辅助生殖技术中早/中期自然流产与生长发育相关印记基因甲基化状态的相关性研究
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摘要
人类辅助生殖技术(assisted reproductive technology, ART)包括常规体外受精(in vitro fertilization, IVF)、卵细胞浆内单精子显微注射技术(intracytoplasmic sperm injection, ICSI)、胚胎植入前遗传学诊断(preimplantation genetics diagnosis,PGD)等。通过辅助生殖技术出生的后代正逐渐成为人口的一个重要组成部分,因此ART技术的安全性成为人们关注的焦点。近年来较多研究认为IVF和ICSI技术可能会增加Angelman综合征(AS)、Beckwith-Wiedemann综合征(BWS)等疾病的发病风险,而基因印记缺陷是其发病的重要病因之一。据文献统计,经ART技术出生的BWS、AS患儿出现基因印记缺陷的比例显著高于自然妊娠。因此ART技术与基因印记缺陷的关系成为目前研究的热点。部分研究认为,ART可能影响表遗传的调控,特别是印记基因的表达,从而影响胚胎植入、胎盘形成、器官形成和胎儿生长等方面,基因印记缺陷则有可能影响正常妊娠的维持。无论是经ART技术受孕抑或是自然受孕,总体自然流产发生率均可占全部妊娠的约15-20%,其发生体现了人类优胜劣汰的一种自我选择方式,减少了畸形儿的出生。本研究收集了经ART受孕及自然受孕的孕早期绒毛组织和中期胎儿组织,通过分析H19、KvDMR1、PEG1及SNRPN四个与BWS发病相关的印记基因的甲基化状态、评估DNA甲基转移酶(DNMT)-3A和3B基因启动子区单核苷酸多态性(SNP)与自然流产发生风险的关系,初步探索印记基因在胚胎停育发生中的作用及ART技术相关的表观遗传学风险,为评估ART技术的安全性提供依据。
第一部分辅助生殖技术及自然妊娠早孕期绒毛中生长发育相关印记基因甲基化状态的研究
[研究目的]
大量动物实验研究表明ART可能影响表遗传的调控,特别是印记基因的表达,从而影响胚胎植入、胎盘形成、器官形成和胎儿生长等方面,而基因印记缺陷则可能会影响早期妊娠的维持。本研究采用焦磷酸测序和重亚硫酸盐测序-PCR技术,分析ART妊娠或自然妊娠来源的早期绒毛组织中印记基因H19、KvDMR1(L1T1), PEG1、SNRPN的DMR区甲基化状态,初步评估ART妊娠早孕期胚胎停止发育中印记异常的发生风险,为ART技术的表观遗传学安全性提供初步依据。
[研究方法]
(1)本研究经医院伦理委员会批准,标本收集经患者知情同意。2008年5月至2011年7月于南方医院妇产科门诊收集：①ART胚胎停育绒毛62例；②自然受孕胚胎停育绒毛73例；③自然受孕早孕绒毛69例；④同期于南方医院生殖医学中心、江西省妇幼保健院、广东省妇幼保健院收集ART早孕减胎绒毛73例。所有绒毛标本均为孕6-11周,患者年龄在18--45岁,无解剖学方面因素,无家族遗传病史。自然流产(胚胎停育)组患者B超检查提示：胚胎停止发育或停经6周后多次超声提示未见胚芽。
(2)将绒毛标本清洗处理、DNA提取、基因组DNA重亚硫酸修饰,后行H19KvDMR1(LIT1), PEG1、SNRPN基因甲基化特异性-PCR(MSP)扩增、琼脂糖凝胶电泳检测甲基化条带(M带)及非甲基化条带(U带),进行甲基化状态的定性分析；
(3)绒毛基因组DNA重亚硫酸修饰后行1119, KvDMR1(LIT1)、PEG1、 SNRPN基因PCR扩增,扩增产物行焦磷酸测序及克隆测序,并得出每份标本的甲基化值。采用SPSS16.0统计软件(Statistical Package for the Social Sciences),数据用均数±标准差(x±S)表示。组间数量资料比较采用t3-test或One-wayANOVA,方差不齐时采用Welch近似方差检验,多重比较方差不齐时采用Dunnett's T3法；组间样本率的比较采用X2检验；采用Boxplot分析数据分布得出极端值；ROC曲线评价甲基化值对自然流产的预测价值。设双侧检验,P<0.05为差异有统计学意义。
[研究结果]
(1) ART组与自然妊娠组相比、胚胎停育组与正常早孕组相比,母龄、孕周均无统计学差异,P>0.05；提示样本收集的均衡性较好。
(2)每次MSP扩增均设纯水模板空白对照,电泳检测结果显示空白对照均未发现假阳性M带或U带出现；人外周血淋巴细胞中,4个印记基因的MSP扩增均显示为正常甲基化模式,即M带和U带均显带。在所有绒毛组织中,均可扩增出4个印记基因的甲基化条带及非甲基化条带,未发现完全的高甲基化或完全的非甲基化状态。
(3)所有检测绒毛组织样本中,重亚硫酸盐转化效率波动于90.1-100%；取每种基因所检测的所有CpG位点之平均值作为该样本的甲基化值,H19、PEG1、KvDMR1、SNRPN基因的平均甲基化值分别为49.6士9.3%(波动范围30.3-91.7%)、55.5士9.7%(波动范围38.5-89.4%)、49.0士9.4%(波动范围33.9-86.4%)、41.3士4.3%(波动范围32.6-61.2%),均未出现完全甲基化(100%)或完全非甲基化(0%),结果与MSP结果相符。四组间的H19、KvDMR1、PEG1、 SNRPN基因甲基化程度均存在统计学差异(采用多样本间单因素Welch近似方差分析,P<0.05), ART妊娠胚胎停育及自然妊娠胚胎停育组绒毛印记基因的甲基化值分别高于ART减胎组和自然妊娠早孕组；Boxplot分析显示,所有的异常甲基化值均发生于胚胎停育(自然流产)样本中,即自然流产样本中印记基因甲基化异常的发生率为6.9%(37/540)；且所有的异常甲基化值均为高甲基化改变。每种基因的异常甲基化值发生率呈现KvDMRl> SNRPN> H19>PEG1,分别为7.2%(20/277)、3.2%(9/277)、2.5%(7/277)、0.4%(1/277),其中6例胚胎停育样本(4.4%,6/135)同时存在两个或以上多个基因的甲基化异常：ART妊娠胚胎停育和自然妊娠胚胎停育各3例。将所有样本分为胚胎停育与正常早孕两组,采用ROC曲线分析4种印记基因甲基化程度与早期自然流产之间的关系,结果提示,绒毛组织中4种印记基因的甲基化程度对自然流产发生的风险预测价值欠佳。而ART组与自然妊娠组相比、IVF组与ICSI组相比,4种印记基因的平均甲基化值、异常甲基化值发生率均无统计学差异(P>0.05)。
[研究结论]
(1)印记基因H19、KvDMR1、PEG1、SNRPN的甲基化状态异常可能与早期妊娠胚胎停止发育育天.且甲基化程度越高发生自然流产的几率可能越大；提示表观遗传学的异常也可能是导致胚胎停育的重要因素之一；
(2)辅助生殖技术可能并不增加印记基因KvDMR1、 PEG1、 SNRPN异常甲基化状态的发生；且不同受精方式之间(IVF与ICSI相比),印记基因的甲基化状态不存在显著性差异；
(3)本研究仅限于对少数印记基因的研究,需要进一步扩大样本量、扩展研究的基因种类,以证实上述的初步研究结论。
第二部分辅助生殖技术及自然妊娠中孕期胎儿组织中生长发育相关印记基因甲基化状态的研究[研究目的]人类胎儿的发育在每个阶段都可能因出现异常而导致妊娠终止,尽管遗传
学诊断手段越来越完善,但是仍有一部分中期妊娠丢失原因不明。随着近年关于胚胎发育与印记基因DNA甲基化相关研究的增加,越来越多的学者认为,表观遗传学也参与了胚胎／胎儿发育及停止发育的过程,尤其对于不明原因的中期妊娠丢失。本研究采用焦磷酸测序和重亚硫酸盐测序PCR技术,分析ART妊娠或自然妊娠来源的中期胎儿组织中印记基因1的DMR区甲基化状态,初步评估ART妊娠中孕期胎儿停止发育中印记异常的发H19、KvDmR1(LIT1)、PEG1、SNRPN生风险,为印记基因DNA甲基化改变与胎儿停止发育的关系及ART技术的表观遗传学安全性提供初步依据。
[研究方法]
(1)本研究经医院伦理委员会批准,标本收集经患者知情同意。2010年8月至2011年7月于南方医院产科病房收集：①ART妊娠中孕期死胎胎儿肌肉组织13例；②自然妊娠中孕期死胎胎儿肌肉组织17例；③同期以自然妊娠正常早孕胎儿肌肉组织13例为对照。胎儿组织来源于孕18-26周,母龄在18-38岁,无解剖学方面因素,无家族遗传病史,排除宫颈因素、感染等导致的流产。于死胎娩出后2小时内,使用手术刀片及剪刀剪取胎儿大腿前方肌肉组织；
(2)将胎儿肌肉组织标本清洗处理、DNA提取、基因组DNA重亚硫酸修饰,后行H19、KvDMR1(LIT1)、PEG1、SNRPN基因甲基化特异性-PCR(MSP)扩增、琼脂糖凝胶电泳检测甲基化条带(M带)及非甲基化条带(U带),进行甲基化状态的定性分析；
(3)胎儿组织基因组DNA重亚硫酸修饰后行1H19、KvDMR1(LIT1)、 PEG1、SNRPN基因PCR扩增,扩增产物行焦磷酸测序及克隆测序,并得出每份标本的甲基化值。采用SPSS16.0统计软件(Statistical Package for the Social Sciences)、数据用均数±标准差(X士S)表示。组间数量资料比较采用t-test或One-way ANOVA,方差齐时多重比较采用Bonnferroni法；组间样本率的比较采用x2检验；采用Boxplot分析数据分布得出极端值；ROC曲线评价甲基化值对自然流产的预测价值。设双侧检验,P<0.05为差异有统计学意义
[研究结果]
(1) ART组与自然妊娠组相比,母龄、孕周均无统计学差异,P>0.05；此外,自然流产组与正常中孕组相比,母龄、孕周亦无显著性差异,P>0.05；提示胎儿组织样本的收集具有较好的均衡性。
(2)每次MSP扩增均设纯水模板空白对照,电泳检测结果显示空白对照均未发现假阳性M带或U带出现；人外周血淋巴细胞中,4个印记基因的MSP扩增均显示为正常甲基化模式,即M带和U带均显带。在所有胎儿组织中,均可扩增出4个印记基因的甲基化条带及非甲基化条带,未发现完全的高甲基化或完全的非甲基化状态。
(3)所有检测胎儿组织样本中,重亚硫酸盐转化效率波动于92.6-100%；取每种基因所检测的所有CpG位点之平均值作为该样本的甲基化值,H19、PEG1、KvDMR1、SNRPN基因的平均甲基化值分别为53.1士3.8%(波动范围46.2-60.9%)、52.7士7.9%(波动范围44.6-85.6%)、48.9士8.7%(波动范围39.3-83.4%)、42.0士3.9%(波动范围36.4-54.8%),均未出现完全甲基化(100%)或完全非甲基化(0%),结果与MSP结果相符。三组间的H19、KvDMR1、PEG1、 SNRPN基因甲基化程度经比较发现(采用多样本间单因素方差分析),仅KvDMR1基因的甲基化程度存在统计学差异(P=0.015);Bonnferroni多重比较后,自然妊娠自然流产显著性高于ART妊娠自然流产及自然妊娠正常中孕组；而其他3种印记基因甲基化程度在三组间不存在统计学差异(P>0.05)。以上结果提示,KvDMR1基因可能较其他基因更容易出现样本间甲基化值的差异,而其他3种基因未发现样本间存在统计学差异则可能是由于样本量较小造成的。Boxplot分析显示,每种基因的异常甲基化值发生率呈现KvDMR1=PEG1> SNRPN> H19,分别为4.7%(2/43)、4.7%(2/43)、2.3%(1/43)、0(0/43)；所有的异常甲基化值均发生于自然流产(死胎)样本中,即自然流产样本中印记基因甲基化异常的发生率为4.2%(5/120),且异常甲基化值亦均为高甲基化改变,1例女性死胎呈现2个基因甲基化异常2.3%(1/43)。将所有样本分为自然流产与正常中孕两组,采用ROC曲线分析4种印记基因甲基化程度与中期自然流产之间的关系,结果提示,KvDMR1、PEG1基因的甲基化程度对自然流产发生的风险有较好的预测价值(P<0.05),而H19、SNRPN基因对自然流产的发生无预测价值。此外,ART组与自然妊娠组相比、IVF组与ICSI组相比,4种印记基因的平均甲基化值、异常甲基化值发生率均无统计学差异(P>0.05)。
