摘要
自1978年首例试管婴儿诞生以来,辅助生殖技术(assisted reproductive technology, ART)用于临床已逾30年,为不孕症的治疗作出了巨大的贡献。目前,发达国家1-4%的新生儿来自ART,随着子代生育高峰的到来,ART子代已逐渐成为世界人口的重要组成部分。ART非自然生殖的特性以及缺乏临床前安全性研究而直接临床应用的背景,日益引起人们对其生殖遗传安全性的关注。虽然目前未发现ART可直接导致子代出现明显畸形,但不断有报道指出,ART子代不良健康风险增加,生长发育问题就是其中之一,胎儿过度生长或低出生体重儿的风险增高,但原因尚不明确。可能源于不孕夫妇自身的遗传背景,也可能与ART的一系列操作相关。
表观遗传学是近年来在国际上迅猛发展的崭新研究领域,它是指通过DNA甲基化、组蛋白翻译后修饰等不改变基因核苷酸序列的方式,将遗传信息传递给子代的过程,包括DNA甲基化、基因组印记、组蛋白修饰等。其中,基因印记使某些基因(印记基因)在个体中只有一个亲本来源的等位基因表达。印记基因在人类基因组中只占少数,但在生长发育过程中却发挥至关重要的作用,一旦出现异常,将导致印记疾病或肿瘤的发生。基因组的印记修饰是可逆的,易受环境因素的干扰而出现可遗传的改变,影响子代表型。基因印记在配子及胚胎发育早期需经历擦除及重建过程,ART恰施于这一表观遗传修饰重编程的关键时期。近年来已有大量研究发现,ART操作可导致印记基因表达、DNA甲基化修饰等改变。流行病学调查发现,ART子代患罕见印记异常疾病,如Beckwith-Wiedemann综合征的风险增加。本课题组之前的研究也发现,ART子代脐血中印记基因表达、印记状态及甲基化状态异常。因此,ART极有可能通过干扰基因印记而影响子代生长发育潜能。
在胚胎期,滋养层细胞较内细胞团对环境因素的干扰更为敏感,因此胎盘组织发生表观遗传修饰异常的风险比胎儿更大。在胎盘中有许多参与母胎间物质转运、影响胎盘、胎儿生长的印记基因表达。其中,父源表达(母源印记)的印记基因促进胎儿生长和营养吸收；母源表达(父源印记)的印记基因抑制胎儿生长。这些基因的缺失或异常激活均可导致胎儿生长发育异常。
ART是否可通过干扰胎盘基因印记而影响子代的生长发育?其干扰的程度如何?相关机制如何?至今不明。
本研究以人类ART及自然妊娠足月、单胎子代的胎盘组织为研究对象,选择其中父源印记的生长抑制基因CDKN1C (cyclin-dependent kinase inhibitor 1C)、PHLDA2 (pleckstrin homology-like domain, family A, member 2)及母源印记的生长促进基因IGF2(insulin-like growth factor 2),用逆转录(reverse transcription, RT)-实时荧光定量PCR (Real time PCR).蛋白印迹(Westernblot).PCR直接测序、RT-PCR直接测序、PCR-聚丙烯酰胺凝胶电泳、亚硫酸盐测序(bisulfite sequencing,BSP)等方法研究其表达、甲基化状态及印记状态,探索ART影响子代生长发育的表观遗传机制,评估ART的生殖遗传安全性。
第一部分ART子代胎盘中印记基因CDKN1C、PHLDA2及IGF2的表达差异
目的：研究ART子代胎盘中生长发育相关印记基因CDKN1C、PHLDA2及IGF2表达与自然妊娠子代是否存在差异,并探索其与表型间的联系。
方法：收集足月、单胎ART子代胎盘39例,自然妊娠子代胎盘40例,比较两组新生儿出生体重。采用实时荧光定量PCR检测胎盘中目的基因nRNA表达水平；采用蛋白印迹检测蛋白表达水平。
结果：ART组新生儿平均出生体重低于对照组,但无显著性差异。ART组胎盘中,CDKN1C、IGF2 mRNA表达水平均显著上调,PHLDA2蛋白表达水平显著上调；PHLDA2 mRNA及CDKN1C、IGF2蛋白表达水平无显著改变。
结论：ART子代胎盘中存在生长发育相关印记基因表达异常,可能影响子代的生长发育潜能。
第二部分ART子代胎盘中印记基因CDKN1C、PHLDA2及IGF2印记调控区和启动子区的DNA甲基化状态研究
目的：研究ART子代胎盘中印记基因CDKN1C、PHLDA2及IGF2印记调控区及启动子区的DNA甲基化状态,比较其与自然妊娠子代间的差异,并探索其与基因表达间的关系。
方法：选择6例ART足月、单胎子代及5例自然妊娠足月、单胎子代胎盘,用亚硫酸盐测序法检测目的基因印记调控区KvDMR1、H19 DMR及CDKN1C、PHLDA2启动子区CpG岛的DNA甲基化状态。
结果：对照组印记调控区KvDMR1、H19 DMR均呈差异甲基化,甲基化率在50%左右。ART组KvDMR1区31个CpG位点的甲基化率均低于对照组,第9位点甲基化率显著下降,其中一例ICSI子代甲基化率明显低于对照组及其他ART标本；H19 DMR区20个CpG位点的甲基化率普遍高于对照组,第7位点甲基化率显著上升,其中一例IVF子代甲基化率明显高于对照组及其他ART标本。对照组及ART组CDKN1C、PHLDA2启动子区均呈低甲基化,甲基化率在0-23%左右。ART组CDKN1C启动子区15个CpG位点中,第7-11位点甲基化率显著下降；PHLDA2启动子区23个CpG位点中,第5位点甲基化率显著下降,第1、2、7、10、11位点甲基化率显著上升。
结论：ART子代胎盘印记基因的印记调控区及启动子区DNA甲基化状态改变,提示ART可能通过干扰调控区的DNA甲基化而影响印记基因表达。
第三部分ART子代胎盘中印记基因CDKN1C、PHLDA2及IGF2的印记状态研究
目的：研究ART子代胎盘中印记基因CDKN1C、PHLDA2及IGF2的等位基因表达状态及CDKN1C、IGF2的亲源表达状态,揭示它们在胎盘中的印记状态是否改变,并探索印记状态与印记调控区DNA甲基化改变及基因表达异常间的关系。
方法：以39例ART足月、单胎子代及40例自然妊娠足月、单胎子代胎盘为研究对象,用PCR-聚丙烯酰胺凝胶电泳、PCR直接测序、RT-PCR直接测序检测胎盘中目的基因的印记状态。
结果：CDKN1C、PHLDA2及IGF2在胎盘中均为单等位基因表达的印记基因。CDKN1C为母源表达、父源印记基因,IGF2为父源表达、母源印记基因。在ART组未发现上述目的基因异常的印记状态。
结论：ART子代胎盘印记调控区及启动子区DNA甲基化状态改变,但未发现印记基因CDKN1C、PHLDA2及IGF2的印记状态不稳定性。提示胎盘的基因组印记可能还受到DNA甲基化以外的修饰调控。
