Department of Obstetrics, Gynecology and Reproductive Sciences, University of Vermont, Burlington, VT 05405, USA.
Hum Reprod. 2011 Sep;26(9):2576-85. doi: 10.1093/humrep/der201. Epub 2011 Jun 17.
Although assisted reproductive technology (ART) is reported to result in abnormal genomic imprinting and/or altered genomic methylation, few if any studies have used high-throughput methods to analyze genomic methylation in ART embryos. We hypothesized that a microarray-based assessment of genomic methylation could be used to reveal differences between ART and normal preimplantation embryos.
In this pilot study, we performed methylation-sensitive amplification of genomic DNA from preimplantation mouse blastocysts, obtained by natural mating and either maintained in vivo until E3.5 (n = 4) or cultured in vitro (n = 4) from E0.5 until E3.5. An oligonucleotide microarray was then used to perform comparative hybridization of amplified DNA, allowing us to assess relative methylation at ~16,000 loci on mouse chromosome 7.
We show that for in vivo derived embryos, the methylation/microarray results were strikingly consistent. In contrast, all four in vitro cultured embryos showed evidence of generalized hypermethylation as well as greater locus-to-locus variability, when compared with in vivo derived embryos. Genomic segments that overlapped exons and CpG islands were most likely to be hypomethylated in both normal and experimental blastocysts. Other sequence features, such as repetitive elements, were not associated with the presence of or the degree of methylation.
We conclude that a general assessment of genomic methylation in blastocyst stage embryos is technically feasible. Data from this small sample suggest that in vitro embryo culture is associated with generalized hypermethylation as well as increased locus-to-locus variability in methylation. However, it is premature to conclude that this is a general property of in vitro cultured blastocysts.
尽管辅助生殖技术（ART）被报道会导致异常的基因组印记和/或改变的基因组甲基化，但很少有研究使用高通量方法来分析 ART 胚胎中的基因组甲基化。我们假设，基于微阵列的基因组甲基化评估可以用于揭示 ART 和正常胚胎之间的差异。
在这项初步研究中，我们对自然交配获得的囊胚进行了基因组 DNA 的甲基化敏感扩增，这些囊胚分别在体内维持到 E3.5（n = 4）或从 E0.5 体外培养到 E3.5（n = 4）。然后，我们使用寡核苷酸微阵列对扩增的 DNA 进行了比较杂交，从而可以评估小鼠染色体 7 上约 16000 个基因座的相对甲基化。
我们表明，对于体内来源的胚胎，甲基化/微阵列结果惊人地一致。相比之下，与体内来源的胚胎相比，所有四个体外培养的胚胎都表现出普遍的过度甲基化，以及更大的基因座间变异性。在正常和实验囊胚中，重叠外显子和 CpG 岛的基因组片段最有可能发生低甲基化。其他序列特征，如重复元件，与甲基化的存在或程度没有关联。
我们得出结论，对囊胚阶段胚胎的基因组甲基化进行全面评估在技术上是可行的。来自这个小样本的数据表明，体外胚胎培养与广泛的过度甲基化以及甲基化的基因座间变异性增加有关。然而，现在就得出这是体外培养囊胚的普遍特性还为时过早。
