辅助生殖技术子代染色体11p15.5区印记控制区甲基化状态的研究
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摘要
研究背景
体外受精-胚胎移植技术(IVF-ET)已有30余年的发展历程,到目前为止全世界范围内经辅助生育技术(assisted reproductive technology, ART)治疗出生的子代已超过三百五十万,且出生率呈逐年增加的趋势,ART子代正成为全球人口的一个重要组成部分。有报道提示在ART子代中健康风险增加,包括自然流产、低体重、极低体重、出生缺陷、脑瘫等,原因尚不明确。关于ART操作安全性的研究已成为当前遗传学界和生殖学界的一个热门研究领域。本研究即是关于ART操作安全性研究的一个分支。
近年来印迹缺陷,包括Beckwith-Wiedemann综合征(Beckwith-Wiedemann Syndrome, BWS)、Angelman综合征(Angelman Syndrome, AS)引起了人们的广泛关注。多项病例对照研究提示在ART子代中印迹缺陷风险增加。印迹基因等位基因特异性表达的破坏与人类遗传疾病、某些肿瘤及神经系统疾病相关,其亲缘特异性表达需要正常水平的DNA甲基化。印迹基因的甲基化在配子发生过程中擦除并重新建立,在种植前后维持甲基化,而ART技术在配子发生、受精及种植前后阶段操作配子和胚胎,这一时期恰恰是印迹基因甲基化重新编程的关键时期,因此可能导致印迹基因甲基化异常。另外,接受ART治疗的不育夫妇,很大比例在自然情况下并不具备孕育子代的能力,其生育能力的缺陷亦有可能会通过ART遗传给下一代,而是否与印记基因有关尚不得知。
目前ART操作与印迹基因印迹状态的关系尚未明确。动物实验提示操作及体外培养胚胎可能破坏基因组印迹,而且在配子形成过程中发生的表观遗传改变在种植后的发育过程中不能纠正,可能与印迹基因表达异常及表型异常相关。但是人类研究尚未发现可检测的印迹基因表达异常。
目前对于ART子代安全性的研究,以大样本的临床调查研究和对患有印记基因疾病病例进行实验室研究为主,而关于正常表型的ART子代印记基因的研究,只是近两年才开始见有散在的小样本报道,有必要扩大对ART子代关键印记控制区的印迹状态进行研究以排查ART操作的风险。
人染色体11p15.5区是一个重要的印迹基因聚集的区域,该区基因失调可能与生长过度、肿瘤易感综合症、BWS、偏侧发育过度、SRS和新生儿短暂性糖尿病等有关。本文从遗传学和表观遗传学角度分析辅助生殖技术的安全性,对部分ART子代的脐血染色体核型进行分析,筛查正常表型ART子代是否可能存在染色体核型的异常,同时选取人染色体11p15.5上关键性的印记控制区1(H19印记控制区)与印记控制区2(KCNQ1印记控制区)进行研究,检测与生长发育密切相关的印记基因IGF2和H19mRNA的表达水平和两印记控制区的印记和甲基化状态,评估辅助生殖技术的安全性。
第一部分辅助生殖技术子代IGF2和H19mRNA表达水平的研究
目的
印记基因IGF2和H19与调控生长发育及印记基因疾病的发生有密切的联系,我们通过研究ART子代脐血印迹基因IGF2与H19mRNA的表达水平以评估ART操作对子代生长发育情况和出生缺陷的影响。
研究方法
研究组为2008年至2009年于南方医院行ART治疗并在南方医院分娩的58例新生儿,包括异卵双胎40例,单胎18例,对照组为以同期自然妊娠分娩于南方医院的32例新生儿,包括30例单胎与2例异卵双胎,采集新生儿脐带血10-15ml (EDTA或肝素钠抗凝),记录新生儿出生体重、分娩孕周、母龄、受孕方式、有无出生缺陷及孕期监测情况等。采用荧光定量PCR技术(SYBR Green染料法)检测印记基因IGF2和H19mRNA的表达情况,数据处理采用2-⊿⊿Ct法,使用SPSS16.0软件进行统计学分析,样本均数比较使用T检验及单向方差分析(One-way ANOVA),方差不齐时使用近似F检验Welch法,两变量相关关系使用双变量相关分析(Bivariate), P<0.05为差异有统计学意义或有显著相关关系。
结果
1 ART组与自然妊娠组比较：ART组与自然妊娠组分娩孕周在34-40周之内,无一例新生儿有表型畸形,ART组中有19例异卵双胎为低出生体重儿(出生体重<2500g),其中16例为早产儿,对照组中有2例为低出生体重儿,其中1例为早产儿。ART组平均母龄高于自然妊娠组,而平均出生体重与分娩周数均低于自然妊娠组,差异均有统计学意义(p<0.05)。ART组IGF2基因与H19基因表达水平均高于自然妊娠组,差异有统计学意义(p<0.05)。ART组两印记基因表达水平的组内变异高于自然妊娠组。
2 ART双胎组、ART单胎组与自然妊娠单胎组比较：ART双胎组与自然妊娠单胎组比较,母龄、分娩周数、出生体重、IGF2基因及H19基因表达水平之间均有有显著性差异(p<0.05); ART单胎组及ART双胎组母龄均高于自然妊娠单胎组(p<0.05)；而出生体重、分娩孕周均显示ART双胎组低于ART单胎组和自然妊娠单胎组(p<0.05)。H19基因与IGF2基因表达水平均为ART双胎组高于自然妊娠单胎组(p<0.05)。ART单胎组H19基因与IGF2基因表达水平的组内变异均大于自然妊娠单胎组。
3 IVF组、ICSI组与自然妊娠组比较：58例ART子代包括采用常规体外授精(IVF)获妊娠分娩子代33例(单胎10例+双胎23例),单精了卵胞浆内注射(ICSI)子代22例(单胎7例+双胎15例),IVF+ICSI子代3例。IVF组母龄与H19基因表达水平高于ICSI组和自然妊娠组(p<0.05), ICSI组与自然妊娠组之间不存在显著性差异(p>0.05)；出生体重、分娩周数则显示IVF组和ICSI组均低于自然妊娠组(p<0.05), IGF2基因表达水平为IVF组和ICSI组均高于自然妊娠组(p<0.05)。IVF组两印记基因H19与IGF2表达水平的组内变异明显高于自然妊娠组,ICSI组IGF2基因表达水平的组内变异亦高于自然妊娠组。
4.冻融胚胎移植组、新鲜胚胎移植组与自然妊娠组比较：58例ART子代中,包括新鲜移植胚胎获妊娠分娩子代44例(16例单胎+28例双胎)和冻融胚胎移植获妊娠分娩子代14例(2例单胎+12例双胎)。新鲜周期组母龄、IGF2基因表达水平高于自然妊娠组(p<0.05)；新鲜周期组和冻融胚胎组分娩周数、出生体重均低于自然妊娠组(p<0.05),H19基因表达在各组间均无显著性差异(p>0.05)。新鲜周期组和冻融胚胎组印记基因H19与IGF2表达水平的组内变异均高于自然妊娠组,冻融胚胎组两印记基因表达水平的组内变异亦有高于新鲜周期组的趋势。
5.相关性分析：出生体重与分娩周数呈显著正相关关系,且相关关系密切(p<0.001,相关系数为0.710)；出生体重与H19基因表达水平有显著负相关关系,但相关关系并不密切(p<0.05,相关系数为-0.267)；分娩周数与IGF2基因表达水平有显著负相关关系,但相关关系并不密切(p<0.05,相关系数为-0.223)；印记基因IGF2与H19表达水平有显著正相关关系,但相关关系并不密切(p<0.001,相关系数为0.394)；母龄与出生体重、分娩周数、IGF2及H19表达水平均无显著相关关系(p>0.05)。
结论
1.印记基因H19及IGF2mRNA的表达对胎儿宫内生长发育起着一定的调节作用,但两基因的表达水平同时又受分娩孕周和出生体重影响。
2.ART子代印迹基因IGF2与H19表达水平高于自然妊娠了代,与ART较高的双胎妊娠引发的早产低体重率有一定的关系。
3.ART单胎组H19与IGF2基因表达水平的组内变异高于自然妊娠单胎组,提示ART操作可能会对印记基因的表达水平产生一定的影响。
4. ICSI显微操作在常规IVF上基础上并未增加对印记基因IGF2与H19表达水平的影响。
5.胚胎冻融操作在新鲜胚胎移植的基础上并未增加对印记基因IGF2与H19表达水平变化的影响。
第二部分辅助生殖技术子代染色体11p15.5印记控制区印记与甲基化状态的研究
目的
人染色体11p15.5区域的印记基因的表达与甲基化状态主要由H19印记控制区和KCNQ1印记控制区两个印记控制区主导控制,该区域印记失调与印记疾病BWS、SRS等有着密切的联系,本研究即是对ART子代的这两个印记控制区的印记和甲基化状态进行探讨,比较ART子代与自然妊娠子代是否在这两个关键印记控制区存在差异性。