[研究结论]
(1)印记基因H19、KvDMR1、PEG1、SNRPN的甲基化状态异常可能与中期妊娠自然流产有关,其中KvDMR1基因甲基化状态的改变可能与中期妊娠自然流产关系最密切,且甲基化程度越高发生中期自然流产的几率可能越大；提示表观遗传学的异常可能是导致中期妊娠丢失的重要因素之一；
(2)辅助生殖技术可能并不增加印记基因KvDMR1、PEG1、SNRPN异常甲基化状态的发生；且不同受精方式之间(IVF与ICSI相比),印记基因的甲基化状态不存在显著性差异；
(3)本研究仅限于对少数印记基因的研究,且样本量较小,进一步的工作需要扩大样本量、扩展研究的基因种类,以证实上述的初步研究结论。
第三部分辅助生殖技术及自然妊娠早孕期绒毛、中孕期胎儿组织DNMT-3基因启动子SNP的研究
[研究目的]
DNA甲基转移酶(DNMT)-3A与DNMT-3B是催化重新甲基化的DNA甲基转移酶,对基因组甲基化模式的形成、胚胎形成及许多病理发生中的甲基化进程起着关键作用。DNMT-3A、3B启动子区一些单核苷酸多态性(single nucleotide polymorphisms,SNPs)可改变启动子的转录活性,使DNMTs表达增加、基因组DNA甲基化模式改变,从而导致各种疾病的发生。本研究以人类ART来源和自然妊娠来源的早孕期绒毛组织、中孕期胎儿肌肉组织为研究对象,采用PCR结合限制性片段长度多态性分析(PCR-RFLP)方法检测这些样本中DNMT-3A和3B基因启动子-448A>G位点和-149C>T位点的单核苷酸多态性,以探讨此两个位点与人类早中期妊娠丢失的相关性,及ART技术对DNMT-3A和3B多态性的影响,为进一步了解早中期妊娠丢失的发生机制提供依据。
[研究方法]
本研究经医院伦理委员会批准,标本收集经患者知情同意。研究对象样本收集同于第一、第二部分,将所有样本分为以下两组：①研究组：ART妊娠及自然妊娠自然流产早孕期绒毛、中孕期死胎胎儿肌肉组织,共计152例；②对照组：ART妊娠及自然妊娠正常早孕期绒毛、中孕期胎儿肌肉组织,共计155例。组织的收集、基因组DNA提取同第一、二部分。采用PCR-RFLP分析DNMT-3A(GenBank accession No. NT_022184.14:g.4381840A/G)、 DNMT-3B(GenBank accession No. AL035071,46359C/T)基因启动子区SNP位点,并选取部分样本采用DNA测序加以验证。采用SPSS16.0统计软件(Statistical Package for the Social Sciences),数据用均数±标准差(x士S)表示。两组间数量资料比较采用t-test;组间样本率的比较采用X2检验,设双侧检验,P<0.05为差异有统计学意义。
[研究结果]
(1) DNMT-3A PCR产物经Taal限制性酶切后的产物片段显示,酶切之后在研究组自然流产者和对照组正常早／中孕组织中均检测到AA变异纯合型、AG变异杂合型和GG野生型3种基因型。研究组中,GG、AG、AA基因型的分布频率分别为45.4%、40.8%、13.8%, A等位基因的频率为34.2%。对照组中,GG、AG、AA基因型的分布频率分别为68.4%、30.3%、1.3%, A等位基因的频率为16.5%。研究组与对照组样本相比,与-448A>G变异相关的自然流产的发生风险增高,AG杂合型较GG野生型发生自然流产的风险高2倍(OR值2.027,95%可信区间1.247-3.293, P=0.004)；而AA变异纯合型较GG野生型发生自然流产的风险高达16倍(OR值16.130,95%可信区间3.665-70.984,P=0.000)。
(2)当进行年龄、孕周分组时发现,在孕周<12w的样本中,与GG野生型相比AA变异纯合型和AG变异杂合型均与自然流产发生风险增高相关,OR值分别为16.897(95%可信区间3.810-74.925)、2.293(95%可信区间1.371-3.834)；在母龄<35岁组中,AA变异纯合型和AG变异杂合型亦与自然流产发生风险增高相关,OR值分别为28.868(95%可信区间3.734-223.156)、1.900(95%可信区间1.111-3.251)。而在孕周>12w和母龄>35岁组的样本中,未发现AA纯合型或AG杂合型与自然流产发生风险增高相关(P>0.05),可能与此组中样本量较小有关。此外,ART来源的样本与自然妊娠来源样本中GG、AG、AA基因型的分布频率亦无统计学差异(P=0.661)。
(3)分析277例人早孕期ART受孕和自然受孕来源的胚胎停育绒毛、正常早孕绒毛(同本文第一部分),比较发生异常甲基化值的样本与未发生异常甲基化值的样本其DNMT-3A的基因型分布：37个异常甲基化值的29例样本中,DNMT-3A启动子区GG野生型仅占13.8%(4/29),而AG杂合变异(44.8%,13/29)和AA纯合子变异(41.4%,12/29)共达86.2%,远高于未发生甲基化异常的样本中AG、AA基因型的分布频率(34.7%、4.0%,P=0.000)
(4) DNMT-3B PCR产物经AvrⅡ限制性酶切后的产物片段显示,酶切之后在研究组自然流产者和对照组正常早／中孕组织的所有样本中均仅检测到TT变异纯合型,未见到CT变异杂合型和CC野生型。
[研究结论]
(1) DNMT-3A基因启动子区-448A>G位点SNP在早／中期妊娠丢失中可能发挥了一定作用,其途径即可能通过改变印记基因的甲基化状态,从而影响印记基因的表达；ART技术并未影响胚胎停育及正常妊娠中该位点基因型的分布；
(2)目前尚无研究报道DNMT-3B基因启动子区-149C/T位点SNP在人早孕期绒毛组织及中孕期胎儿组织中的基因型,通过本研究结果推测,不排除DNMT-3B启动子区该位点突变型可能代表了早中期胎儿生长发育中的正常变异。
(3)本研究仅限于对DNMT-3A、3B基因启动子区DNA水平的研究,进一步的工作需要对DNMT-3A、3B基因表达及酶活力检测,以证实上述的初步研究结论。
Assisted reproductive technologies including in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), in vitro maturation (IVM), embryos freezing, and preimplantation genetics diagnosis (PGD) et al.. The offspring born after ART is becoming an important part of population, thus concerns about the safety of ART have been increasing. Recent studies suggest that in vitro fertilization (IVF)/intracytoplasmic sperm injection may be associated with increased incidence of Angelman syndrome (AS) and Beckwith-Wiedemann syndrome (BWS), and Imprinting defect is one of the most important factors. The majority of ART-conceived children with BWS and AS demonstrated imprinting defects, so Concerns about imprinting disorders linked to ART are therefore increasing. ART may affect epigenetic regulation, particularly the expression of imprinted genes, thus affecting the embryo implantation, placentation, organ formation and fetal growth. The prevalence of miscarriage has been estimated as15-20%of all clinically recognized pregnancies. To some extent, the occurrence of early miscarriage reduced the birth effects. Some studies suggests that genomic imprinting defects affecting the maintenance of early pregnancy. In the present study, we analyzed the DNA methylation patterns of four imprinted genes including H19, KvDMRl, PEG1, and SNRPN in human abortions and stillbirths, comparing ART with spontaneous conception, In addition, the association between DNA-methyltransferase (DNMT)-3A and DNMT-3B promoter genetic variant and the susceptibility to pregnancyloss was evaluated.
Part I Quantitative methylation analysis of developmentally important imprinted genes in human early pregnancy loss after ART and spontaneous conception
[Objective]
