Li Tao, Vu Thanh H, Ulaner Gary A, Littman Eva, Ling Jian-Qun, Chen Hui-Ling, Hu Ji-Fan, Behr Barry, Giudice Linda, Hoffman Andrew R
Medical Service, VA Palo Alto Health Care System, CA 94304, USA.
Mol Hum Reprod. 2005 Sep;11(9):631-40. doi: 10.1093/molehr/gah230. Epub 2005 Oct 11.
Recent studies suggest that IVF and assisted reproduction technologies (ART) may result in abnormal genomic imprinting, leading to an increased frequency of Angelman syndrome (AS) and Beckwith-Weidemann syndrome (BWS) in IVF children. To learn how ART might alter the epigenome, we examined morulas and blastocysts derived from C57BL/6J X M. spretus F1 mice conceived in vivo and in vitro and determined the allelic expression of four imprinted genes: Igf2, H19, Cdkn1c and Slc221L. IVF-derived mouse embryos that were cultured in human tubal fluid (HTF) (Quinn's advantage) media displayed a high frequency of aberrant H19 imprinting, whereas in vivo and IVF embryos showed normal maternal expression of Cdkn1c and normal biallelic expression of Igf2 and Slc221L. Embryonic stem (ES) cells derived from IVF blastocysts also showed abnormal Igf2/H19 imprinting. Allele-specific bisulphite PCR reveals abnormal DNA methylation at a CCCTC-binding factor (CTCF) site in the imprinting control region (ICR), as the normally unmethylated maternal allele acquired a paternal methylation pattern. Chromatin immunoprecipitation (ChIP) assays indicate an increase of lysine 4 methylation (dimethyl Lys4-H3) on the paternal chromatin and a gain in lysine 9 methylation (trimethyl Lys9-H3) on the maternal chromatin at the same CTCF-binding site. Our results indicate that de novo DNA methylation on the maternal allele and allele-specific acquisition of histone methylation lead to aberrant Igf2/H19 imprinting in IVF-derived ES cells. We suggest that ART, which includes IVF and various culture media, might cause imprinting errors that involve both aberrant DNA methylation and histone methylation at an epigenetic switch of the Igf2-H19 gene region.
近期研究表明，体外受精（IVF）及辅助生殖技术（ART）可能导致基因组印记异常，致使IVF出生的儿童中天使综合征（AS）和贝克威思-维德曼综合征（BWS）的发病率增加。为了解ART如何改变表观基因组，我们检测了体内受精和体外受精的C57BL/6J×M. spretus F1小鼠来源的桑椹胚和囊胚，并确定了四个印记基因的等位基因表达：Igf2、H19、Cdkn1c和Slc221L。在人输卵管液（HTF）（Quinn's advantage）培养基中培养的IVF来源的小鼠胚胎显示出H19印记异常的高频率，而体内受精和IVF胚胎显示Cdkn1c的母源表达正常，Igf2和Slc221L的双等位基因表达正常。IVF囊胚来源的胚胎干细胞也显示出Igf2/H19印记异常。等位基因特异性亚硫酸氢盐PCR揭示了印记控制区（ICR）中CCCTC结合因子（CTCF）位点的DNA甲基化异常，因为通常未甲基化的母源等位基因获得了父源甲基化模式。染色质免疫沉淀（ChIP）分析表明，在同一CTCF结合位点，父源染色质上赖氨酸4甲基化（二甲基赖氨酸4-H3）增加，母源染色质上赖氨酸9甲基化（三甲基赖氨酸9-H3）增加。我们的结果表明，母源等位基因上的从头DNA甲基化和组蛋白甲基化的等位基因特异性获得导致IVF来源的胚胎干细胞中Igf2/H19印记异常。我们认为，包括IVF和各种培养基在内的ART可能会导致印记错误，这涉及到Igf2-H19基因区域表观遗传开关处的异常DNA甲基化和组蛋白甲基化。