研究方法
收集2008年至2009年于南方医院行ART治疗并在南方医院分娩的61例新生儿作为研究组,包括异卵双胎43例,单胎18例,以同期自然妊娠分娩于南方医院的30例单胎为对照组,采集新生儿脐带血10-15ml(EDTA或肝素钠抗凝),其中3例ART双胎脐血收集量仅2ml,记录新生儿出生体重、分娩孕周、母龄、受孕方式、有无出生缺陷及孕期监测情况等。采用甲基化特异性PCR (MSP)和结合重亚硫酸盐的限制性内切酶法对61例ART子代和30例自然妊娠单胎H19印记控制区(H19ICR)、KCNQ1印记控制区(KCNQ1 ICR)和IGF2甲基化差异性区域(DMR)的印记与甲基化状态进行检测,使用重亚硫酸盐测序法(BSP)验证20例H19 ICR的甲基化状态和15例KCNQ1 ICR的甲基化状态。两独立样本率的比较使用χ2检验。
结果
1.MSP检测到61例ART子代和30例自然妊娠子代的H19 ICR、KCNQ1 ICR及IGF2DMR的甲基化DNA链与非甲基化DNA链均存在。
2.限制性内切酶TaqI与BstUI检测到3例ICSI双胎出现多个酶切位点的印记缺失,占总检测ART子代的比率为4.9%(3/61),与这3例双胎同胞妊娠的另3例ART子代经酶切检测均表现为H19 ICR的正常印记。病例资料显不ICSI操作缘于父方不育因素。
3.BSP检测的20例样本中,3例ICSI双胎的H19 ICR甲基化程度<10%,处于低度甲基化状态,证实了TaqI与BstUI酶切检测到的相关位点的印记缺失；16例H19 ICR甲基化程度在34%-67%之间,1例为25%。
4.限制性内切酶TaqI检测到16例样本出现H198271位点(基因库序列号AF125183)的C/T多态性(单核苷酸多态性,SNP), ART双胎发生率为自然妊娠单胎的2.5倍,ART子代发生率是自然妊娠子代的2.13倍,但并无统计学差异(p>0.05)。低体重儿较正常出生体重儿更易发生这种SNP,差异有统计学意义(p<0.05)。BSP证实了这种多态性的存在,并发现H198271位点的C/T多态性常和H197966位点的C/T多态性并存出现。
5. TaqI与BstUI未检测到KCNQ1 ICR有相关酶切位点的印记缺失。
6.BSP法检测15例样本的KCNQ1 ICR,其中有6例样本的甲基化程度>70%,包括两例自然妊娠低体重儿、1例ICSI单胎足月正常体重儿、1例ICSI早产低体重双胎和2例IVF早产低体重双胎。
7. H19 ICR与KCNQ1 ICR甲基化程度无显著相关关系(p>0.05),但相关系数为负值。
8. BstUI检测61例ART子代和30例自然妊娠子代的IGF2 DMR均处在正常的甲基化状态。
结论
1.正常表型的ART子代不伴有H19ICR、KCNQ1 ICR与IGF2 DMR印记的完全缺失。
2.3例ICSI双胎表现出H19 ICR多个位点的印记缺失和低度甲基化状态,可能与使用父方提供的异常的精子授精有关。
3.H198271位点完全非甲基化(C/T多态性)的现象易发生于低体重儿,该位点可能与生长发育有一定的关系。
4. KCNQ1 ICR对生长发育起着一定的调控作用,当其表现为高度甲基化时对生长发育有一定的抑制作用。
5.ART子代出现异常的印记与DNA甲基化时并不一定伴有表型的异常。
第三部分辅助生殖技术子代染色体核型分析
目的
染色体核型分析是最常规用于筛查遗传缺陷的检测方法,本研究对ART子代行染色体核型分析以排查ART操作是否会对子代造成直接的遗传风险。
研究方法
研究组为48例使用ART治疗妊娠的子代,包括行常规IVF获妊娠出生者30例,行ICSI获妊娠出生者18例,对照组为20例同期于我院出生母龄匹配的自然妊娠子代,采集脐带血,使用肝素钠抗凝。使用常规淋巴细胞培养方法行染色体核型分析。
结果
除两例中期妊娠引产的ART无脑儿外,其余ART子代和所有的自然妊娠子代均无出生缺陷的发生,染色体核型分析显示48例ART子代与20例自然妊娠子代均为正常核型。
结论
伴有染色体核型异常的病例多在早期胚胎发育阶段就遭到了淘汰,本研究未发现ART子代有染色体核型异常的病例,可能与样本量较小有关。
Introduction
Assisted reproductive technology (ART) have been widely used over the past three decades to help infertile couples conceive. Over 3,500,000 babies have been conceived through ART woridwide, the number is growing. Recent studies on the ARTconceived population have raised concern about the possible risks of these techniques, in particular with regard to increased incidence of growth and developmental disorders. Amplified risks associated with ART has been reported, including spontaneous abortion, low or very low birthweight, small for gestational age, major malformations, cerebral palsy and so on. Study on the safety of ART has becoming to a hot-spot to genetist and researchers working on reproduction.
Recently, imprinting disorders including Beekwith-Wiedemann Syndrome and Angelman Syndrome attract most attention with respect to the safety of ART. Although current evidence does not allow definite conclusions to be drawn,findings of different case series and case-control studies have consistently suggested an inereased risk of imprinting disorders in pregnaneies after ART.Differential expression of the Paternal and matmal alleles of imprinted genes requires regulated DNA methylation.ART procedures, including hormone-induced superovulation, in vitro fertilization and embryo culture, manipulate gametes and embryos during gametogenesis, fertilization and pre-implantation stages, which is crucial for methytation reprogramming of imprinted genes.Therefore, ART is likely to induce in appropriate methylation of imprinted genes and disrupts imprinting.On the other hand, the couples suffering infertility might spread the disabled fertility problem to their offspring by ART treatment.