Animal studies have demonstrated that genomic imprinting defects may affect the maintenance of early pregnancy. ART procedures may affect epigenetic regulation, particularly the expression of imprinted genes, thus affecting the embryo implantation, placentation, organ formation and fetal growth. In the present study, we analyzed the DNA methylation patterns of four imprinted genes including H19, KvDMR1, PEG1, and SNRPN in human chrionic villus from early pregnancy loss, comparing ART with spontaneous conception, primarily evaluating epigenetic risks linked to pregnancy loss.
[Materials&Methods]
(1) Chorionic villus samples (CVS) were collected from women who underwent abortion procedures in Department of Gynecology and Obstetrics in Nanfang hospital from May2008to July2011. The duration of pregnancy ranged from6to11weeks, and the maternal age ranged from18to45years old. These patients had no known anatomic or genetic abnormalities.
(2) Four groups were tested:spontaneous abortion after ART (SN, n=62), multi-fetal reduction after ART (FA, n=73), spontaneous abortion of natural pregnancies (SN, n=73), and induced abortion of natural pregnancies (IN, n=69).
(3) Genomic DNA was extracted after sample collection by proteinase K digestion. Bisulfite treatment of genomic DNA was performed with the EpiTect Bisulfite Kit (Qiagen). The DNA methylation patterns of H19, LIT1, and SNRPN were analyzed using methylation speeific PCR (MSP) and pyrosequencing, which was performed using PyroMark Q96ID System (Qiagen) and pyrosequencing reactions were performed according to the manufacturer's instructions. The degree of methylation at each CpG site was determined by Allele Quantification (AQ) softwase-All samples were analyzed in triplicate. Bisulfite sequencing polymerase chain reaction (BSP) was then perform to confirm the pyrosequencing results.
(4) SPSS16.0statistical analysis program was used to analyze the percentage of methylation in chorionic villus from the four groups. Box plots were generated using the program's default parameters. The receiver operating characteristic (ROC) curve method was used to analyze the potential association between imprinted genes' methylation percentage and the incidence of early spontaneous abortion. The study protocol was approved by the Institutional Ethics Committee of Nanfang Hospital, and informed consent was obtained from all patients.
[Results]
(1) None of the277chorionic villus samples showed a complete loss of maternal or paternal alleles of H149, KvDMR1, PEG1, and SNPRN. The PCR products from methylated maternal chromosome and those from unmethylated paternal chromosome have been detected in all samples.
(2) The gestation and maternal age between samples from ART and spontaneous conceptions were not significantly different, neither were the samples from spontaneous abortions and normal early pregnancies.
(3) Bisulfite conversion efficiency values for all samples were90.1%-100%. The mean methylation values for H19, KvDMR1, PEG1, and SNPRN were49.6±9.3%(range,30.3-91.7%),49.0±9.4%(range,33.9-86.4%),55.5±9.7%(range,38.5-89.4%),41.3±4.3%(range,32.6-61.2%), respectively. Clear hypomethylation (0%) or hypermethylation (100%) was not detected. The percentage methylation of all the studied genes for the four groups showed significant differences (P<0.05). The percentage methylation in spontaneous abortion groups (SA and SN groups) was significantly higher than in the non-spontaneous abortion (normal early pregnancy) groups (FA and IN groups)(P<0.05).
(4) Box plot analysis showed that in the135spontaneous abortions,37of540(6.9%) analyzed DMR methylation values (for the four studied genes) represented outliers. In contrast, the142normal early pregnancies exhibited none outliers. Furthermore, the imprinted KvDMR1gene (20outliers,14.8%) exhibited significantly more potentially abnormal methylation values in spontaneous abortions than the other studied genes. Six of135(4.4%) spontaneous abortions displayed hypermethylated DMRs in two to three genes. It is interesting to note that all the outliers represented hypermethylated and no hypomethylated DMRs.
(5) However, ROC curve analysis could not predict an increased risk of early spontaneous abortion related to the methylation percentage of the four genes in chorionic villus. In addition, The mean percentage methylation did not differ significantly between samples from ART group and from non-ART group (P>0.05), neither were samples from in vitro fertilization and intracytoplasmic sperm injection (P>0.05). There's no evidence for an increased rate of potentially abnormal methylation values in the ART group.
[Conclusions]
(1) As some studies have suggested, imprinting errors of imprinted genes may contribute to early spontaneous abortion. The higher the methylation percentage, the greater the chance of early miscarriage.
(2) ART procedures or different semination methods might not affect the methylation patterns of imprinted genes, which were potentially related to the early pregnancy loss.
(3) The further work is to enlarge the sample size and gene quantity, confirming the hypothesis above.