Since the birth of Louise Brown in 1978, assisted reproductive technology (ART), as a worldwidely accepted technology, has enabled many infertile couples to enjoy parenthood in the past 30 years. The babies conceived by ART have accounted for 1-4% of all births in developed countries now and gradually become an important part of the world population. ART induces an increasing worry about its risk in reproductive genetics because of its artificial character and the absence of sufficient risk evaluation before clinical use. Till now no evidence has found that ART is directly related to obvious malformation in offspring. But a series of epidemiological studies have revealed that ART babies tend to have health problems, such as growth and development problems. However, little is known about the exact underlying mechanisms. It is also unclear whether the genetic disorders of the infertile parents or the manipulations of ART raise the risk.
Epigenetics means changes in phenotype that are heritable but do not involve DNA mutation, including DNA methylation, genomic imprinting, histone modifications and so on. Genomic imprinting is an epigenetic process by which the male and female germ line are confered a sex-specific mark (imprint) on certain chromosomal regions. As a consequence, the imprinted gene is expressed from only one of the paternal or maternal chromosomes while the other copy is silent. There are only a few genes in human genome found to be imprinted, but they play important roles in growth and development and the abnormality will induce imprinting defects or tumors. Genomic imprinting is reversible and is vulnerable to environmental stress which will lead to heritable disturbance and change the phenotype of offspring. Genomic imprinting will undergo erasing and re-establishment during gamete and preimlantation embryo development. It is high time that ART manipulations take place. A series of studies have found that ART would lead to aberrant imprinted genes expression, DNA methylation, and so on. A number of reports have suggested that ART might be associated with an increased risk of imprinting defects, such as Beckwith-Wiedemann syndrome (BWS). The previous research of our team also found aberrant imprinted genes expression and DNA methylation in the umbilical blood of ART offspring. Therfore, ART probably influence the growth and development potential of offspring by disturbing the genomic imprinting.
Trophectoderm cells might be more sensitive to environmental stress than inner cell mass, so that the risk of epigenetic changes is higher in placenta than in fetus. There are many imprinted genes in placenta known to be crucial for placental and fetal growth. In general, the paternally expressed (maternally imprinted) genes enhance fetal growth and the maternally expressed (paternally imprinted) genes restrict fetal growth. Loss or aberrant activation of them will cause abnormalities in fetal growth and development.
