【作者】 李雷；

【导师】 金帆；

【作者基本信息】 浙江大学 ， 妇产科学， 2011， 博士

【摘要】 第一部分IVF出生小鼠及其之间交配出生后代行为学、形态学及全基因组甲基化模式的研究目的：通过IVF小鼠模型的建立,完成以IVF为代表的ART出生群体之间婚育安全性动物模型方面的评估,揭示ART的对人类未来可能的表观遗传学效应。材料和方法：1.建立IVF小鼠模型,获得IVF出生F1代小鼠；6-7周龄时,IVF F1代小鼠雌雄性合笼,自然交配受孕,获其F2子代；2.,以自然出生小鼠为对照,分别于6-7周龄时,进行F1代,F2代小鼠的水迷宫实验,评价其学习及记忆能力；3.以自然出生小鼠为对照,开展F1代,F2代小鼠的体重、器官测量,外表结构畸形观察、组织器官的大体和组织形态学检测和分析；4.应用小鼠全基因组甲基化芯片,对F1代及F2代小鼠及自然妊娠出生小鼠各四只进行全基因组水平的甲基化的检测,比较三组全基因组水平的甲基化的模式；5.选取8个在F1、F2代同时发生高甲基化的启动子位点Cryga、Fgf1、Nos3、Mb、Myog、Notch3、Th釉Vavl以及4个只有在F2代发生高甲基化的启动子位点：Col9a2、Fgf6、Lck和Slc5al进行亚硫酸氢盐测序,对芯片的部分结果进行验证；6.采用荧光定量PCR方法检测经亚硫酸氢盐测序证实在F1代阳性的位点Fgf1、Nos3、Notch3、Th和Vavl以及在F2代的阳性位点Col9a2、Fgf1、Fgf6、Nos3、Notch3、Slc5a1、Th及Vavl的表达,明确甲基化状态其对其mRNA表达情况的影响。结果：1.成功建立IVF F1代小鼠模型及F2代模型；2.水迷宫实验显示IVF F1、F2代组与自然妊娠出生小鼠相比在潜伏期、游泳距离等方面无显著性差异；3. IVF F1、F2代的体重、器官比重及组织形态学与自然妊娠出生小鼠相比无显著性差异；4.小鼠脑组织全基因组甲基化芯片结果显示,在F1代中共有225个CpG岛及191启动子区域发生了高甲基化；22个CpG岛和28启动子区域发生了低甲基化。F2代有196个CpG岛和213个启动子区域高甲基化；69个CpG岛,56个启动子发生了低甲基化。F1代与F2代相比,共有113个启动子和143个CpG岛发生了共同的高甲基化；5.在经亚硫酸氢盐测序验证的8个启动子中,Fgf1、Nos3, Notch3、Th及Vavl在F1、F2代甲基化的程度较自然妊娠组高,差异有显著性,符合芯片发现的结果。4个只在F2代发生高甲基化的位点中,有3个发生了高甲基化,符合芯片的发现；6.荧光定量PCR方法显示Fgf1、Nos3、Notch3、Lck, Co19a2、Fgf6及Slc5a1的表达受到其甲基化状态得影响,其mRNA的表达有不同程度的降低,但Th、Vavl没有显示这种趋势。结论：1. IVF对F1、F2代小鼠的学习和记忆能力、生长发育及器官形态等没有明显的影响；2.IVF出生小鼠脑组织基因组DNA甲基化修饰存在改变,尤以高甲基化异常明显,并可影响到基因的表达；3. IVF出生小鼠的部分基因组DNA甲基化修饰异常可以传递给其相互交配出生的后代,同时这些后代也有新的DNA甲基化修饰异常发生,此类异常甲基化同样可影响基因的表达。第二部分IVF所致异常甲基化向后代传递的遗传方式研究目的：分析IVF出生小鼠基因组DNA异常甲基化的子代传递方式,为探索异常甲基化发生机制和可能阻断途径提供研究基础。材料和方法：1.将IVF出生的雌性及雄性F1代小鼠分别与野生型雄性及雌性小鼠交配获得母源和父源IVF F2代小鼠；2.选取3个前期研究中发现在F1、F2代同时发生高甲基化位点：Fgfl、Nos3、Notch3以及3个只有在F2代发生高甲基化位点：Col9a2、Fgf6、Slc5a4,分别在母源和父源IVFF2代小鼠考察上述启动子区域的甲基化模式；3.荧光定量PCR方法检测Fgf1、Nos3、Notch3、Col9a2、Fgf6及Slc5al在母源和父源IVF F2代小鼠中枢神经系统中的表达,明确甲基化状态其对其mRNA表达的影响。结果：1.2个在IVF F1和F2代同时发生高甲基化的位点：Fgf1、Notch3以及3个只有在IVF F2代发生高甲基化位点：Col9a2. Fgf6和Slc5a1,在母源IVF F2代小鼠脑组织中,其甲基化程度与自然妊娠出生小鼠无显著差异。但Nos3仍表现出高甲基化,且荧光定量PCR结果显示Nos3基因的表达也存在不同程度的降低；2.3个在IVF F1和F2代同时发生高甲基化的位点：Fgf1、Nos3、Notch3以及3个只有在F2代发生高甲基化位点：Col9a2、Fgf6及Slc5al,在父源IVF F2代小鼠的脑组织中,其甲基化程度与自然妊娠出生小鼠均无显著差异。结论：1. IVF F1代雄性小鼠发生的基因组DNA甲基化修饰异常,在其后代可得以纠正；2. IVF F1代雌性小鼠发生的甲基化修饰异常,存在传递给其后代的风险；3. IVF出生小鼠相互交配出生的F2代小鼠的异常甲基化可能主要是通过母源传递。第三部分Nos3启动子在母源F3代的甲基化状态及IVF出生双胎子代脐血印迹基因调控区KvDMRl、H19/IGF2 DMR及PEG1的甲基化状况目的：进一步考察在母源IVF F2代小鼠中发生异常的Nos3位点是否可以进一步传递；研究IVF双胎子代脐血中几个重要印记基因调控区的甲基化状况,分析IVF双胎子代中印迹状态异常风险。材料和方法：1.将论文第二部分的母源IVF F2代进一步与野生型的C57/BL6J雄性小鼠之间交配,获的母源IVF F3代,以自然妊娠子代为对照；同时荧光定量RT-PCR进一步检测Nos3所调控基因的mRNA表达水平2.共收集59对双胎脐血标本,其中29对IVF双胎组成研究组,30对自然妊娠双胎组成对照组,采用亚硫酸氢盐测序对两个母源性甲基化印记区域(KvDMR1、PEG1)及一个父源性甲基化印记区域(H19/IGF2 DMR)进行甲基化模式检测和比较。结果：1.在母源IVF F2代小鼠中异常甲基化的Nos3位点在母源IVF F3代与自然妊娠出生的小鼠相比已无差别,Nos3基因表达水平到母源F3代已经无显著性变化；2.IVF和自然妊娠双胎子代均未发现有PEG1基因的印记缺失,二组甲基化程度无显著性差异(P=0.103)。3例IVF双胎子代中存在KvDMR1低甲基化,甲基化程度在21%左右,发生率为5.08%(3/58),1例自然妊娠双胎子代存在低甲基化,发生率为1.67%(1/60),二者相比,无统计学差异(P=0.611)。IVF和自然妊娠双胎子代各有1例存在H19/IGF2 DMR的高甲基化,二组甲基化程度没有显著性差异(P=0.103)。结论：1.在F1代和母源IVF F2代发生异常高甲基化的Nos3启动子位点在母源IVF F3代得到了纠正；2.虽然没有发现IVF和自然妊娠双胎子代在KvDMR1, H19/IGF2 DMR及PEG1的甲基化模式和程度的统计学差异,但IVF子代较多的异常甲基化人数仍然提示IVF可能存在一定的表观遗传风险,有关结论优待增加样本的进一步研究。更多还原