Part Ⅱ Quantitative methylation analysis of developmentally important imprinted genes in human stillbirths from ART and spontaneous conceptions
[Objective]
The development of fetal may end at any time during the whole pregnancy satge. Although the genetic diagnosis methods were more and more excellent, part of the pregnancy loss in second trimester were still with unknown aetiology. Recent studies showed that among many reasons for such miscarriages, gene imprinting defects are some of the possible causes. Animal and human studies suggested in vitro culture of embryos can cause methylation defects in individual genes, which might affect subsequent embryonic development and contribute to pregnancy loss. To evaluate the correlation between methylation of imprinted genes and pregnancy loss in second trimester, the methylation status of H19, KvDMR1, PEG1, and SNPRN were assessed using pyrosequencing and Bisulfite sequencing polymerase chain reaction (BSP).
[Materials&Methods]
(1) Muscle samples (MS) were obtained from aborted fetuses and stillbirths in the Department of Gynecology and Obstetrics in Nanfang Hospital from August2010 to July2011. Gestational age of each fetus was determined by last menstrual period. The sex was identified by examination of the external genitalia. The duration of pregnancy ranged from18to26weeks, and the maternal age ranged from18to38years old. These patients had no known anatomic or genetic abnormalities.
(2) Three groups were tested:stillbirths from ART pregnancy (n=13), stillbirths from natural pregnancy (n=17), and induced aborted fetuses from natural pregnancy (n=13).
(3) Muscle smples were dissected within2hours after abortion and genomic DNA was extracted after sample collection by proteinase K digestion. Bisulfite treatment of genomic DNA was performed with the EpiTect Bisulfite Kit (Qiagen). The DNA methylation patterns of H19, KvDMR1, PEG1and SNRPN were analyzed using methylation specific PCR (MSP) and pyrosequencing. All samples were analyzed in triplicate. Bisulfite sequencing polymerase chain reaction (BSP) was then perform to confirm the pyrosequencing results.
(4) SPSS16.0statistical analysis program was used to analyze the percentage of methylation in chorionic villus from the four groups. Box plots were generated using the program's default parameters. The receiver operating characteristic (ROC) curve method was used to analyze the potential association between imprinted genes' methylation percentage and the incidence of early spontaneous abortion. The study protocol was approved by the Institutional Ethics Committee of Nanfang Hospital, and informed consent was obtained from all patients.
[Results]
(1) None of the43muscle samples showed a complete loss of maternal or paternal alleles of H19, KvDMR1, PEG1, and SNPRN. The PCR products from methylated maternal chromosome and those from unmethylated paternal chromosome have been detected in all samples.
(2) The gestation and maternal age between samples from ART and spontaneous conceptions were not significantly different, neither were the samples from spontaneous abortions and normal early pregnancies.
(3) Bisulfite conversion efficiency values for all samples were92.6%-100%. The mean methylation values for H19, KvDMRl, PEG1, and SNPRN were53.1±3.8%(range,46.2-60.9%),48.9±8.7%(range,39.3-83.4%),53.1±3.8%(range,46.2-60.9%),42.0±3.9%(range,36.4-54.8%), respectively. Clear hypomethylation (0%) or hypermethylation (100%) was not detected. The percentage methylation of KvDMR1in stillbirths from natural pregnancy was significantly higher than in the stillbirths from ART pregnancy and induced aborted fetuses from natural pregnancy (P<0.05). For the other three genes, no significant differences were found in the methylation percentage among three groups (P>0.05).
(4) Box plot analysis showed that in the30stillbirths,5of120(4.2%) analyzed DMR methylation values (for the four studied genes) represented outliers. In contrast, the13normal pregnancies in second trimester exhibited none outliers. And the outlier rates in H19, KvDMR1, PEG1, and SNPRN were4.7%(2/43),2.3%(1/43),4.7%(2/43), and0(0/43), respectively. All the outliers represented hypermethylated and no hypomethylated DMRs.1of43(2.3%) stillbirths displayed hypermethylated DMRs in two genes.
(5) In addition, ROC curve analysis showed a positive correlation between the methylation percentage of KvDMR1and PEG1in muscle samples and rates of spontaneous abortion in second trimenster:the higher the methylation percentage, the greater the chance of miscarriage (P<0.05). The mean percentage methylation did not differ significantly between samples from ART group and from non-ART group (P>0.05), neither were samples from in vitro fertilization and intracytoplasmic sperm injection (P>0.05). There's no evidence for an increased rate of potentially abnormal methylation values in the ART group.
[Conclusions]
(1) Imprinting errors of imprinted genes may contribute to spontaneous abortion in second trimester. Especially for KvDMR1gene, the higher the methylation percentage, the greater the chance of miscarriage.
(2) ART procedures or different semination methods might not affect the methylation patterns of imprinted genes, which were potentially related to the early pregnancy loss.
(3) To confirm the hypothesis above, further work is also needed, such as enlarging the sample size, gene quantity, and different kind of tissue type.
Part III The DNMT-3A and DNMT-3B promoter polyphisms and risk of human pregnancy loss
[Objective]
Methylation of gene promoters is one of the major regulatory mechanisms of gene expression and abnormal methylation patterns of imprinted genes have been demonstrated to be associated with pregnancy loss. The establishment of DNA methylation patterns requires de novo methylation that occurs predominantly during early development and gametogenesis. DNA-methyltransferase (DNMT)-3A and DNMT-3B are both essential for de novo methylation and play an important role in the development of embryogenesis and the generation of aberrant methylation in carcinogenesis. Here we hypothesized that genetic variants of the genes that are responsible for regulating genomic methylation are associated with increased risk of human pregnancy loss.
[Materials&Methods]
(1) Two types of tissue were collected from women who underwent abortion procedures in Department of Gynecology and Obstetrics in Nanfang hospital from May2008to July2011:(a) chorionic villus samples (CVS) and muscle samples (MS) from spontaneous abortions conceived by assisted reproductive technology (ART) and spontaneous way (study group), n=152;(b) CVS and MS from normal early pregnancy and second trimester (control group), n=155. The duration of pregnancy ranged from6to26weeks, and the maternal age ranged from18-45years old.
(2) We selected one of the single nucleotide polymorphisms (SNPs)-448A>G in the DNMT-3A promoter region and one of the SNP-149C/T in the DNMT-3B promoter region, then genotyped subjects for the DNMT-3A and3B promoter polymorphism to determine the association between this genetic variant and risk of pregnancy loss. The distribution of-448A>G and-149C/T polymorphisms was detected in all samples.
(3) Genomic DNA was extracted after sample collection by proteinase K digestion. The transition of A>G of DNMT-3A SNP creates a TaaⅠ restriction site, and for DNMT-3B the variant T allele had an AvrⅡl restriction site. PCR-restriction fragment length polymorphism (PCR-RFLP) was used to detect this A-G transition in the promoter of DNMT-3A at-448A>G (GenBank accession No. NT_022184.14:g.4381840) and the C-T transition in the promoter of DNMT-3B at-149C/T (GenBank accession No. AL035071,46359C/T). DNA sequencing analysis was performed to confirm the PCR-RFLP analysis. The study protocol was approved by the Institutional Ethics Committee of Nanfang Hospital, and informed consent was obtained from all patients.
[Results]
(1) The DNMT-3A genotypes AA, AG, and GG were detected in the spontaneous abortions and the controls. The genotyping by PCR-RFLP analysis was completely confirmed by DNA sequencing analysis. The allele frequency of-448A among pregnancy loss group and control group was34.2%(52/152) versus16.5%(25.5/155), respectively. Overall, we found that, compared with GG homozygotes, the DNMT-3A-448AA homozygotes had an about16fold increased risk of pregnancy loss [OR=16.130,95%confidence interval (CI),3.665-70.984] and AG homozygotes an OR of2.027(95%CI,1.247-3.293).
(2) When the analyses of DNMT-3A were stratified by the maternal age and gestation, the AA and AG genotypes were associated with an increased risk of pregnancy loss (for AA, OR16.897,95%CI=3.810-74.925; for AG, OR2.293,95%CI=1.371-3.834) at<12weeks, and the AA and AG genotypes were also associated with an increased risk of pregnancy loss (for AA, OR28.868,95%CI=3.734-223.156; for AG, OR1.900,95%CI=1.111-3.251) at<35years. However, the distribution of-448A>G in individuals derived from ART pregnancy was not statistically significantly compared with those derived from spontaneous pregnancy (P=0.661).
(3) The DNMT-3A genotypes AA, AG, and GG were then detected in the135chrionic villus samples from spontaneous abortions and142from controls (study population in Part I). We found that the distribution of-448A>G in samples with outlier (n=29)was much higher than that in samples with non-outlier (25/29,86.2%vs.96/248,38.7%)(p=0.000).