Do the growth and development problems of ART babies have something to do with the aberrant genomic imprinting in placenta caused by ART? How seriously can it be disturbed by ART? What are the underlying mechanisms?
In the present study, we focused on paternally imprinted growth restricting genes CDKN1C (cyclin-dependent kinase inhibitor 1C), PHLDA2 (pleckstrin homology-like domain, family A, member 2) and maternally imprinted growth promoting gene IGF2 (insulin-like growth factor 2) in placenta. We conducted reverse transcription (RT)-Real time PCR, Westernblot, direct sequencing, PCR-polyacrylamide gel electrophoresis and bisulfite sequencing (BSP) to investigate the expression and imprinted status of these three genes and DNA methylation patterns of their impinting control regions and promoters in term and singleton placentas derived from ART and spontaneous pregnancy in order to elucidate the underlying epigenetic mechanisms for the growth and development problems of the ART conceived babies and evaluate the epigenetic risk of ART.
Part I The expression of imprinted genes CDKN1C, PHLDA2 and IGF2 changed in human placentas derived from ART
Objective:To investigate the mRNA and protein expression differences of CDKN1C, PHLDA2 and IGF2 between ART and spontaneously conceived placentas.
Methods:We collected 39 ART conceived, term and singleton placentas and 40 comparative spontaneously conceived placentas. We compared the birth weights of babies between two groups, used RT-Real time PCR to analyze the mRNA expression levels and used Westernblot to analyze the protein expression levels of the target genes.
Results:The average birth weight of ART group was lower than that of control group, but without significant difference. In ART conceived placenta, CDKN1C and IGF2 were significantly up-regulated in mRNA levels and PHLDA2 was significantly up-regulated in protein level. There were no significant differences in PHLDA2 mRNA and CDKN1C, IGF2 protein expressions between two groups.
Conclusion:The expression of several growth and development related imprinted genes changed in ART conceived placentas. It may influence the growth and development potential of ART babies.
PartⅡDNA methylation analysis of imprinting control regions and promoters of imprinted genes CDKN1C, PHLDA2 and IGF2 in human placentas derived from ART
Objective:To investigate the change of DNA methylation patterns in imprinting control regions and promoters of CDKN1C, PHLDA2 and IGF2 between ART and spontaneously conceived placentas, and elucidate the relationship between aberrant DNA methylation and abnormal gene expression.
Methods:Six ART conceived and 5 spontaneously conceived term and singleton placentas were subjected to bisulfite sequencing. DNA methylation patterns in CpG islands of imprinting control regions KvDMRl, H19 DMR and promoters of CDKN1C and PHLDA2 were analyzed.
Results:All spontaneously conceived placentas were differentially methylated in KvDMR1 and H19 DMR. The methylation rates were both around 50%. The average methylation rate of every CpG site in KvDMRl was lower in ART group and there was a significant decrease in methylation at CpG site 9. One case of ICSI conceived placentas was extremely hypomethylated in this region. The average methylation rates of most CpG sites in H19 DMR were higher in ART group and there was a significant increase in methylation at CpG site 7. One case of IVF conceived placentas was extremely hypermethylated in this region. Both ART and control group were hypomethylated in the promoters of CDKN1C and PHLDA2. The methylation rates were 0-23%. However, in ART group, there was a significant decrease in methylation at CpG site 7-11 in CDKN1C promoter while a significant increase at CpG site 1,2,7, 10,11 and a significant decrease at CpG site 5 in PHLDA2 promoter.
Conclusion:The aberrant DNA methylation patterns in the imprinting control regions and promoters may contribute to the abnormal expressions of imprinted genes in ART conceived placentas.
PartⅢImprinted status analysis of imprinted genes CDKN1C, PHLDA2 and IGF2 in human placentas derived from ART
Objective:To investigate the change of allele-specific expression of CDKN1C, PHLDA2 and IGF2 and parent-specific expression of CDKN1C and IGF2 in ART conceived placentas and elucidate the relationship among imprinted status, aberrant DNA methylation and abnormal gene expression.
Methods:Thirty-nine ART conceived and 40 spontaneously conceived term and singleton placentas were involved. PCR-polyacrylamide gel electrophoresis, PCR-direct sequencing and RT-PCR-direct sequencing were applied to detect the imprinted status of target genes in placentas.
Results:CDKN1C, PHLDA2 and IGF2 maintained monoallelic expression in all samples analyzed, so that they are all imprinted genes in placenta. CDKN1C is a maternally expressed and paternally imprinted gene. IGF2 is a paternally expressed and maternally imprinted gene. We found no alteration in imprinted status of target genes in ART conceived placentas.
Conclusion:We found aberrant DNA methylation patterns in imprinting control regions and promoters of ART conceived placentas but detect no instability of imprinted status in target genes. It suggests that other modifications besides DNA methylation may also contribute to the genomic imprinting in placenta.
引文
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