The relationship between ART procedures and expression of imprinted genes in offspring still remains unelear.Experimental evidences in animals and embryonic stem cells indicate that manipulation and invitro embryo culture may lead to disrupted genomie imprinting.However, several studies in human found no detectable alteration of imprinted gene expression, indicating that the imprints, which have been set during gametogenesis, are stabely maintained during the ART procedure.
Till now,we can see most of studies on ART offspring are focusing on clinical case control cohort report and imprinting disorders, but few studies on imprinted genes of phenotypic normal ART offspring have been done, so more molecular biology studies are suggested to confirm the safety of ART.
Human chromosome band 11p15.5 includes a cluster of genes that are imprinted.Dysregulation of this gene cluster is associated with the overgrowth and tumor predisposition syndrome, BWS, isolated hemihyperplasia, SRS, and transient neonatal diabetes mellitus.Here we investigate the expression levels of IGF2 and H19, which are very important in the control of fetal and postnatal growth,and test imprinting and methylation status of two important imprinting control regions at chromosome 11p15.5, which regulate the two main domains of 11p15.5, to evaluate the safety of ART.
Part1 Expression levels of IGF2 and H19 of the offspring conceived throught assisted reproductive technology
Objective:To explore the expression levels of IGF2 and H19 gene of ART offspring and natural conceived offspring, evaluate the growth and developmental condition and safety of ART offspring.
Methods:A total of 58 newborns conceived by ART and 32 newborns conceived naturally from 2008 to 2009 at the Nanfang Hospital, Guangzhou, China, were chosen for our studies. Cord-blood samples (10-15 mL)were collected in EDTA tubes from the umbilical cords of the infants. Maternal weight, height, age, past and present medical history, babies'gender, birth weight (BW), record of birth (including mode and indication for delivery) and gestational age were recorded. Testing their expression levels of imprinted genes IGF2 and H19 by real-time PCR. One-way ANOVA and SPSS 16.0 were used to do satistical work.
Results:1.A major difference between the two sample sets concerns the frequency of twin pregnancies. Due to multiple embryo transfer following ART twin pregnancies are very frequent in ART group, while only one twin pregnancy was among the samples obtained from the spontaneously conceived group (p<0.05). Accordingly, children born after ART had a slightly lower gestational age and were lighter and smaller at birth compared to the spontaneously conceived children (p<0.05). In addition, maternal age was higher in the ART groups (p<0.05)
2. There existed significant difference between ART group and naturally conceived group with the expression levels of IGF2 (p<0.05);the difference of the expression levels of H19 was also significantly (p<0.05).which might be both due to twin pregnancy since there were no significant difference between naturally conceived singletons and ART singletons with the expression levels of IGF2 and H19 gene (p>0.05)
3. There exsited no significant difference between IVF group and ICSI group with the expression levels of IGF2,so did fresh cycle group and frozen/thawn group (p>0.05).But as to H19 gene, the former was significant (p<0.05),the latter was not (p>0.05)
4. The correlation between birth weight and expression levels of H19 gene was significantly (p<0.05), so was gestational age and expression levels of IGF2 gene (p<0.05).The correlation of expression levels between the two imprinted genes was significantly too (p<0.05)
Conclusion:High twin pregnancy rate of ART is likely to result in preterm birth and low birth weight, which might has some connection with changing of expression levels of imprinted genes as well, although birth defects haven't been found in this study, we still need further study to focus on the safety of ART in the long run.
Part2 Imprinting and methylation status of two imprinting control regions at chromosome 11p15.5 in offspring conceived through assisted technology
Objective:To explore the imprinting and methylation status of two important control regions at human chromosome 11p15.5, evaluate the probability of abnormal imprinting and methylation happening to ART offspring.
Methods:A total of 61 newborns conceived by ART and 30 newborns conceived naturally from 2008 to 2009 at the Nanfang Hospital, Guangzhou, China, were chosen for our studies. Cord-blood samples (10-15 mL)were collected in EDTA tubes or heparin sodium tubes from the umbilical cords of the infants. Maternal weight, height, age, past and present medical history, babies'gender, birth weight (BW), record of birth (including mode and indication for delivery) and gestational age were recorded.Methylation specific PCR(MSP) was used to detect methylated and unmethylated alleles. Combined bisulphite restriction analysis(COBRA) and bisulphite sequencing(BSP) were chosen for detecting imprinting and methylation status of the two imprinting control regions.
Results:1 Abnormal imprinting and hypo-methylation at H19 ICR were found to happen to three dizygotic twins conceived after intracytoplasmic sperm injection (ICSI).Near upon 66%(14/22) of cases with low birth-weight were found to have a single C/T polymorphism(SNP) (H19 8271, GenBank accession no. AF125183) at H19 ICR, calculated with nearly 25%(13/43) of ART twins compared with 10%(3/30) of naturally conceived singletons and 0%(0/18) of ART singletons.There existed significant difference between cases with low birthweight and cases with normal birthweight (p<0.05)
2.COBRA did found no cases with apparent abnormal imprinting of KCNQ1 ICR while BSP found 6 cases with low birthweight(including 2 naturally conceived singletons) and 1 ICSI singleton with normal birthweight in hyper-methylation status of KCNQ1 ICR.
3.All of the cases showed normal imprinting at IGF2 DMR which detected by MSP and COBRA.
Conclusion:The abnormal imprinting/methylation probably results from an imprint erasure defect in the paternal germ line and not the in vitro fertilization/ICSI procedure. Imprinting/methylation test on paternal sperm is suggested to confirm our imagination. Additional larger study is considered to determine whether the C/T SNP detected in our study is associated with growth regulation.
Part3 Karotype of offspring conceived by assisted reproductive technology
Objective:To investigate the karyotype of ART offsping, evaluate genetic risk of this group.
Methords:48 ART offspring including 30 conceived through IVF,18 conceived through ICSI were recruited as study group,20 naturally conceived newborns were collected as compared group.Karyotype was detected in a standard way.
Results:All cases had normal karyotype,including 35 male cases with 46,XY and 33 female cases with 46,XX.
Conclusion:We consider that the children conceived by ART with no birth defects have few chances to carry abnormal karyotype, a larger number of case study is suggested to support our imagination.