【Abstract】 Part I Behavioral, morphological and genome-wide DNA methylation evaluation in IVF-conceived mouse and their cross-bred descendantsObjective:By establishing the IVF-conceived mouse model, we want to gain a prospective understanding of the marriage safety between ART-born children in human population, reveal the possible effect of IVF on the epigenetic inheritance, and privide theoretical foundation for empirical study in human population.Materials and methods:1. Mice model derived from in vitro fertilizationand embryo transfer (IVF-ET) were built and crossed. Naturally conceived (NC) mice were used as the control group. Effects of IVF on the spatial learning and memory capability at Fl generation were examined by Morris water maze at 6 weeks old. After Morris water maze were finished, Fl male mice were bred to F1 female mice to get the Fl generation;2. Effects of IVF on the spatial learning and memory capability at F2 generation were detected as did in F1 generation. After Morris water maze test, organs, including brain, heart, liver, lung, stomach, intestine, kidney, spleen, testis and ovary were excised, weighted and stained with hematoxilyn and eosin (H&E). The effects of IVF on development were detected by checking the specific gravity of the organs from male and in the female mice at 7 week;3. Genome-wide DNA methylation status were investigated in central nervous system (CNS) at F1,2 generation and naturally conceived mice by using the MM8 CpG promoter microarray;4. Eight concomitant hypermethylated promoters in F1 and F2 generation (Cryga, Fgfl, NosS, Mb, Myog, Notch3, Th, Vavl) and four unique hypermethylated promoters (Col9a2, Fgf6, Lck, SlcSa1) only in F2 generation, which associated with organ development function were further validated by bisulfite genomic sequencing;5. Expression of bisulfite sequencing confirmed genes(Fgfl, Nos3, Notch3, Th, Vavl in Fl, F2 generation and Col9a2, Fgf6, Slc5al in F2 generation) were analyzed by quantitative real-time RT-PCR in order to evaluate the relationship between promoter methylation and gene expression.Result(s):1. IVF-born mice model were successfully established;2. No differences in learning and memory ability including incubation period and the swimming distances were detected by the water maze test between IVF-born mice (F1), their cross-bred F2 generation and naturally conceived littermates;3. No statistically significant reduction or increase in the weight of total body. Obvious phenotypic abnormalities and defects, such as small eye, brachyury, short ear extra toes from first week to 6-7week old were not fount in F1 and F2 generation, but significant decrease in gravity of spleen in Fl generation mice was observed when compared with naturally conceived littermates;4.225 CpG islands and 191 promoters were hypermethylated at Fl generation. In contrast, only 22 CpG islands and 28 promoters showed a trend towards hypomethylation.196 CpG islands and 213 promoters were hypermethylated and 69 CpG islands,56 promoters were hypomethylated in F2 generation. A comparison of F1 