Institute of Human Genetics, Julius Maximilians University, 97074 Würzburg, Germany.
Department of Bioinformatics, Julius Maximilians University, 97074 Würzburg, Germany.
Hum Reprod. 2017 Aug 1;32(8):1761-1769. doi: 10.1093/humrep/dex209.
Does ICSI induce specific DNA methylation changes in the resulting offspring?
Although several thousand analyzed CpG sites (throughout the genome) displayed significant between-group methylation differences, both ICSI and spontaneously conceived children varied within the normal range of methylation variation.
Children conceived by ART have increased risks for medical problems at birth and to the extent of present knowledge also in later life (i.e. impaired metabolic and cardiovascular functions). One plausible mechanism mediating these ART effects are epigenetic changes originating in the germ cells and/or early embryos and persisting during further development.
STUDY DESIGN, SIZE, DURATION: We compared the cord blood methylomes and candidate gene methylation patterns of newborns conceived through ICSI or spontaneously.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Umbilical cord bloods were obtained from healthy newborn singletons conceived spontaneously (53 samples), through ICSI (89) or IVF (34). Bisulfite-converted DNA samples of 48 ICSI and 46 control pregnancies were used for genome-wide analyses with Illumina's 450K methylation arrays. Candidate genes from the methylation screen were analyzed in all three groups by bisulfite pyrosequencing.
Altogether, 4730 (0.11%) of 428 227 analyzed CpG sites exhibited significant between-group methylation differences, but all with small (β < 10%) or very small (β < 1%) effect size. ICSI children showed a significantly decreased DNA methylation age at birth, lagging approximately half a week behind the controls. ART-susceptible CpGs were enriched in CpG islands with low methylation values (0-20%) and in imprinting control regions (ICRs). Eighteen promoter regions (six in microRNA and SNORD RNA genes), four CpG islands (three in genes including one long non-coding RNA), and two ICRs contained multiple significant sites. Three differentially methylated regions were studied in more detail by bisulfite pyrosequencing. ATG4C and SNORD114-9 could be validated in an independent ICSI group, following adjustment for maternal age and other confounding factors. ATG4C was also significant in the IVF group.
N/A.
LIMITATIONS, REASONS FOR CAUTION: The observed epigenetic effects are small and there are numerous potential confounding factors such as parental age and infertility. Although our study meets current standards for epigenetic screens, sample size is still two orders of magnitude below that of genome-wide association studies.
Our study suggests an impact of ICSI on the offspring's epigenome(s), which may contribute to phenotypic variation and disease susceptibility in ART children. Epigenetic regulation of gene expression by different classes of non-coding RNAs may be a key mechanism for developmental programming through ART.
STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a research grant (no. 692185) from the European Union (ERA of ART). There are no competing interests.
ICSI 是否会在后代中引起特定的 DNA 甲基化变化？
尽管在整个基因组中分析了数千个 CpG 位点（通过全基因组分析），显示出组间甲基化差异有统计学意义，但 ICSI 和自然受孕的儿童在正常的甲基化变异范围内存在差异。
通过 ART 受孕的儿童在出生时出现医疗问题的风险增加，并且就目前的知识而言，在以后的生活中也会出现问题（即代谢和心血管功能受损）。介导这些 ART 效应的一个合理机制是源自生殖细胞和/或早期胚胎的表观遗传变化，并在进一步发育过程中持续存在。
研究设计、大小、持续时间：我们比较了通过 ICSI 或自然受孕的新生儿的脐带血甲基组和候选基因甲基化模式。
参与者/材料、设置、方法：从自然受孕的健康新生儿（53 例）、通过 ICSI（89 例）或 IVF（34 例）受孕的单胎新生儿中获得脐带血。用 Illumina 的 450K 甲基化阵列对 48 例 ICSI 和 46 例对照妊娠的经 bisulfite 转化的 DNA 样本进行全基因组分析。通过 bisulfite 焦磷酸测序在所有三组中分析候选基因的甲基化情况。
总共，在 428227 个分析的 CpG 位点中，有 4730 个（0.11%）显示出组间甲基化差异有统计学意义，但所有差异的效应大小都很小（β<10%）或非常小（β<1%）。ICSI 儿童的出生时 DNA 甲基化年龄明显降低，比对照组滞后约半周。ART 易感的 CpG 位点在低甲基化值（0-20%）和印迹控制区（ICR）的 CpG 岛上富集。18 个启动子区域（6 个在 microRNA 和 SNORD RNA 基因中）、4 个 CpG 岛（3 个在包括一个长非编码 RNA 的基因中）和 2 个 ICR 含有多个有统计学意义的位点。通过 bisulfite 焦磷酸测序对三个差异甲基化区域进行了更详细的研究。在调整了母亲年龄和其他混杂因素后，在另一个 ICSI 组中可以验证 ATG4C 和 SNORD114-9。ATG4C 在 IVF 组中也有意义。
无。
局限性、谨慎的原因：观察到的表观遗传效应很小，并且存在许多潜在的混杂因素，如父母年龄和不孕。尽管我们的研究符合当前的表观遗传学筛查标准，但样本量仍然是全基因组关联研究的两个数量级以下。
我们的研究表明 ICSI 对后代的表观基因组有影响，这可能导致 ART 儿童的表型变异和疾病易感性。不同类别的非编码 RNA 对基因表达的表观遗传调控可能是通过 ART 进行发育编程的关键机制。
研究资金/竞争利益：这项工作得到了欧盟 ERA of ART（编号 692185）研究资助的支持。没有竞争利益。