体外受精-胚胎移植技术(IVF-ET)已有30余年的发展历程,到目前为止全世界范围内经辅助生育技术(assisted reproductive technology, ART)治疗出生的子代已超过三百五十万,且出生率呈逐年增加的趋势,ART子代正成为全球人口的一个重要组成部分。有报道提示在ART子代中健康风险增加,包括自然流产、低体重、极低体重、出生缺陷、脑瘫等,原因尚不明确。关于ART操作安全性的研究已成为当前遗传学界和生殖学界的一个热门研究领域。本研究即是关于ART操作安全性研究的一个分支。
近年来印迹缺陷,包括Beckwith-Wiedemann综合征(Beckwith-Wiedemann Syndrome, BWS)、Angelman综合征(Angelman Syndrome, AS)引起了人们的广泛关注。多项病例对照研究提示在ART子代中印迹缺陷风险增加。印迹基因等位基因特异性表达的破坏与人类遗传疾病、某些肿瘤及神经系统疾病相关,其亲缘特异性表达需要正常水平的DNA甲基化。印迹基因的甲基化在配子发生过程中擦除并重新建立,在种植前后维持甲基化,而ART技术在配子发生、受精及种植前后阶段操作配子和胚胎,这一时期恰恰是印迹基因甲基化重新编程的关键时期,因此可能导致印迹基因甲基化异常。另外,接受ART治疗的不育夫妇,很大比例在自然情况下并不具备孕育子代的能力,其生育能力的缺陷亦有可能会通过ART遗传给下一代,而是否与印记基因有关尚不得知。
目前ART操作与印迹基因印迹状态的关系尚未明确。动物实验提示操作及体外培养胚胎可能破坏基因组印迹,而且在配子形成过程中发生的表观遗传改变在种植后的发育过程中不能纠正,可能与印迹基因表达异常及表型异常相关。但是人类研究尚未发现可检测的印迹基因表达异常。
目前对于ART子代安全性的研究,以大样本的临床调查研究和对患有印记基因疾病病例进行实验室研究为主,而关于正常表型的ART子代印记基因的研究,只是近两年才开始见有散在的小样本报道,有必要扩大对ART子代关键印记控制区的印迹状态进行研究以排查ART操作的风险。
人染色体11p15.5区是一个重要的印迹基因聚集的区域,该区基因失调可能与生长过度、肿瘤易感综合症、BWS、偏侧发育过度、SRS和新生儿短暂性糖尿病等有关。本文从遗传学和表观遗传学角度分析辅助生殖技术的安全性,对部分ART子代的脐血染色体核型进行分析,筛查正常表型ART子代是否可能存在染色体核型的异常,同时选取人染色体11p15.5上关键性的印记控制区1(H19印记控制区)与印记控制区2(KCNQ1印记控制区)进行研究,检测与生长发育密切相关的印记基因IGF2和H19mRNA的表达水平和两印记控制区的印记和甲基化状态,评估辅助生殖技术的安全性。
第一部分辅助生殖技术子代IGF2和H19mRNA表达水平的研究
目的
印记基因IGF2和H19与调控生长发育及印记基因疾病的发生有密切的联系,我们通过研究ART子代脐血印迹基因IGF2与H19mRNA的表达水平以评估ART操作对子代生长发育情况和出生缺陷的影响。
研究方法
研究组为2008年至2009年于南方医院行ART治疗并在南方医院分娩的58例新生儿,包括异卵双胎40例,单胎18例,对照组为以同期自然妊娠分娩于南方医院的32例新生儿,包括30例单胎与2例异卵双胎,采集新生儿脐带血10-15ml (EDTA或肝素钠抗凝),记录新生儿出生体重、分娩孕周、母龄、受孕方式、有无出生缺陷及孕期监测情况等。采用荧光定量PCR技术(SYBR Green染料法)检测印记基因IGF2和H19mRNA的表达情况,数据处理采用2-⊿⊿Ct法,使用SPSS16.0软件进行统计学分析,样本均数比较使用T检验及单向方差分析(One-way ANOVA),方差不齐时使用近似F检验Welch法,两变量相关关系使用双变量相关分析(Bivariate), P<0.05为差异有统计学意义或有显著相关关系。
结果
1 ART组与自然妊娠组比较：ART组与自然妊娠组分娩孕周在34-40周之内,无一例新生儿有表型畸形,ART组中有19例异卵双胎为低出生体重儿(出生体重<2500g),其中16例为早产儿,对照组中有2例为低出生体重儿,其中1例为早产儿。ART组平均母龄高于自然妊娠组,而平均出生体重与分娩周数均低于自然妊娠组,差异均有统计学意义(p<0.05)。ART组IGF2基因与H19基因表达水平均高于自然妊娠组,差异有统计学意义(p<0.05)。ART组两印记基因表达水平的组内变异高于自然妊娠组。
2 ART双胎组、ART单胎组与自然妊娠单胎组比较：ART双胎组与自然妊娠单胎组比较,母龄、分娩周数、出生体重、IGF2基因及H19基因表达水平之间均有有显著性差异(p<0.05); ART单胎组及ART双胎组母龄均高于自然妊娠单胎组(p<0.05)；而出生体重、分娩孕周均显示ART双胎组低于ART单胎组和自然妊娠单胎组(p<0.05)。H19基因与IGF2基因表达水平均为ART双胎组高于自然妊娠单胎组(p<0.05)。ART单胎组H19基因与IGF2基因表达水平的组内变异均大于自然妊娠单胎组。
3 IVF组、ICSI组与自然妊娠组比较：58例ART子代包括采用常规体外授精(IVF)获妊娠分娩子代33例(单胎10例+双胎23例),单精了卵胞浆内注射(ICSI)子代22例(单胎7例+双胎15例),IVF+ICSI子代3例。IVF组母龄与H19基因表达水平高于ICSI组和自然妊娠组(p<0.05), ICSI组与自然妊娠组之间不存在显著性差异(p>0.05)；出生体重、分娩周数则显示IVF组和ICSI组均低于自然妊娠组(p<0.05), IGF2基因表达水平为IVF组和ICSI组均高于自然妊娠组(p<0.05)。IVF组两印记基因H19与IGF2表达水平的组内变异明显高于自然妊娠组,ICSI组IGF2基因表达水平的组内变异亦高于自然妊娠组。
4.冻融胚胎移植组、新鲜胚胎移植组与自然妊娠组比较：58例ART子代中,包括新鲜移植胚胎获妊娠分娩子代44例(16例单胎+28例双胎)和冻融胚胎移植获妊娠分娩子代14例(2例单胎+12例双胎)。新鲜周期组母龄、IGF2基因表达水平高于自然妊娠组(p<0.05)；新鲜周期组和冻融胚胎组分娩周数、出生体重均低于自然妊娠组(p<0.05),H19基因表达在各组间均无显著性差异(p>0.05)。新鲜周期组和冻融胚胎组印记基因H19与IGF2表达水平的组内变异均高于自然妊娠组,冻融胚胎组两印记基因表达水平的组内变异亦有高于新鲜周期组的趋势。
5.相关性分析：出生体重与分娩周数呈显著正相关关系,且相关关系密切(p<0.001,相关系数为0.710)；出生体重与H19基因表达水平有显著负相关关系,但相关关系并不密切(p<0.05,相关系数为-0.267)；分娩周数与IGF2基因表达水平有显著负相关关系,但相关关系并不密切(p<0.05,相关系数为-0.223)；印记基因IGF2与H19表达水平有显著正相关关系,但相关关系并不密切(p<0.001,相关系数为0.394)；母龄与出生体重、分娩周数、IGF2及H19表达水平均无显著相关关系(p>0.05)。
结论
1.印记基因H19及IGF2mRNA的表达对胎儿宫内生长发育起着一定的调节作用,但两基因的表达水平同时又受分娩孕周和出生体重影响。
2.ART子代印迹基因IGF2与H19表达水平高于自然妊娠了代,与ART较高的双胎妊娠引发的早产低体重率有一定的关系。
3.ART单胎组H19与IGF2基因表达水平的组内变异高于自然妊娠单胎组,提示ART操作可能会对印记基因的表达水平产生一定的影响。
4. ICSI显微操作在常规IVF上基础上并未增加对印记基因IGF2与H19表达水平的影响。
5.胚胎冻融操作在新鲜胚胎移植的基础上并未增加对印记基因IGF2与H19表达水平变化的影响。
第二部分辅助生殖技术子代染色体11p15.5印记控制区印记与甲基化状态的研究
目的
人染色体11p15.5区域的印记基因的表达与甲基化状态主要由H19印记控制区和KCNQ1印记控制区两个印记控制区主导控制,该区域印记失调与印记疾病BWS、SRS等有着密切的联系,本研究即是对ART子代的这两个印记控制区的印记和甲基化状态进行探讨,比较ART子代与自然妊娠子代是否在这两个关键印记控制区存在差异性。
研究方法
收集2008年至2009年于南方医院行ART治疗并在南方医院分娩的61例新生儿作为研究组,包括异卵双胎43例,单胎18例,以同期自然妊娠分娩于南方医院的30例单胎为对照组,采集新生儿脐带血10-15ml(EDTA或肝素钠抗凝),其中3例ART双胎脐血收集量仅2ml,记录新生儿出生体重、分娩孕周、母龄、受孕方式、有无出生缺陷及孕期监测情况等。采用甲基化特异性PCR (MSP)和结合重亚硫酸盐的限制性内切酶法对61例ART子代和30例自然妊娠单胎H19印记控制区(H19ICR)、KCNQ1印记控制区(KCNQ1 ICR)和IGF2甲基化差异性区域(DMR)的印记与甲基化状态进行检测,使用重亚硫酸盐测序法(BSP)验证20例H19 ICR的甲基化状态和15例KCNQ1 ICR的甲基化状态。两独立样本率的比较使用χ2检验。
结果
1.MSP检测到61例ART子代和30例自然妊娠子代的H19 ICR、KCNQ1 ICR及IGF2DMR的甲基化DNA链与非甲基化DNA链均存在。
2.限制性内切酶TaqI与BstUI检测到3例ICSI双胎出现多个酶切位点的印记缺失,占总检测ART子代的比率为4.9%(3/61),与这3例双胎同胞妊娠的另3例ART子代经酶切检测均表现为H19 ICR的正常印记。病例资料显不ICSI操作缘于父方不育因素。
3.BSP检测的20例样本中,3例ICSI双胎的H19 ICR甲基化程度<10%,处于低度甲基化状态,证实了TaqI与BstUI酶切检测到的相关位点的印记缺失；16例H19 ICR甲基化程度在34%-67%之间,1例为25%。