(4) For DNMT-3B, we observed genotype frequencies of100%(TT) in study populations and control groups. The genotyping by PCR-RFLP analysis was also completely confirmed by DNA sequencing analysis.
[Conclusions]
(1) The DNMT-3A-448A>G polymorphism is a novel functional SNP and contributes to its genetic susceptibility to pregnancy loss, especially for early pregnancy loss (<12w) in younger women (<35years). ART did not affect the distribution of of-448A>G in pregnancy loss and normal pregnancy.
(2) At present, we cannot exclude the possibility that the observed TT genotype of DNMT-3B represent normal variation during fetal development.
(3) Our study about the SNP of DNMT-3A and3B is restricted to the DNA level. Additional studies on the underlying molecular mechanism of this polymorphism and the expression of DNMTs are warranted.
第一部分辅助生殖技术及自然妊娠早孕期绒毛中生长发育相关印记基因甲基化状态的研究
[研究目的]
大量动物实验研究表明ART可能影响表遗传的调控,特别是印记基因的表达,从而影响胚胎植入、胎盘形成、器官形成和胎儿生长等方面,而基因印记缺陷则可能会影响早期妊娠的维持。本研究采用焦磷酸测序和重亚硫酸盐测序-PCR技术,分析ART妊娠或自然妊娠来源的早期绒毛组织中印记基因H19、KvDMR1(L1T1), PEG1、SNRPN的DMR区甲基化状态,初步评估ART妊娠早孕期胚胎停止发育中印记异常的发生风险,为ART技术的表观遗传学安全性提供初步依据。
[研究方法]
(1)本研究经医院伦理委员会批准,标本收集经患者知情同意。2008年5月至2011年7月于南方医院妇产科门诊收集：①ART胚胎停育绒毛62例；②自然受孕胚胎停育绒毛73例；③自然受孕早孕绒毛69例；④同期于南方医院生殖医学中心、江西省妇幼保健院、广东省妇幼保健院收集ART早孕减胎绒毛73例。所有绒毛标本均为孕6-11周,患者年龄在18--45岁,无解剖学方面因素,无家族遗传病史。自然流产(胚胎停育)组患者B超检查提示：胚胎停止发育或停经6周后多次超声提示未见胚芽。
(2)将绒毛标本清洗处理、DNA提取、基因组DNA重亚硫酸修饰,后行H19KvDMR1(LIT1), PEG1、SNRPN基因甲基化特异性-PCR(MSP)扩增、琼脂糖凝胶电泳检测甲基化条带(M带)及非甲基化条带(U带),进行甲基化状态的定性分析；
(3)绒毛基因组DNA重亚硫酸修饰后行1119, KvDMR1(LIT1)、PEG1、 SNRPN基因PCR扩增,扩增产物行焦磷酸测序及克隆测序,并得出每份标本的甲基化值。采用SPSS16.0统计软件(Statistical Package for the Social Sciences),数据用均数±标准差(x±S)表示。组间数量资料比较采用t3-test或One-wayANOVA,方差不齐时采用Welch近似方差检验,多重比较方差不齐时采用Dunnett's T3法；组间样本率的比较采用X2检验；采用Boxplot分析数据分布得出极端值；ROC曲线评价甲基化值对自然流产的预测价值。设双侧检验,P<0.05为差异有统计学意义。
[研究结果]
(1) ART组与自然妊娠组相比、胚胎停育组与正常早孕组相比,母龄、孕周均无统计学差异,P>0.05；提示样本收集的均衡性较好。
(2)每次MSP扩增均设纯水模板空白对照,电泳检测结果显示空白对照均未发现假阳性M带或U带出现；人外周血淋巴细胞中,4个印记基因的MSP扩增均显示为正常甲基化模式,即M带和U带均显带。在所有绒毛组织中,均可扩增出4个印记基因的甲基化条带及非甲基化条带,未发现完全的高甲基化或完全的非甲基化状态。
(3)所有检测绒毛组织样本中,重亚硫酸盐转化效率波动于90.1-100%；取每种基因所检测的所有CpG位点之平均值作为该样本的甲基化值,H19、PEG1、KvDMR1、SNRPN基因的平均甲基化值分别为49.6士9.3%(波动范围30.3-91.7%)、55.5士9.7%(波动范围38.5-89.4%)、49.0士9.4%(波动范围33.9-86.4%)、41.3士4.3%(波动范围32.6-61.2%),均未出现完全甲基化(100%)或完全非甲基化(0%),结果与MSP结果相符。四组间的H19、KvDMR1、PEG1、 SNRPN基因甲基化程度均存在统计学差异(采用多样本间单因素Welch近似方差分析,P<0.05), ART妊娠胚胎停育及自然妊娠胚胎停育组绒毛印记基因的甲基化值分别高于ART减胎组和自然妊娠早孕组；Boxplot分析显示,所有的异常甲基化值均发生于胚胎停育(自然流产)样本中,即自然流产样本中印记基因甲基化异常的发生率为6.9%(37/540)；且所有的异常甲基化值均为高甲基化改变。每种基因的异常甲基化值发生率呈现KvDMRl> SNRPN> H19>PEG1,分别为7.2%(20/277)、3.2%(9/277)、2.5%(7/277)、0.4%(1/277),其中6例胚胎停育样本(4.4%,6/135)同时存在两个或以上多个基因的甲基化异常：ART妊娠胚胎停育和自然妊娠胚胎停育各3例。将所有样本分为胚胎停育与正常早孕两组,采用ROC曲线分析4种印记基因甲基化程度与早期自然流产之间的关系,结果提示,绒毛组织中4种印记基因的甲基化程度对自然流产发生的风险预测价值欠佳。而ART组与自然妊娠组相比、IVF组与ICSI组相比,4种印记基因的平均甲基化值、异常甲基化值发生率均无统计学差异(P>0.05)。
[研究结论]
(1)印记基因H19、KvDMR1、PEG1、SNRPN的甲基化状态异常可能与早期妊娠胚胎停止发育育天.且甲基化程度越高发生自然流产的几率可能越大；提示表观遗传学的异常也可能是导致胚胎停育的重要因素之一；
(2)辅助生殖技术可能并不增加印记基因KvDMR1、 PEG1、 SNRPN异常甲基化状态的发生；且不同受精方式之间(IVF与ICSI相比),印记基因的甲基化状态不存在显著性差异；
(3)本研究仅限于对少数印记基因的研究,需要进一步扩大样本量、扩展研究的基因种类,以证实上述的初步研究结论。
第二部分辅助生殖技术及自然妊娠中孕期胎儿组织中生长发育相关印记基因甲基化状态的研究[研究目的]人类胎儿的发育在每个阶段都可能因出现异常而导致妊娠终止,尽管遗传
学诊断手段越来越完善,但是仍有一部分中期妊娠丢失原因不明。随着近年关于胚胎发育与印记基因DNA甲基化相关研究的增加,越来越多的学者认为,表观遗传学也参与了胚胎／胎儿发育及停止发育的过程,尤其对于不明原因的中期妊娠丢失。本研究采用焦磷酸测序和重亚硫酸盐测序PCR技术,分析ART妊娠或自然妊娠来源的中期胎儿组织中印记基因1的DMR区甲基化状态,初步评估ART妊娠中孕期胎儿停止发育中印记异常的发H19、KvDmR1(LIT1)、PEG1、SNRPN生风险,为印记基因DNA甲基化改变与胎儿停止发育的关系及ART技术的表观遗传学安全性提供初步依据。
[研究方法]
(1)本研究经医院伦理委员会批准,标本收集经患者知情同意。2010年8月至2011年7月于南方医院产科病房收集：①ART妊娠中孕期死胎胎儿肌肉组织13例；②自然妊娠中孕期死胎胎儿肌肉组织17例；③同期以自然妊娠正常早孕胎儿肌肉组织13例为对照。胎儿组织来源于孕18-26周,母龄在18-38岁,无解剖学方面因素,无家族遗传病史,排除宫颈因素、感染等导致的流产。于死胎娩出后2小时内,使用手术刀片及剪刀剪取胎儿大腿前方肌肉组织；
(2)将胎儿肌肉组织标本清洗处理、DNA提取、基因组DNA重亚硫酸修饰,后行H19、KvDMR1(LIT1)、PEG1、SNRPN基因甲基化特异性-PCR(MSP)扩增、琼脂糖凝胶电泳检测甲基化条带(M带)及非甲基化条带(U带),进行甲基化状态的定性分析；
(3)胎儿组织基因组DNA重亚硫酸修饰后行1H19、KvDMR1(LIT1)、 PEG1、SNRPN基因PCR扩增,扩增产物行焦磷酸测序及克隆测序,并得出每份标本的甲基化值。采用SPSS16.0统计软件(Statistical Package for the Social Sciences)、数据用均数±标准差(X士S)表示。组间数量资料比较采用t-test或One-way ANOVA,方差齐时多重比较采用Bonnferroni法；组间样本率的比较采用x2检验；采用Boxplot分析数据分布得出极端值；ROC曲线评价甲基化值对自然流产的预测价值。设双侧检验,P<0.05为差异有统计学意义
[研究结果]
(1) ART组与自然妊娠组相比,母龄、孕周均无统计学差异,P>0.05；此外,自然流产组与正常中孕组相比,母龄、孕周亦无显著性差异,P>0.05；提示胎儿组织样本的收集具有较好的均衡性。
(2)每次MSP扩增均设纯水模板空白对照,电泳检测结果显示空白对照均未发现假阳性M带或U带出现；人外周血淋巴细胞中,4个印记基因的MSP扩增均显示为正常甲基化模式,即M带和U带均显带。在所有胎儿组织中,均可扩增出4个印记基因的甲基化条带及非甲基化条带,未发现完全的高甲基化或完全的非甲基化状态。
(3)所有检测胎儿组织样本中,重亚硫酸盐转化效率波动于92.6-100%；取每种基因所检测的所有CpG位点之平均值作为该样本的甲基化值,H19、PEG1、KvDMR1、SNRPN基因的平均甲基化值分别为53.1士3.8%(波动范围46.2-60.9%)、52.7士7.9%(波动范围44.6-85.6%)、48.9士8.7%(波动范围39.3-83.4%)、42.0士3.9%(波动范围36.4-54.8%),均未出现完全甲基化(100%)或完全非甲基化(0%),结果与MSP结果相符。三组间的H19、KvDMR1、PEG1、 SNRPN基因甲基化程度经比较发现(采用多样本间单因素方差分析),仅KvDMR1基因的甲基化程度存在统计学差异(P=0.015);Bonnferroni多重比较后,自然妊娠自然流产显著性高于ART妊娠自然流产及自然妊娠正常中孕组；而其他3种印记基因甲基化程度在三组间不存在统计学差异(P>0.05)。以上结果提示,KvDMR1基因可能较其他基因更容易出现样本间甲基化值的差异,而其他3种基因未发现样本间存在统计学差异则可能是由于样本量较小造成的。Boxplot分析显示,每种基因的异常甲基化值发生率呈现KvDMR1=PEG1> SNRPN> H19,分别为4.7%(2/43)、4.7%(2/43)、2.3%(1/43)、0(0/43)；所有的异常甲基化值均发生于自然流产(死胎)样本中,即自然流产样本中印记基因甲基化异常的发生率为4.2%(5/120),且异常甲基化值亦均为高甲基化改变,1例女性死胎呈现2个基因甲基化异常2.3%(1/43)。将所有样本分为自然流产与正常中孕两组,采用ROC曲线分析4种印记基因甲基化程度与中期自然流产之间的关系,结果提示,KvDMR1、PEG1基因的甲基化程度对自然流产发生的风险有较好的预测价值(P<0.05),而H19、SNRPN基因对自然流产的发生无预测价值。此外,ART组与自然妊娠组相比、IVF组与ICSI组相比,4种印记基因的平均甲基化值、异常甲基化值发生率均无统计学差异(P>0.05)。
[研究结论]
(1)印记基因H19、KvDMR1、PEG1、SNRPN的甲基化状态异常可能与中期妊娠自然流产有关,其中KvDMR1基因甲基化状态的改变可能与中期妊娠自然流产关系最密切,且甲基化程度越高发生中期自然流产的几率可能越大；提示表观遗传学的异常可能是导致中期妊娠丢失的重要因素之一；
