School of Agriculture and Food Science and Lyons Research Farm, University College Dublin, Belfield, Dublin 4, Ireland.
Biomedical Sciences Research Institute, University of Ulster, Coleraine, UK.
BMC Genomics. 2018 Jun 5;19(1):438. doi: 10.1186/s12864-018-4818-3.
Assisted reproductive technologies (ART) are widely used to treat fertility issues in humans and for the production of embryos in mammalian livestock. The use of these techniques, however, is not without consequence as they are often associated with inauspicious pre- and postnatal outcomes including premature birth, intrauterine growth restriction and increased incidence of epigenetic disorders in human and large offspring syndrome in cattle. Here, global DNA methylation profiles in the trophectoderm and embryonic discs of in vitro produced (IVP), superovulation-derived (SOV) and unstimulated, synchronised control day 17 bovine conceptuses (herein referred to as AI) were interrogated using the EmbryoGENE DNA Methylation Array (EDMA). Pyrosequencing was used to validate four loci identified as differentially methylated on the array and to assess the differentially methylated regions (DMRs) of six imprinted genes in these conceptuses. The impact of embryo-production induced DNA methylation aberrations was determined using Ingenuity Pathway Analysis, shedding light on the potential functional consequences of these differences.
Of the total number of differentially methylated loci identified (3140) 77.3 and 22.7% were attributable to SOV and IVP, respectively. Differential methylation was most prominent at intragenic sequences within the trophectoderm of IVP and SOV-derived conceptuses, almost a third (30.8%) of the differentially methylated loci mapped to intragenic regions. Very few differentially methylated loci were detected in embryonic discs (ED); 0.16 and 4.9% of the differentially methylated loci were located in the ED of SOV-derived and IVP conceptuses, respectively. The overall effects of SOV and IVP on the direction of methylation changes were associated with increased methylation; 70.6% of the differentially methylated loci in SOV-derived conceptuses and 57.9% of the loci in IVP-derived conceptuses were more methylated compared to AI-conceptuses. Ontology analysis of probes associated with intragenic sequences suggests enrichment for terms associated with cancer, cell morphology and growth.
By examining (1) the effects of superovulation and (2) the effects of an in vitro system (oocyte maturation, fertilisation and embryo culture) we have identified that the assisted reproduction process of superovulation alone has the largest impact on the DNA methylome of subsequent embryos.
辅助生殖技术（ART）广泛用于治疗人类的生育问题，并用于哺乳动物家畜的胚胎生产。然而，这些技术的使用并非没有后果，因为它们通常与不良的产前和产后结果有关，包括早产、宫内生长受限以及人类中表观遗传障碍的发生率增加和牛的大胚胎综合征。在这里，使用胚胎基因 DNA 甲基化阵列（EDMA）检测体外生产（IVP）、超数排卵衍生（SOV）和未刺激同步控制第 17 天牛胚胎（在此称为 AI）的滋养层和胚胎盘的全基因组 DNA 甲基化谱。焦磷酸测序用于验证在阵列上鉴定为差异甲基化的四个基因座，并评估这些胚胎中六个印迹基因的差异甲基化区域（DMR）。使用 Ingenuity 通路分析确定胚胎生产引起的 DNA 甲基化异常的影响，揭示这些差异的潜在功能后果。
在所鉴定的差异甲基化基因座总数（3140 个）中，SOV 和 IVP 分别占 77.3%和 22.7%。IVP 和 SOV 衍生胚胎的滋养层中基因内序列的差异甲基化最为明显，30.8%的差异甲基化基因座映射到基因内区域。在胚胎盘（ED）中检测到的差异甲基化基因座很少；SOV 衍生和 IVP 衍生胚胎的 ED 中分别有 0.16%和 4.9%的差异甲基化基因座位于 ED 中。SOV 和 IVP 对甲基化变化方向的总体影响与甲基化增加有关；与 AI 胚胎相比，SOV 衍生胚胎中 70.6%的差异甲基化基因座和 IVP 衍生胚胎中 57.9%的基因座甲基化程度更高。与基因内序列相关的探针的本体论分析表明，与癌症、细胞形态和生长相关的术语富集。
通过检查（1）超数排卵的影响和（2）体外系统的影响（卵母细胞成熟、受精和胚胎培养），我们发现，仅超数排卵的辅助生殖过程对随后胚胎的 DNA 甲基组学有最大的影响。