4.限制性内切酶TaqI检测到16例样本出现H198271位点(基因库序列号AF125183)的C/T多态性(单核苷酸多态性,SNP), ART双胎发生率为自然妊娠单胎的2.5倍,ART子代发生率是自然妊娠子代的2.13倍,但并无统计学差异(p>0.05)。低体重儿较正常出生体重儿更易发生这种SNP,差异有统计学意义(p<0.05)。BSP证实了这种多态性的存在,并发现H198271位点的C/T多态性常和H197966位点的C/T多态性并存出现。
5. TaqI与BstUI未检测到KCNQ1 ICR有相关酶切位点的印记缺失。
6.BSP法检测15例样本的KCNQ1 ICR,其中有6例样本的甲基化程度>70%,包括两例自然妊娠低体重儿、1例ICSI单胎足月正常体重儿、1例ICSI早产低体重双胎和2例IVF早产低体重双胎。
7. H19 ICR与KCNQ1 ICR甲基化程度无显著相关关系(p>0.05),但相关系数为负值。
8. BstUI检测61例ART子代和30例自然妊娠子代的IGF2 DMR均处在正常的甲基化状态。
结论
1.正常表型的ART子代不伴有H19ICR、KCNQ1 ICR与IGF2 DMR印记的完全缺失。
2.3例ICSI双胎表现出H19 ICR多个位点的印记缺失和低度甲基化状态,可能与使用父方提供的异常的精子授精有关。
3.H198271位点完全非甲基化(C/T多态性)的现象易发生于低体重儿,该位点可能与生长发育有一定的关系。
4. KCNQ1 ICR对生长发育起着一定的调控作用,当其表现为高度甲基化时对生长发育有一定的抑制作用。
5.ART子代出现异常的印记与DNA甲基化时并不一定伴有表型的异常。
第三部分辅助生殖技术子代染色体核型分析
目的
染色体核型分析是最常规用于筛查遗传缺陷的检测方法,本研究对ART子代行染色体核型分析以排查ART操作是否会对子代造成直接的遗传风险。
研究方法
研究组为48例使用ART治疗妊娠的子代,包括行常规IVF获妊娠出生者30例,行ICSI获妊娠出生者18例,对照组为20例同期于我院出生母龄匹配的自然妊娠子代,采集脐带血,使用肝素钠抗凝。使用常规淋巴细胞培养方法行染色体核型分析。
结果
除两例中期妊娠引产的ART无脑儿外,其余ART子代和所有的自然妊娠子代均无出生缺陷的发生,染色体核型分析显示48例ART子代与20例自然妊娠子代均为正常核型。
结论
伴有染色体核型异常的病例多在早期胚胎发育阶段就遭到了淘汰,本研究未发现ART子代有染色体核型异常的病例,可能与样本量较小有关。
Introduction
Assisted reproductive technology (ART) have been widely used over the past three decades to help infertile couples conceive. Over 3,500,000 babies have been conceived through ART woridwide, the number is growing. Recent studies on the ARTconceived population have raised concern about the possible risks of these techniques, in particular with regard to increased incidence of growth and developmental disorders. Amplified risks associated with ART has been reported, including spontaneous abortion, low or very low birthweight, small for gestational age, major malformations, cerebral palsy and so on. Study on the safety of ART has becoming to a hot-spot to genetist and researchers working on reproduction.
Recently, imprinting disorders including Beekwith-Wiedemann Syndrome and Angelman Syndrome attract most attention with respect to the safety of ART. Although current evidence does not allow definite conclusions to be drawn,findings of different case series and case-control studies have consistently suggested an inereased risk of imprinting disorders in pregnaneies after ART.Differential expression of the Paternal and matmal alleles of imprinted genes requires regulated DNA methylation.ART procedures, including hormone-induced superovulation, in vitro fertilization and embryo culture, manipulate gametes and embryos during gametogenesis, fertilization and pre-implantation stages, which is crucial for methytation reprogramming of imprinted genes.Therefore, ART is likely to induce in appropriate methylation of imprinted genes and disrupts imprinting.On the other hand, the couples suffering infertility might spread the disabled fertility problem to their offspring by ART treatment.
The relationship between ART procedures and expression of imprinted genes in offspring still remains unelear.Experimental evidences in animals and embryonic stem cells indicate that manipulation and invitro embryo culture may lead to disrupted genomie imprinting.However, several studies in human found no detectable alteration of imprinted gene expression, indicating that the imprints, which have been set during gametogenesis, are stabely maintained during the ART procedure.
Till now,we can see most of studies on ART offspring are focusing on clinical case control cohort report and imprinting disorders, but few studies on imprinted genes of phenotypic normal ART offspring have been done, so more molecular biology studies are suggested to confirm the safety of ART.
Human chromosome band 11p15.5 includes a cluster of genes that are imprinted.Dysregulation of this gene cluster is associated with the overgrowth and tumor predisposition syndrome, BWS, isolated hemihyperplasia, SRS, and transient neonatal diabetes mellitus.Here we investigate the expression levels of IGF2 and H19, which are very important in the control of fetal and postnatal growth,and test imprinting and methylation status of two important imprinting control regions at chromosome 11p15.5, which regulate the two main domains of 11p15.5, to evaluate the safety of ART.