(2)辅助生殖技术可能并不增加印记基因KvDMR1、PEG1、SNRPN异常甲基化状态的发生；且不同受精方式之间(IVF与ICSI相比),印记基因的甲基化状态不存在显著性差异；
(3)本研究仅限于对少数印记基因的研究,且样本量较小,进一步的工作需要扩大样本量、扩展研究的基因种类,以证实上述的初步研究结论。
第三部分辅助生殖技术及自然妊娠早孕期绒毛、中孕期胎儿组织DNMT-3基因启动子SNP的研究
[研究目的]
DNA甲基转移酶(DNMT)-3A与DNMT-3B是催化重新甲基化的DNA甲基转移酶,对基因组甲基化模式的形成、胚胎形成及许多病理发生中的甲基化进程起着关键作用。DNMT-3A、3B启动子区一些单核苷酸多态性(single nucleotide polymorphisms,SNPs)可改变启动子的转录活性,使DNMTs表达增加、基因组DNA甲基化模式改变,从而导致各种疾病的发生。本研究以人类ART来源和自然妊娠来源的早孕期绒毛组织、中孕期胎儿肌肉组织为研究对象,采用PCR结合限制性片段长度多态性分析(PCR-RFLP)方法检测这些样本中DNMT-3A和3B基因启动子-448A>G位点和-149C>T位点的单核苷酸多态性,以探讨此两个位点与人类早中期妊娠丢失的相关性,及ART技术对DNMT-3A和3B多态性的影响,为进一步了解早中期妊娠丢失的发生机制提供依据。
[研究方法]
本研究经医院伦理委员会批准,标本收集经患者知情同意。研究对象样本收集同于第一、第二部分,将所有样本分为以下两组：①研究组：ART妊娠及自然妊娠自然流产早孕期绒毛、中孕期死胎胎儿肌肉组织,共计152例；②对照组：ART妊娠及自然妊娠正常早孕期绒毛、中孕期胎儿肌肉组织,共计155例。组织的收集、基因组DNA提取同第一、二部分。采用PCR-RFLP分析DNMT-3A(GenBank accession No. NT_022184.14:g.4381840A/G)、 DNMT-3B(GenBank accession No. AL035071,46359C/T)基因启动子区SNP位点,并选取部分样本采用DNA测序加以验证。采用SPSS16.0统计软件(Statistical Package for the Social Sciences),数据用均数±标准差(x士S)表示。两组间数量资料比较采用t-test;组间样本率的比较采用X2检验,设双侧检验,P<0.05为差异有统计学意义。
[研究结果]
(1) DNMT-3A PCR产物经Taal限制性酶切后的产物片段显示,酶切之后在研究组自然流产者和对照组正常早／中孕组织中均检测到AA变异纯合型、AG变异杂合型和GG野生型3种基因型。研究组中,GG、AG、AA基因型的分布频率分别为45.4%、40.8%、13.8%, A等位基因的频率为34.2%。对照组中,GG、AG、AA基因型的分布频率分别为68.4%、30.3%、1.3%, A等位基因的频率为16.5%。研究组与对照组样本相比,与-448A>G变异相关的自然流产的发生风险增高,AG杂合型较GG野生型发生自然流产的风险高2倍(OR值2.027,95%可信区间1.247-3.293, P=0.004)；而AA变异纯合型较GG野生型发生自然流产的风险高达16倍(OR值16.130,95%可信区间3.665-70.984,P=0.000)。
(2)当进行年龄、孕周分组时发现,在孕周<12w的样本中,与GG野生型相比AA变异纯合型和AG变异杂合型均与自然流产发生风险增高相关,OR值分别为16.897(95%可信区间3.810-74.925)、2.293(95%可信区间1.371-3.834)；在母龄<35岁组中,AA变异纯合型和AG变异杂合型亦与自然流产发生风险增高相关,OR值分别为28.868(95%可信区间3.734-223.156)、1.900(95%可信区间1.111-3.251)。而在孕周>12w和母龄>35岁组的样本中,未发现AA纯合型或AG杂合型与自然流产发生风险增高相关(P>0.05),可能与此组中样本量较小有关。此外,ART来源的样本与自然妊娠来源样本中GG、AG、AA基因型的分布频率亦无统计学差异(P=0.661)。
(3)分析277例人早孕期ART受孕和自然受孕来源的胚胎停育绒毛、正常早孕绒毛(同本文第一部分),比较发生异常甲基化值的样本与未发生异常甲基化值的样本其DNMT-3A的基因型分布：37个异常甲基化值的29例样本中,DNMT-3A启动子区GG野生型仅占13.8%(4/29),而AG杂合变异(44.8%,13/29)和AA纯合子变异(41.4%,12/29)共达86.2%,远高于未发生甲基化异常的样本中AG、AA基因型的分布频率(34.7%、4.0%,P=0.000)
(4) DNMT-3B PCR产物经AvrⅡ限制性酶切后的产物片段显示,酶切之后在研究组自然流产者和对照组正常早／中孕组织的所有样本中均仅检测到TT变异纯合型,未见到CT变异杂合型和CC野生型。
[研究结论]
(1) DNMT-3A基因启动子区-448A>G位点SNP在早／中期妊娠丢失中可能发挥了一定作用,其途径即可能通过改变印记基因的甲基化状态,从而影响印记基因的表达；ART技术并未影响胚胎停育及正常妊娠中该位点基因型的分布；
(2)目前尚无研究报道DNMT-3B基因启动子区-149C/T位点SNP在人早孕期绒毛组织及中孕期胎儿组织中的基因型,通过本研究结果推测,不排除DNMT-3B启动子区该位点突变型可能代表了早中期胎儿生长发育中的正常变异。
(3)本研究仅限于对DNMT-3A、3B基因启动子区DNA水平的研究,进一步的工作需要对DNMT-3A、3B基因表达及酶活力检测,以证实上述的初步研究结论。
Assisted reproductive technologies including in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), in vitro maturation (IVM), embryos freezing, and preimplantation genetics diagnosis (PGD) et al.. The offspring born after ART is becoming an important part of population, thus concerns about the safety of ART have been increasing. Recent studies suggest that in vitro fertilization (IVF)/intracytoplasmic sperm injection may be associated with increased incidence of Angelman syndrome (AS) and Beckwith-Wiedemann syndrome (BWS), and Imprinting defect is one of the most important factors. The majority of ART-conceived children with BWS and AS demonstrated imprinting defects, so Concerns about imprinting disorders linked to ART are therefore increasing. ART may affect epigenetic regulation, particularly the expression of imprinted genes, thus affecting the embryo implantation, placentation, organ formation and fetal growth. The prevalence of miscarriage has been estimated as15-20%of all clinically recognized pregnancies. To some extent, the occurrence of early miscarriage reduced the birth effects. Some studies suggests that genomic imprinting defects affecting the maintenance of early pregnancy. In the present study, we analyzed the DNA methylation patterns of four imprinted genes including H19, KvDMRl, PEG1, and SNRPN in human abortions and stillbirths, comparing ART with spontaneous conception, In addition, the association between DNA-methyltransferase (DNMT)-3A and DNMT-3B promoter genetic variant and the susceptibility to pregnancyloss was evaluated.
Part I Quantitative methylation analysis of developmentally important imprinted genes in human early pregnancy loss after ART and spontaneous conception
[Objective]
Animal studies have demonstrated that genomic imprinting defects may affect the maintenance of early pregnancy. ART procedures may affect epigenetic regulation, particularly the expression of imprinted genes, thus affecting the embryo implantation, placentation, organ formation and fetal growth. In the present study, we analyzed the DNA methylation patterns of four imprinted genes including H19, KvDMR1, PEG1, and SNRPN in human chrionic villus from early pregnancy loss, comparing ART with spontaneous conception, primarily evaluating epigenetic risks linked to pregnancy loss.
[Materials&Methods]
(1) Chorionic villus samples (CVS) were collected from women who underwent abortion procedures in Department of Gynecology and Obstetrics in Nanfang hospital from May2008to July2011. The duration of pregnancy ranged from6to11weeks, and the maternal age ranged from18to45years old. These patients had no known anatomic or genetic abnormalities.
(2) Four groups were tested:spontaneous abortion after ART (SN, n=62), multi-fetal reduction after ART (FA, n=73), spontaneous abortion of natural pregnancies (SN, n=73), and induced abortion of natural pregnancies (IN, n=69).