to F2 epigenome identified 113 concomitant hypermethylated promoters and 143 hypermethylated CpG islands;5. Fgfl, Nos3, Notch3, Col9a2, Fgf6 and Slc5al were found with a gain in DNA methylation as validated by bisulfite genomic sequencing in F1 and F2 generation, which confirmed the results obtained by MeDIP-CHIP. No significant differences in Cryga, Mb, Myog in F1, F2 generation and Lck in F2 generation were found, which suggest they were false positive loci; 6. Fgfl, Nos3, Notch3, Col9a2, Fgf6 and Slc5al displayed lower levels of expression, which further validated their methylation status, but expression of Th and Vavl were not disturbed by their hypermethylated promoters, indicating that some genes were not susceptible to perturbation by promoter methylation.Conclusion(s):1. Learning and memory ability in IVF-conceived mice and their cross-bred F2 generation were not affected;2. Even the growth and development of Fl, F2 generation were not disturbed, IVF can slightly modify the epigenome at the central nerve system in F1 generation;3. IVF-induced DNA methylation aberration can be transmitted from Fl to F2 generation, and de novo aberrant DNA methylation occurred in F2 generation;4. Our findings suggest that there are some epigenetic risks in the marital events between ART-born children in human population and the exact mechanism need further investigation. Part II The inheritance of IVF-induced aberrant DNA methylation in IVF descendantsObjective:To determine the mode of epigenetic inheritance in IVF-induced aberrant DNA methylation and find the way they got transmitted to their following descendants, which could pave a road for investigating the precipitating factor of epigenetic disorders in human.Materials and methods:1. Female and male IVF-born F1 generation mice were mated to wild-type male and female mice to get the female-and male-line-derived F2 generation;2. Three concomitantly hypermethylated promoters loci (Fgfl, Nos3, Notch3) that were previously found in IVF-born mice and their F2 generation and three loci (Col9a2, Fgf6, Slc5a1) that found hypermethylated only in F2 generation were examined in female-line and male-line derived F2 generation;3. Expressions of these genes(Fgfl, Nos3, Notch3, Col9a2, Fgf6 and Slc5al) in male-and female-line-derived F2 generation were analyzed by quantitative real-time RT-PCR.Results:1. In female-line derived F2 generation, Fgfl, Notch3 and three de novo hypermethylated promoters indicated normal methylation status in central nerve system, but Nos3 was still hypermethylated and displayed disturbed levels of mRNA;2. In male-line-derived F2 generation, Fgfl, Nos3, Notch3 and three de novo hypermethylated promoters (Col9a2, Fgf6, Slc5a1) have come to normal methylation status, and all of the six loci did no show significant difference in the methylation values and their expression were not disturbed.Conclusions:1. Hypermethylated have been repaired by some pathways in male-line-derived F2 generation at the following phases of development in order to