Part1 Expression levels of IGF2 and H19 of the offspring conceived throught assisted reproductive technology
Objective:To explore the expression levels of IGF2 and H19 gene of ART offspring and natural conceived offspring, evaluate the growth and developmental condition and safety of ART offspring.
Methods:A total of 58 newborns conceived by ART and 32 newborns conceived naturally from 2008 to 2009 at the Nanfang Hospital, Guangzhou, China, were chosen for our studies. Cord-blood samples (10-15 mL)were collected in EDTA tubes from the umbilical cords of the infants. Maternal weight, height, age, past and present medical history, babies'gender, birth weight (BW), record of birth (including mode and indication for delivery) and gestational age were recorded. Testing their expression levels of imprinted genes IGF2 and H19 by real-time PCR. One-way ANOVA and SPSS 16.0 were used to do satistical work.
Results:1.A major difference between the two sample sets concerns the frequency of twin pregnancies. Due to multiple embryo transfer following ART twin pregnancies are very frequent in ART group, while only one twin pregnancy was among the samples obtained from the spontaneously conceived group (p<0.05). Accordingly, children born after ART had a slightly lower gestational age and were lighter and smaller at birth compared to the spontaneously conceived children (p<0.05). In addition, maternal age was higher in the ART groups (p<0.05)
2. There existed significant difference between ART group and naturally conceived group with the expression levels of IGF2 (p<0.05);the difference of the expression levels of H19 was also significantly (p<0.05).which might be both due to twin pregnancy since there were no significant difference between naturally conceived singletons and ART singletons with the expression levels of IGF2 and H19 gene (p>0.05)
3. There exsited no significant difference between IVF group and ICSI group with the expression levels of IGF2,so did fresh cycle group and frozen/thawn group (p>0.05).But as to H19 gene, the former was significant (p<0.05),the latter was not (p>0.05)
4. The correlation between birth weight and expression levels of H19 gene was significantly (p<0.05), so was gestational age and expression levels of IGF2 gene (p<0.05).The correlation of expression levels between the two imprinted genes was significantly too (p<0.05)
Conclusion:High twin pregnancy rate of ART is likely to result in preterm birth and low birth weight, which might has some connection with changing of expression levels of imprinted genes as well, although birth defects haven't been found in this study, we still need further study to focus on the safety of ART in the long run.
Part2 Imprinting and methylation status of two imprinting control regions at chromosome 11p15.5 in offspring conceived through assisted technology
Objective:To explore the imprinting and methylation status of two important control regions at human chromosome 11p15.5, evaluate the probability of abnormal imprinting and methylation happening to ART offspring.
Methods:A total of 61 newborns conceived by ART and 30 newborns conceived naturally from 2008 to 2009 at the Nanfang Hospital, Guangzhou, China, were chosen for our studies. Cord-blood samples (10-15 mL)were collected in EDTA tubes or heparin sodium tubes from the umbilical cords of the infants. Maternal weight, height, age, past and present medical history, babies'gender, birth weight (BW), record of birth (including mode and indication for delivery) and gestational age were recorded.Methylation specific PCR(MSP) was used to detect methylated and unmethylated alleles. Combined bisulphite restriction analysis(COBRA) and bisulphite sequencing(BSP) were chosen for detecting imprinting and methylation status of the two imprinting control regions.
Results:1 Abnormal imprinting and hypo-methylation at H19 ICR were found to happen to three dizygotic twins conceived after intracytoplasmic sperm injection (ICSI).Near upon 66%(14/22) of cases with low birth-weight were found to have a single C/T polymorphism(SNP) (H19 8271, GenBank accession no. AF125183) at H19 ICR, calculated with nearly 25%(13/43) of ART twins compared with 10%(3/30) of naturally conceived singletons and 0%(0/18) of ART singletons.There existed significant difference between cases with low birthweight and cases with normal birthweight (p<0.05)
2.COBRA did found no cases with apparent abnormal imprinting of KCNQ1 ICR while BSP found 6 cases with low birthweight(including 2 naturally conceived singletons) and 1 ICSI singleton with normal birthweight in hyper-methylation status of KCNQ1 ICR.
3.All of the cases showed normal imprinting at IGF2 DMR which detected by MSP and COBRA.
Conclusion:The abnormal imprinting/methylation probably results from an imprint erasure defect in the paternal germ line and not the in vitro fertilization/ICSI procedure. Imprinting/methylation test on paternal sperm is suggested to confirm our imagination. Additional larger study is considered to determine whether the C/T SNP detected in our study is associated with growth regulation.
Part3 Karotype of offspring conceived by assisted reproductive technology
Objective:To investigate the karyotype of ART offsping, evaluate genetic risk of this group.
Methords:48 ART offspring including 30 conceived through IVF,18 conceived through ICSI were recruited as study group,20 naturally conceived newborns were collected as compared group.Karyotype was detected in a standard way.
Results:All cases had normal karyotype,including 35 male cases with 46,XY and 33 female cases with 46,XX.
Conclusion:We consider that the children conceived by ART with no birth defects have few chances to carry abnormal karyotype, a larger number of case study is suggested to support our imagination.
引文
1. ICMART. (International Committee Monitoring ART):Presentation of preliminary data for 2004. In:24th Annual Meeting of the ESHRE. Barcelona, Spain:Hum Reprod,2008.
2. Centers for Disease Control and Prevention, American Society for Reproductive Medicine and Society for Assisted Reproductive Technology.2005 Assisted Reproductive Technology Success Rates:National Summary and Fertility Clinic Reports. Atlanta:Centers for Disease Control and Prevention,2007.
3. Andersen AN, Goossens V, Ferraretti AP, et al. Assisted reproductive technology in Europe,2004:results generated from European registers by ESHRE [J]. Hum Reprod,2008,23(4):756-771.
4. Helmerhorst FM, PerquinDA, DonkerD, etal. Perinatal outeome of singletons and twins after assisted conception:a systematic review of controlled studies[J]. BMJ,2004,328 (7434):261.
5. Stromberg B, Dahlquist G, Ericson A, et al. Neurological sequelae in children born after in-vitro fertilization:a population-based study[J]. Lancet,2002,359 (9305):461-465.
6. Hansen M, Colvin L, Petterson B, et al. Twins born following assisted reproductive technology:perinatal outcome and admission to hospital[J]. Hum Reprod,2009,24 (9):2321-2331.
7. Hansen M, Bower C, Milne E, et al. Assisted reproductive technologies and the risk of birth defects-a systematic review[J]. Hum Reprod,2005,20(2):328-338.
8.黎真,傅衍,牛冬,等.遗传印记--一种对孟德尔定律的发展与扩充的新现象[J].生物学通报,2008,38(12)：3-7.
9. Wu C T, Morris J R. Genes, genetics and epigenetics:a correspondence [J]. Science,2001,293:1103-1105.
10. Ehrlinch M, Gama-Sosa MA, Huang LH, et al. Amount and distribution of 5-methylcytosine in human DNA from different types of tissues or cells[J]. Nucleic Acids Res,1982,10(8):2709-2721.
11.刘鸿禧,詹海勇,陈孔屏.表遗传学研究若干进展[J].国际遗传学杂志,2006,29(1)：35-39.