(3) Genomic DNA was extracted after sample collection by proteinase K digestion. Bisulfite treatment of genomic DNA was performed with the EpiTect Bisulfite Kit (Qiagen). The DNA methylation patterns of H19, LIT1, and SNRPN were analyzed using methylation speeific PCR (MSP) and pyrosequencing, which was performed using PyroMark Q96ID System (Qiagen) and pyrosequencing reactions were performed according to the manufacturer's instructions. The degree of methylation at each CpG site was determined by Allele Quantification (AQ) softwase-All samples were analyzed in triplicate. Bisulfite sequencing polymerase chain reaction (BSP) was then perform to confirm the pyrosequencing results.
(4) SPSS16.0statistical analysis program was used to analyze the percentage of methylation in chorionic villus from the four groups. Box plots were generated using the program's default parameters. The receiver operating characteristic (ROC) curve method was used to analyze the potential association between imprinted genes' methylation percentage and the incidence of early spontaneous abortion. The study protocol was approved by the Institutional Ethics Committee of Nanfang Hospital, and informed consent was obtained from all patients.
[Results]
(1) None of the277chorionic villus samples showed a complete loss of maternal or paternal alleles of H149, KvDMR1, PEG1, and SNPRN. The PCR products from methylated maternal chromosome and those from unmethylated paternal chromosome have been detected in all samples.
(2) The gestation and maternal age between samples from ART and spontaneous conceptions were not significantly different, neither were the samples from spontaneous abortions and normal early pregnancies.
(3) Bisulfite conversion efficiency values for all samples were90.1%-100%. The mean methylation values for H19, KvDMR1, PEG1, and SNPRN were49.6±9.3%(range,30.3-91.7%),49.0±9.4%(range,33.9-86.4%),55.5±9.7%(range,38.5-89.4%),41.3±4.3%(range,32.6-61.2%), respectively. Clear hypomethylation (0%) or hypermethylation (100%) was not detected. The percentage methylation of all the studied genes for the four groups showed significant differences (P<0.05). The percentage methylation in spontaneous abortion groups (SA and SN groups) was significantly higher than in the non-spontaneous abortion (normal early pregnancy) groups (FA and IN groups)(P<0.05).
(4) Box plot analysis showed that in the135spontaneous abortions,37of540(6.9%) analyzed DMR methylation values (for the four studied genes) represented outliers. In contrast, the142normal early pregnancies exhibited none outliers. Furthermore, the imprinted KvDMR1gene (20outliers,14.8%) exhibited significantly more potentially abnormal methylation values in spontaneous abortions than the other studied genes. Six of135(4.4%) spontaneous abortions displayed hypermethylated DMRs in two to three genes. It is interesting to note that all the outliers represented hypermethylated and no hypomethylated DMRs.
(5) However, ROC curve analysis could not predict an increased risk of early spontaneous abortion related to the methylation percentage of the four genes in chorionic villus. In addition, The mean percentage methylation did not differ significantly between samples from ART group and from non-ART group (P>0.05), neither were samples from in vitro fertilization and intracytoplasmic sperm injection (P>0.05). There's no evidence for an increased rate of potentially abnormal methylation values in the ART group.
[Conclusions]
(1) As some studies have suggested, imprinting errors of imprinted genes may contribute to early spontaneous abortion. The higher the methylation percentage, the greater the chance of early miscarriage.
(2) ART procedures or different semination methods might not affect the methylation patterns of imprinted genes, which were potentially related to the early pregnancy loss.
(3) The further work is to enlarge the sample size and gene quantity, confirming the hypothesis above.
Part Ⅱ Quantitative methylation analysis of developmentally important imprinted genes in human stillbirths from ART and spontaneous conceptions
[Objective]
The development of fetal may end at any time during the whole pregnancy satge. Although the genetic diagnosis methods were more and more excellent, part of the pregnancy loss in second trimester were still with unknown aetiology. Recent studies showed that among many reasons for such miscarriages, gene imprinting defects are some of the possible causes. Animal and human studies suggested in vitro culture of embryos can cause methylation defects in individual genes, which might affect subsequent embryonic development and contribute to pregnancy loss. To evaluate the correlation between methylation of imprinted genes and pregnancy loss in second trimester, the methylation status of H19, KvDMR1, PEG1, and SNPRN were assessed using pyrosequencing and Bisulfite sequencing polymerase chain reaction (BSP).
[Materials&Methods]
(1) Muscle samples (MS) were obtained from aborted fetuses and stillbirths in the Department of Gynecology and Obstetrics in Nanfang Hospital from August2010 to July2011. Gestational age of each fetus was determined by last menstrual period. The sex was identified by examination of the external genitalia. The duration of pregnancy ranged from18to26weeks, and the maternal age ranged from18to38years old. These patients had no known anatomic or genetic abnormalities.