assure the integrity of the epigenome and IVF-induced aberrant DNA methylation might not be transmitted through male-line gametes;2. Epigenetic disturbances induced by IVF, such as aberrant methylation, can not be completely eliminated in the maternal germ line in mice, which were responsible for the epigenetic inheritance to their following generation. The risk of animals with maternally transmitted methylation aberrations depends on the efficiency of maternal epigenome repair following after fertilization. PartⅢNos3 methylationo in female-line F3 generation and evaluation of DNA methylation status at differentially methylated regions (DMRs) in IVF-conceived newborn twinsObjective:To study the aberrant methylated Nos3 in female-line F3 generation. The effects of in-vitro fertilization (IVF) on the stability of DNA methylation at differentially methylated regions (DMRs) in IVF-conceived twins were investigated.Materials and methods:1. Nos3, which was found hypermethylated in IVF-born mice and can be transmitted through female gamete, was further investigated in female-line F3 generation; Its expressions was detected by real-time RT-PCR;2.59 pairs of twins were recruited, including 29 pairs conceived through IVF and 30 pairs naturally conceived twins. Umbilical cord blood samples were collection after cesarean section. DNA was extracted from umbilical cord blood. Two maternally methylated regions (KvDMR1 and PEG1) and one paternally methylated region (H19/IGF2 DMR) were analyzed using bisulfite-based technologies.Results:1. The methylation tendency of Nos3 has come to normal pattern at F3 generation. No differences were detected in the expressions of Nos3 in F3 generation;2. The median methylation percentages of IVF-conceived at PEG1 were 5 comparable to naturally conceived controls, and no significant differences were found (P=0.103) There was a trend toward hypomethylation in 3 children from 3 separate pairs of twins out of 29 (5.08%) pairs of IVF children who had methylation levels slightly lower than 21%, but 1 child out of 30 pairs (1.67%) of control children also displayed hypomethylation (Fisher’s exact test,_P=0.611). One IVF child had an H19/IGF2 DMR methylation level of 68%. However, one naturally conceived case with a methylation level slightly higher than 70%was also found. Statistical analysis did not reveal significant differences in the methylation percentages of the H19/IGF2 DMR (P=0.103).Conclusion:1. Aberrant hypermethylated Nos3, which was found in IVF-born Fl generation and their female line F2 generation, has been corrected at F3 generation;2. Our results suggest no significant increase in imprint variability at these DMRs, but KvDMRl showed slightly more variable levels of methylation in IVF cases than in spontaneous cases, and the greater variance in the IVF kids has a biologically meaningful consequence and is still sources of concern for future investigation. Large samples of study are needed to systematically assess the potential epigenetic risk in IVF-conceived twins.更多还原