12. Robertson KD. DNA methylation, methyltransferase and cancer[J].Oncogene, 2001,20(24):3139-3155.
13. Zhang Y, Ng HH, Erdjument-Bromage H, et al. Analysis of the NuRD Subunits reveals a histone deacetylase core complex and a connection with DNA methylation[J]. Genes Dev,1999,13(15):1924-1935.
14. Weiss A, Keshet I, Razin A, et al. DNA demethylation in vitro:involvement of RNA[J]. Cell,1996,86(5):709-718.
15. Dahl C, Guldberg P. DNA methylation analysis techniques[J]. Biogerontology, 2003,4(4):233-250.
16.董玉玮,侯进慧,朱必才,等.表观遗传学的相关概念和研究进展[J].生命的化学,2005,22(1)：1-3.
17.戴毅敏,胡娅莉.表遗传重建和辅助生育技术的风险[J].生殖与避孕,2006,26(11)：689-93.
18. Feinberg A P, Tycko B. The history of cancer epigenetic[J]. Nat Rev Cancer,2004, 4(2):143-153.
19. Thorburn MJ, Wright ES, Miller CG, et al. Exomphalos macroglossia-gigantism syndrome in Jamaican infants[J]. Am J Dis Child,1970,119(4):316-321.
20. Elliott M, Bayly R, Cole T, et al. Clinical features and natural history of Beckwith-Wiedemann syndrome:presentation of 74 new cases[J]. Clin Genet, 1994,46(2):168-174.
21. Junien C. Beckwith-Wiedemann syndrome, tumourignesis and imprinting[J].Curr Opin Genet Dev,1992,2(3):431-438.
22. Weksberg R, Smith AC, Squire J, et al. Beckwith-Wiedemann syndrome demonstrates a role for epigenetic control of normal development[J]. Hum Mol Genet,2003,12(Spec 1):R61-R68.
23. Weksberg R, Shuman C, Smith AC. Beckwith-Wiedemann Syndrome [J]. Am J Med Genet C Semin Med Genet,2005,137C(1):12-23.
24. DeBaun MR, Niemitz EL, Feinberg AP. Association of in vitro fertilization with Beckwith-Wiedemann syndrome and epigenetic alterations of LIT1 and H19[J]. Am J Hum Genet,2003,72(1):156-160.
25. Maher ER, Brueton LA, Bowdin SC, et al. Beckwith-Wiedemann syndrome and assisted reproductive technology (ART) [J]. J Med Genet,2003,40(1):62-64.
26.Gicquel C, Gaston V, Mandelbaum J, et al. In vitro fertilization may increase the risk of Beckwith-Wiedemann syndrome related to the abnormal imprinting of the KCN1OT gene[J]. Am J Hum Genet,2003,72(5):1338-1341.
27. Halliday J, Oke K, Breheny S, et al. Beckwith-Weidemann syndrome and IVF:a case-control study[J]. Am J Hum Genet,2004,75(3):526-528.
28.Chang AS, Moley KH, Wangler M, et al. Association between Beckwith-Wiedemann syndrome and assisted reproductive technology:a case series of 19 patients[J]. Fertil Steril,2005,83(2):349-354.
29. Sutcliffe AG, Peters CJ, Bowdin S, et al. Assisted reproductive therapies and imprinting disorders-a preliminary British survey [J]. Hum Reprod,2006,21(4): 1009-1011.
30. Bowdin S, Allen C, Kirby G, et al. A survey of assisted reproductive technology births and imprinting disorders[J].Hum Reprod,2007,22(12):3237-3240.
31. Doornbos ME, Maas SM, McDonnell J, et al. Infertility, assisted reproduction technologies and imprinting disturbances:a Dutch study[J]. Hum Reprod,2007, 22(9):2476-80.
32. Lidegaard 0, Pinborg A, Andersen AN. Imprinting diseases and IVF:Danish National IVF cohort study[J]. Hum Reprod,2005,20(4):950-954.
33. Kallen B, Finnstrom 0, Nygren KG, et al. In vitro fertilization (IVF) in Sweden: risk for congenital malformations after different IVF methods[J]. Birth Defects Res A Clin Mol Teratol,2005,73(3):162-169.
34. Owen CM, Segars JH Jr. Imprinting disorders and assisted reproductive[J]. Semin Reprod Med,2009,27(5):417-428.
35. Gicquel C, Rossignol S, Cabrol S, et al. Epimutation of the telomeric imprinting center region on chromosome 11p15 in Silver-Russell syndrome[J]. Nat Genet, 2005,37(9):1003-1007.
36. Bliek J, Terhal P, van den Bogaard MJ, et al. Hypomethylation of the H19 Gene Causes Not Only Silver-Russell Syndrome (SRS) but Also Isolated Asymmetry or an SRS-Like Phenotype[J].Am J Hum Genet,2006,78(4):604-614.
37. Bartholdi D, Krajewska-Walasek M, Ounap K, et al. Epigenetic mutations of the imprinted IGF2-H19 domain in Silver-Russell syndrome (SRS):results from a large cohort of patients with SRS and SRS-like phenotypes[J]. J Med Genet, 2009,46(3):192-197.
38. Penaherrera MS, Weindler S, Van Allen MI, et al. Methylation profiling in individuals with Russell-Silver syndrome[J]. Am J Med Genet A,2010,152A (2):347-355.
39. Svensson J, Bjornstahl A, Ivarsson SA. Increases risk of Silver-Russell syndrome after in vitro fertilization? [J] Acta Paediatr,2005,94(8):1163-1165.
40. Kishino T, Lalande M, Wagstaff J. UBE3A/E6-AP mutations cause Angelman syndrome[J]. Nat Genet,1997,15(1):70-73.
41. Mann MR, Bartolomei MS. Towards a molecular understanding of Prader-Willi and Angelman syndromes[J]. Hum Mol Genet,1999,8(10):1867-1873.
42. Steffenburg S, Gillberg CL, Steffenburg U, et al. Autism in Angelman syndrome: a population-based study [J]. Pediatr Neurol,1996,14(2):131-136.
43. Cox GF, Burger J, Lip V, et al. Intracytoplasmic sperm injection may increase the risk of imprinting defects[J]. Am J Hum Genet,2002,71(1):162-164.
44. Orstavik KH, Eiklid K, Van Der Hagen CB, et al. Another case of imprinting defect in a girl with Angelman syndrome who was conceived by intracytoplasmic sperm injection[J].Am J Hum Genet,2003,72(1):218-219.
45. Ludwig M, Katalinic A, Gross S,et al. Increased prevalence of imprinting defects in patients with Angelman syndrome born to subfertile couples[J]. J Med Genet, 2005,42(4):289-291.
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2. Centers for Disease Control and Prevention, American Society for Reproductive Medicine and Society for Assisted Reproductive Technology.2005 Assisted Reproductive Technology Success Rates:National Summary and Fertility Clinic Reports. Atlanta:Centers for Disease Control and Prevention,2007.
3. Andersen AN, Goossens V, Ferraretti AP, et al. Assisted reproductive technology in Europe,2004:results generated from European registers by ESHRE [J]. Hum Reprod,2008,23(4):756-771.
4. Helmerhorst FM, PerquinDA, DonkerD, etal. Perinatal outeome of singletons and twins after assisted conception:a systematic review of controlled studies[J]. BMJ,2004,328 (7434):261.