(2) Three groups were tested:stillbirths from ART pregnancy (n=13), stillbirths from natural pregnancy (n=17), and induced aborted fetuses from natural pregnancy (n=13).
(3) Muscle smples were dissected within2hours after abortion and genomic DNA was extracted after sample collection by proteinase K digestion. Bisulfite treatment of genomic DNA was performed with the EpiTect Bisulfite Kit (Qiagen). The DNA methylation patterns of H19, KvDMR1, PEG1and SNRPN were analyzed using methylation specific PCR (MSP) and pyrosequencing. All samples were analyzed in triplicate. Bisulfite sequencing polymerase chain reaction (BSP) was then perform to confirm the pyrosequencing results.
(4) SPSS16.0statistical analysis program was used to analyze the percentage of methylation in chorionic villus from the four groups. Box plots were generated using the program's default parameters. The receiver operating characteristic (ROC) curve method was used to analyze the potential association between imprinted genes' methylation percentage and the incidence of early spontaneous abortion. The study protocol was approved by the Institutional Ethics Committee of Nanfang Hospital, and informed consent was obtained from all patients.
[Results]
(1) None of the43muscle samples showed a complete loss of maternal or paternal alleles of H19, KvDMR1, PEG1, and SNPRN. The PCR products from methylated maternal chromosome and those from unmethylated paternal chromosome have been detected in all samples.
(2) The gestation and maternal age between samples from ART and spontaneous conceptions were not significantly different, neither were the samples from spontaneous abortions and normal early pregnancies.
(3) Bisulfite conversion efficiency values for all samples were92.6%-100%. The mean methylation values for H19, KvDMRl, PEG1, and SNPRN were53.1±3.8%(range,46.2-60.9%),48.9±8.7%(range,39.3-83.4%),53.1±3.8%(range,46.2-60.9%),42.0±3.9%(range,36.4-54.8%), respectively. Clear hypomethylation (0%) or hypermethylation (100%) was not detected. The percentage methylation of KvDMR1in stillbirths from natural pregnancy was significantly higher than in the stillbirths from ART pregnancy and induced aborted fetuses from natural pregnancy (P<0.05). For the other three genes, no significant differences were found in the methylation percentage among three groups (P>0.05).
(4) Box plot analysis showed that in the30stillbirths,5of120(4.2%) analyzed DMR methylation values (for the four studied genes) represented outliers. In contrast, the13normal pregnancies in second trimester exhibited none outliers. And the outlier rates in H19, KvDMR1, PEG1, and SNPRN were4.7%(2/43),2.3%(1/43),4.7%(2/43), and0(0/43), respectively. All the outliers represented hypermethylated and no hypomethylated DMRs.1of43(2.3%) stillbirths displayed hypermethylated DMRs in two genes.
(5) In addition, ROC curve analysis showed a positive correlation between the methylation percentage of KvDMR1and PEG1in muscle samples and rates of spontaneous abortion in second trimenster:the higher the methylation percentage, the greater the chance of miscarriage (P<0.05). The mean percentage methylation did not differ significantly between samples from ART group and from non-ART group (P>0.05), neither were samples from in vitro fertilization and intracytoplasmic sperm injection (P>0.05). There's no evidence for an increased rate of potentially abnormal methylation values in the ART group.
[Conclusions]
(1) Imprinting errors of imprinted genes may contribute to spontaneous abortion in second trimester. Especially for KvDMR1gene, the higher the methylation percentage, the greater the chance of miscarriage.
(2) ART procedures or different semination methods might not affect the methylation patterns of imprinted genes, which were potentially related to the early pregnancy loss.
(3) To confirm the hypothesis above, further work is also needed, such as enlarging the sample size, gene quantity, and different kind of tissue type.
Part III The DNMT-3A and DNMT-3B promoter polyphisms and risk of human pregnancy loss
[Objective]
Methylation of gene promoters is one of the major regulatory mechanisms of gene expression and abnormal methylation patterns of imprinted genes have been demonstrated to be associated with pregnancy loss. The establishment of DNA methylation patterns requires de novo methylation that occurs predominantly during early development and gametogenesis. DNA-methyltransferase (DNMT)-3A and DNMT-3B are both essential for de novo methylation and play an important role in the development of embryogenesis and the generation of aberrant methylation in carcinogenesis. Here we hypothesized that genetic variants of the genes that are responsible for regulating genomic methylation are associated with increased risk of human pregnancy loss.
[Materials&Methods]
(1) Two types of tissue were collected from women who underwent abortion procedures in Department of Gynecology and Obstetrics in Nanfang hospital from May2008to July2011:(a) chorionic villus samples (CVS) and muscle samples (MS) from spontaneous abortions conceived by assisted reproductive technology (ART) and spontaneous way (study group), n=152;(b) CVS and MS from normal early pregnancy and second trimester (control group), n=155. The duration of pregnancy ranged from6to26weeks, and the maternal age ranged from18-45years old.
(2) We selected one of the single nucleotide polymorphisms (SNPs)-448A>G in the DNMT-3A promoter region and one of the SNP-149C/T in the DNMT-3B promoter region, then genotyped subjects for the DNMT-3A and3B promoter polymorphism to determine the association between this genetic variant and risk of pregnancy loss. The distribution of-448A>G and-149C/T polymorphisms was detected in all samples.
(3) Genomic DNA was extracted after sample collection by proteinase K digestion. The transition of A>G of DNMT-3A SNP creates a TaaⅠ restriction site, and for DNMT-3B the variant T allele had an AvrⅡl restriction site. PCR-restriction fragment length polymorphism (PCR-RFLP) was used to detect this A-G transition in the promoter of DNMT-3A at-448A>G (GenBank accession No. NT_022184.14:g.4381840) and the C-T transition in the promoter of DNMT-3B at-149C/T (GenBank accession No. AL035071,46359C/T). DNA sequencing analysis was performed to confirm the PCR-RFLP analysis. The study protocol was approved by the Institutional Ethics Committee of Nanfang Hospital, and informed consent was obtained from all patients.
[Results]
(1) The DNMT-3A genotypes AA, AG, and GG were detected in the spontaneous abortions and the controls. The genotyping by PCR-RFLP analysis was completely confirmed by DNA sequencing analysis. The allele frequency of-448A among pregnancy loss group and control group was34.2%(52/152) versus16.5%(25.5/155), respectively. Overall, we found that, compared with GG homozygotes, the DNMT-3A-448AA homozygotes had an about16fold increased risk of pregnancy loss [OR=16.130,95%confidence interval (CI),3.665-70.984] and AG homozygotes an OR of2.027(95%CI,1.247-3.293).
(2) When the analyses of DNMT-3A were stratified by the maternal age and gestation, the AA and AG genotypes were associated with an increased risk of pregnancy loss (for AA, OR16.897,95%CI=3.810-74.925; for AG, OR2.293,95%CI=1.371-3.834) at<12weeks, and the AA and AG genotypes were also associated with an increased risk of pregnancy loss (for AA, OR28.868,95%CI=3.734-223.156; for AG, OR1.900,95%CI=1.111-3.251) at<35years. However, the distribution of-448A>G in individuals derived from ART pregnancy was not statistically significantly compared with those derived from spontaneous pregnancy (P=0.661).
(3) The DNMT-3A genotypes AA, AG, and GG were then detected in the135chrionic villus samples from spontaneous abortions and142from controls (study population in Part I). We found that the distribution of-448A>G in samples with outlier (n=29)was much higher than that in samples with non-outlier (25/29,86.2%vs.96/248,38.7%)(p=0.000).
(4) For DNMT-3B, we observed genotype frequencies of100%(TT) in study populations and control groups. The genotyping by PCR-RFLP analysis was also completely confirmed by DNA sequencing analysis.
[Conclusions]
(1) The DNMT-3A-448A>G polymorphism is a novel functional SNP and contributes to its genetic susceptibility to pregnancy loss, especially for early pregnancy loss (<12w) in younger women (<35years). ART did not affect the distribution of of-448A>G in pregnancy loss and normal pregnancy.
(2) At present, we cannot exclude the possibility that the observed TT genotype of DNMT-3B represent normal variation during fetal development.
(3) Our study about the SNP of DNMT-3A and3B is restricted to the DNA level. Additional studies on the underlying molecular mechanism of this polymorphism and the expression of DNMTs are warranted.
引文
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