Gomes M V, Huber J, Ferriani R A, Amaral Neto A M, Ramos E S
Department of Genetics, School of Medicine of Ribeirão Preto, University of São Paulo, Avenida Bandeirantes, 3900, Monte Alegre, Ribeirão Preto 14049-900, São Paulo, Brazil.
Mol Hum Reprod. 2009 Aug;15(8):471-7. doi: 10.1093/molehr/gap038. Epub 2009 Jun 3.
Genomic imprinting alterations have been shown to be associated with assisted reproductive technologies (ARTs) in animals. At present, data obtained in humans are inconclusive; however, some epidemiological studies have demonstrated an increased incidence of imprinting disorders in children conceived by ARTs. In the present study, we focused on the effect of ARTs [IVF and intracytoplasmic sperm injection (ICSI)] on the epigenetic reprogramming of the maternally methylated imprinting control region KvDMR1 in clinically normal children. Qualitative and quantitative methylation at KvDMR1 were assessed by the methylation-specific PCR approach and by the methylation-sensitive enzymatic digestion associated with real-time PCR method, respectively. DNA was obtained from peripheral blood of 12/18 and umbilical cord blood and placenta of 6/18 children conceived by IVF or ICSI. The methylation patterns observed in this group were compared with the patterns observed in 30 clinically normal naturally conceived children (negative controls) and in 3 naturally conceived Beckwith-Wiedemann syndrome patients (positive controls). Hypomethylation at KvDMR1 was observed in 3/18 clinically normal children conceived by ARTs (2 conceived by IVF and 1 by ICSI). A discordant methylation pattern was observed in the three corresponding dizygotic twins. Our findings corroborate the hypothesis of vulnerability of maternal imprinting to ARTs. Furthermore, the discordant methylation at KvDMR1 observed between dizygotic twins could be consequent to one of the following possibilities: (i) a differential vulnerability of maternal imprints among different embryos; or (ii) epimutations that occurred during gametogenesis resulting in the production of oocytes without the correct primary imprint at KvDMR1.
基因组印记改变已被证明与动物辅助生殖技术（ARTs）有关。目前，在人类中获得的数据尚无定论；然而，一些流行病学研究表明，通过ARTs受孕的儿童中印记障碍的发病率有所增加。在本研究中，我们重点关注ARTs（体外受精和卵胞浆内单精子注射[ICSI]）对临床正常儿童中母源甲基化印记控制区域KvDMR1的表观遗传重编程的影响。分别通过甲基化特异性PCR方法和与实时PCR方法相关的甲基化敏感酶切来评估KvDMR1的定性和定量甲基化。DNA取自18名通过体外受精或ICSI受孕儿童中的12名儿童的外周血以及另外6名儿童的脐带血和胎盘。将该组中观察到的甲基化模式与30名临床正常自然受孕儿童（阴性对照）和3名自然受孕的贝克威思-维德曼综合征患者（阳性对照）中观察到的模式进行比较。在18名通过ARTs受孕的临床正常儿童中有3名（2名通过体外受精，1名通过ICSI）观察到KvDMR1低甲基化。在三对相应的双卵双胞胎中观察到不一致的甲基化模式。我们的研究结果证实了母源印记对ARTs易感性的假设。此外，在双卵双胞胎之间观察到的KvDMR1甲基化不一致可能是由于以下可能性之一：（i）不同胚胎之间母源印记存在差异易感性；或（ii）配子发生过程中发生的表观突变导致产生的卵母细胞在KvDMR1处没有正确的原始印记。
