Aberrant DNA methylation of imprinted loci in human spontaneous abortions after assisted reproduction techniques and natural conception.
Aberrant DNA methylation of imprinted loci in human spontaneous abortions after assisted reproduction techniques and natural conception.
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DOI：
10.1093/humrep/des358
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发表时间：
2013
影响因子：
6.1
通讯作者：
Hai-yan Zheng;Yan Tang;J. Niu;Pu Li;D. Ye;Xin Chen;Xiao-yun Shi;Li Li-Li;Shi-ling Chen
中科院分区：
文献类型：
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作者：
Hai-yan Zheng;Yan Tang;J. Niu;Pu Li;D. Ye;Xin Chen;Xiao-yun Shi;Li Li-Li;Shi-ling Chen
辅助生殖技术（ART）是否影响印迹基因的DNA甲基化？印迹基因的异常甲基化是否导致人类自然流产（SA）的发生？我们的研究结果表明，印迹基因的印迹错误可能有助于人类SA，ART妊娠中印迹基因异常甲基化的发生与自然妊娠相当。动物数据和人类研究表明，胚胎的体外培养会导致单个基因的甲基化缺陷，这可能会影响随后的胚胎发育并导致SA。然而，我们以前的研究表明，在人类流产绒毛样本（CVS）的PEG 1异常甲基化模式，但从ART衍生的妊娠CVS异常甲基化的发生率增加没有观察到。研究设计、大小及持续时间CVS收集自2008年5月至2011年7月在南方医院妇产科行人工流产手术的妇女。从流产胎儿和死胎中获得肌肉样品（MS）。将样品分为四个实验组：（A）ART后SA/死产（n = 75），（B）ART后多胎减胎（n = 73），（C）自然妊娠的SA/死产（n = 90）和（D）自然妊娠的人工流产（IA）（n = 82）。对象/材料、地点和方法患者的平均年龄± SD为31.0 ± 4.1岁（范围：18-45岁）。采用焦磷酸测序和亚硫酸氢盐测序PCR技术分析CVS和MS中1个父系甲基化（H19）和2个母系甲基化（LIT 1和SNRPN）基因的DNA甲基化模式。在LIT 1和SNRPN中未检测到明显的低甲基化（90%），但在H19中观察到两个高甲基化区域（91.7%和91.4%）。SA组（A组和C组）的平均甲基化百分比高于IA组（B组和D组; P<0.05）。箱形图分析显示，在165个SA样品中，三个基因的40/495（8.1%）差异甲基化区域的甲基化值代表离群值。LIT 1的离群值发生率最高（13.3%，22/165）。相比之下，在155个IA样本中未发现离群值。受试者工作特征曲线分析显示，三个基因甲基化百分比与SA发病率呈正相关（P<0.05）。此外，受孕模式（自然与ART）和ART中使用的受精方法（IVF和ICSI）并不影响印迹基因的甲基化模式。在ART样品中未发现异常甲基化率的增加。局限性和排除的原因所研究的基因座只代表了发育重要基因的一小部分。需要进一步的研究来评估可能导致SA的其他基因的表达和甲基化状态的变化。研究结果的更广泛意义研究结果为人类SA的病因学提供了新的见解。不能排除异常甲基化是导致SA的缺陷的结果的可能性。研究资金/竞争利益（S）没有作者有任何竞争利益。本研究得到了国家自然科学基金项目（81170574）、国家重点基础研究发展计划（973计划）项目（2007 CB 948104）、广东省与中国科学院综合性战略科学合作项目（04020416）和广州市科技计划重点项目（11 C22120737）的资助。
STUDY QUESTION Do assisted reproduction techniques (ARTs) affect DNA methylation of imprinted genes and does aberrant methylation of imprinted genes account for the incidence of human spontaneous abortion (SA)? SUMMARY ANSWER Our results show that imprinting errors of imprinted genes may contribute to human SA, and the occurrence of aberrant methylation of imprinted genes in ART pregnancies was comparable with that in natural pregnancies. WHAT IS KNOWN ALREADY Animal data and human studies demonstrated that in vitro culture of embryos can cause methylation defects in individual genes, which might affect subsequent embryonic development and contribute to SA. However, our previous studies showed an abnormal methylation pattern of PEG1 in human aborted chrionic villus samples (CVS) but an increased occurrence of aberrant methylation in CVS from ART-derived pregnancies was not observed. STUDY DESIGN, SIZE AND DURATION CVS were collected from women who underwent abortion procedures in the Department of Gynecology and Obstetrics in Nanfang Hospital from May 2008 to July 2011. Muscle samples (MS) were obtained from aborted fetuses and stillbirths. The samples were divided into four experimental groups: (A) SA/stillbirth after ART (n = 75), (B) multi-fetal reduction after ART (n = 73), (C) SA/stillbirth of natural pregnancies (n = 90) and (D) induced abortion (IA) of natural pregnancies (n = 82). PARTICIPANTS/MATERIALS, SETTING AND METHODS The mean ± SD age of patients was 31.0 ± 4.1 (range: 18-45 years). The DNA methylation patterns of one paternally methylated (H19) and two maternally methylated (LIT1 and SNRPN) genes were analyzed in CVS and MS using pyrosequencing and bisulfite sequencing PCR. MAIN RESULTS AND THE ROLE OF CHANCE Clear hypo-methylation (90%) were not detected in LIT1 and SNRPN but two regions of hyper-methylation (91.7 and 91.4%) were observed in H19. The mean percentage of methylation in the SA samples (groups A and C) was higher than that in the IA samples (groups B and D; P<0.05). Box plot analyses showed that in the 165 SA samples, methylation values for 40/495 (8.1%) differentially methylated regions of the three genes represented outliers. The incidence of outlier was highest for LIT1 (13.3%, 22/165). In contrast, no outliers were found in the 155 IA samples. The receiver operating characteristic curve analyses showed a positive correlation between percentage methylation of all three genes and incidence of SA (P<0.05). In addition, the conception modes (natural versus ART) and the fertilization methods used in ART (IVF and ICSI) did not affect the methylation patterns of the imprinted genes. No increase in the rate of abnormal methylation was found in the ART samples. LIMITATIONS AND REASONS FOR CAUTION The studied loci represent only a small fraction of developmentally important genes. Further studies are needed to evaluate changes in the expression and the methylation status of other genes that may lead to SA. WIDER IMPLICATIONS OF THE FINDINGS The findings provide new insights into the etiology of human SA. The possibility that the abnormal methylation seen is a consequence of the defect that led to the SA cannot be excluded. STUDY FUNDING/COMPETING INTEREST(S) None of the authors has any competing interest. This study was supported by National Natural Science Foundation of China (81170574), The National Key Basic Research Development Plan of China (973 Program) (2007CB948104), Comprehensive strategic sciences cooperation projects of Guangdong Province and Chinese Academy (04020416) and Guangzhou Science and Technology Program key projects (11C22120737).