5. Stromberg B, Dahlquist G, Ericson A, et al. Neurological sequelae in children born after in-vitro fertilization:a population-based study[J]. Lancet,2002,359 (9305):461-465.
6. Hansen M, Colvin L, Petterson B, et al. Twins born following assisted reproductive technology:perinatal outcome and admission to hospital[J]. Hum Reprod,2009,24 (9):2321-2331.
7. Hansen M, Bower C, Milne E, et al. Assisted reproductive technologies and the risk of birth defects-a systematic review[J]. Hum Reprod,2005,20(2):328-338.
8.黎真,傅衍,牛冬,等.遗传印记--一种对孟德尔定律的发展与扩充的新现象[J].生物学通报,2008,38(12)：3-7.
9. Wu C T, Morris J R. Genes, genetics and epigenetics:a correspondence [J]. Science,2001,293:1103-1105.
10. Ehrlinch M, Gama-Sosa MA, Huang LH, et al. Amount and distribution of 5-methylcytosine in human DNA from different types of tissues or cells[J]. Nucleic Acids Res,1982,10(8):2709-2721.
11.刘鸿禧,詹海勇,陈孔屏.表遗传学研究若干进展[J].国际遗传学杂志,2006,29(1)：35-39.
12. Robertson KD. DNA methylation, methyltransferase and cancer[J].Oncogene, 2001,20(24):3139-3155.
13. Zhang Y, Ng HH, Erdjument-Bromage H, et al. Analysis of the NuRD Subunits reveals a histone deacetylase core complex and a connection with DNA methylation[J]. Genes Dev,1999,13(15):1924-1935.
14. Weiss A, Keshet I, Razin A, et al. DNA demethylation in vitro:involvement of RNA[J]. Cell,1996,86(5):709-718.
15. Dahl C, Guldberg P. DNA methylation analysis techniques[J]. Biogerontology, 2003,4(4):233-250.
16.董玉玮,侯进慧,朱必才,等.表观遗传学的相关概念和研究进展[J].生命的化学,2005,22(1)：1-3.
17.戴毅敏,胡娅莉.表遗传重建和辅助生育技术的风险[J].生殖与避孕,2006,26(11)：689-93.
18. Feinberg A P, Tycko B. The history of cancer epigenetic[J]. Nat Rev Cancer,2004, 4(2):143-153.
19. Thorburn MJ, Wright ES, Miller CG, et al. Exomphalos macroglossia-gigantism syndrome in Jamaican infants[J]. Am J Dis Child,1970,119(4):316-321.
20. Elliott M, Bayly R, Cole T, et al. Clinical features and natural history of Beckwith-Wiedemann syndrome:presentation of 74 new cases[J]. Clin Genet, 1994,46(2):168-174.
21. Junien C. Beckwith-Wiedemann syndrome, tumourignesis and imprinting[J].Curr Opin Genet Dev,1992,2(3):431-438.
22. Weksberg R, Smith AC, Squire J, et al. Beckwith-Wiedemann syndrome demonstrates a role for epigenetic control of normal development[J]. Hum Mol Genet,2003,12(Spec 1):R61-R68.
23. Weksberg R, Shuman C, Smith AC. Beckwith-Wiedemann Syndrome [J]. Am J Med Genet C Semin Med Genet,2005,137C(1):12-23.
24. DeBaun MR, Niemitz EL, Feinberg AP. Association of in vitro fertilization with Beckwith-Wiedemann syndrome and epigenetic alterations of LIT1 and H19[J]. Am J Hum Genet,2003,72(1):156-160.
25. Maher ER, Brueton LA, Bowdin SC, et al. Beckwith-Wiedemann syndrome and assisted reproductive technology (ART) [J]. J Med Genet,2003,40(1):62-64.
26.Gicquel C, Gaston V, Mandelbaum J, et al. In vitro fertilization may increase the risk of Beckwith-Wiedemann syndrome related to the abnormal imprinting of the KCN1OT gene[J]. Am J Hum Genet,2003,72(5):1338-1341.
27. Halliday J, Oke K, Breheny S, et al. Beckwith-Weidemann syndrome and IVF:a case-control study[J]. Am J Hum Genet,2004,75(3):526-528.
28.Chang AS, Moley KH, Wangler M, et al. Association between Beckwith-Wiedemann syndrome and assisted reproductive technology:a case series of 19 patients[J]. Fertil Steril,2005,83(2):349-354.
29. Sutcliffe AG, Peters CJ, Bowdin S, et al. Assisted reproductive therapies and imprinting disorders-a preliminary British survey [J]. Hum Reprod,2006,21(4): 1009-1011.
30. Bowdin S, Allen C, Kirby G, et al. A survey of assisted reproductive technology births and imprinting disorders[J].Hum Reprod,2007,22(12):3237-3240.
31. Doornbos ME, Maas SM, McDonnell J, et al. Infertility, assisted reproduction technologies and imprinting disturbances:a Dutch study[J]. Hum Reprod,2007, 22(9):2476-80.
32. Lidegaard 0, Pinborg A, Andersen AN. Imprinting diseases and IVF:Danish National IVF cohort study[J]. Hum Reprod,2005,20(4):950-954.
33. Kallen B, Finnstrom 0, Nygren KG, et al. In vitro fertilization (IVF) in Sweden: risk for congenital malformations after different IVF methods[J]. Birth Defects Res A Clin Mol Teratol,2005,73(3):162-169.
34. Owen CM, Segars JH Jr. Imprinting disorders and assisted reproductive[J]. Semin Reprod Med,2009,27(5):417-428.
35. Gicquel C, Rossignol S, Cabrol S, et al. Epimutation of the telomeric imprinting center region on chromosome 11p15 in Silver-Russell syndrome[J]. Nat Genet, 2005,37(9):1003-1007.
36. Bliek J, Terhal P, van den Bogaard MJ, et al. Hypomethylation of the H19 Gene Causes Not Only Silver-Russell Syndrome (SRS) but Also Isolated Asymmetry or an SRS-Like Phenotype[J].Am J Hum Genet,2006,78(4):604-614.
37. Bartholdi D, Krajewska-Walasek M, Ounap K, et al. Epigenetic mutations of the imprinted IGF2-H19 domain in Silver-Russell syndrome (SRS):results from a large cohort of patients with SRS and SRS-like phenotypes[J]. J Med Genet, 2009,46(3):192-197.
38. Penaherrera MS, Weindler S, Van Allen MI, et al. Methylation profiling in individuals with Russell-Silver syndrome[J]. Am J Med Genet A,2010,152A (2):347-355.
39. Svensson J, Bjornstahl A, Ivarsson SA. Increases risk of Silver-Russell syndrome after in vitro fertilization? [J] Acta Paediatr,2005,94(8):1163-1165.
40. Kishino T, Lalande M, Wagstaff J. UBE3A/E6-AP mutations cause Angelman syndrome[J]. Nat Genet,1997,15(1):70-73.
41. Mann MR, Bartolomei MS. Towards a molecular understanding of Prader-Willi and Angelman syndromes[J]. Hum Mol Genet,1999,8(10):1867-1873.
42. Steffenburg S, Gillberg CL, Steffenburg U, et al. Autism in Angelman syndrome: a population-based study [J]. Pediatr Neurol,1996,14(2):131-136.
43. Cox GF, Burger J, Lip V, et al. Intracytoplasmic sperm injection may increase the risk of imprinting defects[J]. Am J Hum Genet,2002,71(1):162-164.
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