embryo morphokinetics. The experiments were performed during the cold and the hot seasons. Cumulus-oocyte complexes were aspirated from ovaries, in-vitro-matured, and fertilized. Putative zygotes were cultured in an incubator equipped with a time-lapse system. The cleavage and blastocyst formation rates were lower in the hot vs. the cold season (p < 0.01). The kinetics of the embryos differed between seasons, reflected by a delay in the second cleavage in the hot vs. the cold season (p < 0.03). The distribution of the embryos into different morphological grades (good, fair, and poor) throughout the first three cleavages differed between seasons, with a higher proportion of good-grade embryos in the hot season (p < 0.03). Cleaved embryos were categorized as either normal or abnormal, based on their first cleavage pattern. Normal cleavage was defined as when the first cleavage resulted in two equal blastomeres and further classified as either synchronous or asynchronous, according to their subsequent cleavages. Abnormal cleavage was defined as when the embryo directly cleaved into more than two blastomeres, it cleaved unequally into two unevenly sized blastomeres, or when the fusion of already divided blastomeres occurred. The proportion of abnormally cleaved embryos was higher in the hot season vs. the cold one (p < 0.01), reflected by a higher proportion of unequally cleaved embryos (p < 0.02). In the cold season, abnormally cleaved embryos had a lower potential to develop into blastocysts relative to their normally cleaved counterparts (p < 0.001). Blastocysts that developed in the cold and the hot seasons differed in the expression of genes that related to the cell cycle (STAT1; p < 0.01), stress (HSF1; p < 0.03), and embryo development (ZP3; p < 0.05). A higher expression level was recorded for the STAT1 and UHRF1 genes in blastocysts that developed from unequally vs. the synchronously cleaved embryos (p < 0.04). We provide the first evidence for a seasonal effect on embryo morphokinetics, which might explain the reduced embryo development during the hot season.

夏季热应激是造成着床前胚胎发育能力下降的关键诱因。但目前针对季节因素对胚胎形态动力学（embryo morphokinetics）影响的相关研究仍相对较少。本研究采用可对胚胎进行连续监测的无创延时成像系统（time-lapse system），探究季节对胚胎形态动力学的作用。实验分别在寒冷季节与炎热季节开展：从卵巢中抽吸获取卵丘-卵母细胞复合体（cumulus-oocyte complexes），经体外成熟与体外受精处理后，将推定合子（putative zygotes）置于搭载延时成像系统的培养箱中进行培养。
炎热季节的卵裂率与囊胚形成率均显著低于寒冷季节（p<0.01）。不同季节的胚胎动力学存在显著差异，具体表现为炎热季节胚胎的第二次卵裂出现延迟（p<0.03）。前三次卵裂阶段，胚胎的形态分级（优质、中等、劣质）分布在不同季节存在差异，其中炎热季节优质胚胎的占比更高（p<0.03）。
研究人员根据首次卵裂模式将卵裂胚胎分为正常卵裂与异常卵裂两类：正常卵裂指首次卵裂产生两个等大卵裂球的情况，并根据后续卵裂模式进一步分为同步卵裂与异步卵裂；异常卵裂则包括胚胎直接分裂为3个及以上卵裂球、卵裂产生两个大小不均的卵裂球，以及已分裂的卵裂球发生融合三种情形。炎热季节异常卵裂胚胎的占比显著高于寒冷季节（p<0.01），其中大小不均卵裂胚胎的占比升高尤为明显（p<0.02）。相较于正常卵裂胚胎，寒冷季节的异常卵裂胚胎发育为囊胚的潜能更低（p<0.001）。
寒冷与炎热季节形成的囊胚，其与细胞周期（STAT1）、应激反应（HSF1）及胚胎发育（ZP3）相关的基因表达水平存在显著差异（p值分别为<0.01、<0.03、<0.05）。由大小不均卵裂胚胎发育而来的囊胚，其STAT1与UHRF1基因的表达水平高于同步卵裂胚胎来源的囊胚（p<0.04）。
本研究首次证实季节因素对胚胎形态动力学存在调控作用，该发现或可解释炎热季节胚胎发育能力下降的内在机制。