辅助生殖技术对子代基因组稳定性的影响及其相关机制研究

发布时间：2018-04-13 11:19

本文选题：体外受精 + 动态突变　；参考：《浙江大学》2014年博士论文

【摘要】：第一部分辅助生殖技术对动态突变频率的影响目的：调查ART出生儿动态突变的发生频率及其改变幅度,比较其与自然妊娠出生儿之间的差异,探讨ART对动态突变基因三核苷酸重复拷贝数突变频率和幅度的影响,为ART对DNA稳定性的效应研究提供证据。材料和方法：收集2008-2012年在浙江大学医学院附属妇产科医院完成分娩的ART家系147例,其中包括75例IVF家系和72例ICSI家系。同时收集在该院完成分娩的99例自然妊娠家系,作为正常对照。采集新生儿的脐带血和父母亲的静脉血,提取DNA。以齿状核红核苍白球丘脑下部核萎缩(dentatoubral and pallidoluysian atrophy, DRPLA),脊髓延髓肌萎缩(spinal and bular muscular atrophy,SBMA),脊髓小脑共济失调Ⅰ型(spinocerebellar ataxia,SCA1),马赫多-约瑟夫病(Machado-Joseph,MJD),亨廷顿病(Huntington's disease gene,HD),肌强直性肌萎缩(myotonic dystrophy,DM),脆性X综合征(Fragile X syndrome,FraX)7种动态突变疾病的致病基因：ATN1,AR,ATXN1,ATXN3,Huntington,DMPK,FMR-1基因为目的基因。对所收集的246个家系(783个成员),PCR扩增上述7个基因的三核苷酸重复序列区,琼脂糖凝胶电泳,8%非变性聚丙烯酰胺凝胶电泳、DNA的测序分析完成各家系各基因位点三核苷酸重复拷贝数检测,统计分析,比较各组间三核苷酸重复序列动态突变频率和拷贝数改变的幅度。结果： ART子代中共检测2466个等位基因中,有52个等位基因发生突变,突变率为2.11%,与对照组子代突变率0.77%(10/1300)相比,差异具有统计学意义。其中IVF子代突变率为2.48%(32/1288),ICSI子代突变率为1.70%(20/1178),与对照组相比较,差异具有统计学意义,但是IVF与ICSI组之间无显著性差异。在所有的这些被检测家系中,各动态突变位点的三核苷酸重复拷贝数既有增加,也有减少,但均在其正常范围内。根据不孕不育背景,我们再将IVF和ICSI再分成以下各小组：女性因素包括输卵管性不孕,子宫内膜异位症,排卵障碍；男性因素包括少精,弱精,畸精及梗阻性无精子症；男女双方因素；不明原因不孕及其他因素。我们发现在ICSI组中,不孕因素为女方因素(3.90%)及男女双方因素(2.59%)的发生动态突变的频率要高于单纯男方因素(0.76%),差异具有统计学意义。结论 1.ART子代动态突变频率高于自然妊娠子代。 2.ART技术及其不孕不育背景可能影响子代动态突变频率。第二部分ART对子代胎盘DNA损伤修复基因的研究目的：研究ART子代胎盘组织中DNA损伤修复相关基因表达,揭示ART子代基因组不稳定性原因,并探索相关基因启动子区域DNA甲基化率改变与基因表达异常间的关系。材料和方法：收集2009年-2013年间在浙江大学医学院附属妇产科医院剖宫产分娩的52例足月、单胎ART子代胎盘组织,其中包括32例常规IVF子代及20例ICSI子代。对照组为同期在我院剖宫产的32例自然妊娠子代。记录母亲年龄,出生体重,孕周等一般信息。依据本论文第一部分结果显示ART子代动态突变频率增高这一结果,选择动态突变相关DNA损伤修复基因为目标基因：OGG1:8-oxoguanine DNA glycosylase; MSH6:mutS homolog6; MSH2:mutS homolog2; MSH3:mutS homolog3; MLH1: MutL homolog1; MLH3:MutL homolog3; PMS2:postmeiotic segregation increased2; PMS1:postmeiotic segregation increased1;XPA:xeroderma pigmentosum, complementation group A; APEXI:apurinic/apyrimidinic endonuclease1; RPA1: replication protein Al,选取胎盘为靶组织,进行ART子代动态突变不稳定性的发生机制研究。 (1)ART子代胎盘中DNA损伤修复相关基因表达分析利用荧光定量RT-PCR检测ART子代胎盘中DNA损伤修复相关基因OGG1, MSH6, MSH2, MSH3, MLH1, MLH3, PMS2, PMS1,XPA, APEX1和RPA1的表达变化。 (2)ART子代胎盘中DNA损伤修复相关基因修饰分析应用焦磷酸测序技术检测OGG1,RPA1,PMS2,MSH6和XPA这五个基因启动子区域甲基化状态。 (3)ART子代胎盘中DNA损伤修复相关基因蛋白表达分析通过western-blot检测MLH1,OGG1,RPA1,PMS2,MSH2,MSH6和XPA各蛋白在胎盘组织的表达情况。结果： (1) mRNA水平：IVF与ICSI组中,PMS2,RPA1,XPA,MSH2,MSH6基因相比较对照组存在高表达,差异具有统计学意义。IVF组中的MLH1与对照组和ICSI组比较存在显著性高表达；另外,与IVF组相比,ICSI组的MSH2,XPA,OGG1和RPA1基因也存在显著性高表达,差异具有统计学意义。 (2)DNA水平：OGG1,RPA1,PMS2,MSH6和XPA的CpG岛的甲基化位点在IVF与ICSI组均与体内组存在甲基化率的差异,并有统计学意义,与基因mRNA水平改变一致。 (3)蛋白水平：ICSI组的PMS2,MSH6和MLH1蛋白表达水平显著高于IVF组及对照组,差异具有统计学意义。结论： 1.ART操作及不孕不育背景可能子代胎盘组织DNA损伤修复相关基因的表达水平异常有关。 2.ART操作及不孕不育背景可能影响DNA损伤修复相关基因的DNA甲基化修饰。 3.DNA损伤修复相关基因的mRNA水平与蛋白水平不一致,可能与转录后调控有关。第三部分ART对出生小鼠老年时期中枢神经系统DNA氧化损伤及退行性改变的影响目的：研究ART出生小鼠在老年时期脑组织氧化损伤,DNA损伤修复基因及神经退行性疾病相关基因,评估ART对出生小鼠老年时期中枢神经系统DNA氧化损伤及退行性改变的影响。材料和方法：应用已建好的ART老年C57BL/6J小鼠模型包括：8只IVF小鼠、8只ICSI小鼠、8只体内受精小鼠(每组小鼠雌雄比率相似),选择8-OHdG为DNA氧化损伤标记物,选择DNA损伤修复基因Msh2, Apex1,Msh6, Ogg1, Pcna, Xpa和Mlh1；神经退行性疾病相关Mapt, Bacel,Psen1,Psen2, App, Apoe和相关microRNA以及Insulin/IGF-1信号通路有关基因为目标基因。选取脑组织为靶组织,研究ART对出生小鼠老年时期中枢神经系统DNA氧化损伤及退行性改变风险的影响。 (1)ART老年小鼠8-OHdG水平分析利用ELISA检测ART老年小鼠脑组织8-OHdG水平。 (2)ART老年DNA损伤修复相关基因表达分析利用荧光定量RT-PCR检测ART老年小鼠脑组织Msh2, Apex1,Msh6,Oggl, Pcna, Xpa和Mlhl的表达变化。 (3)ART老年神经退行性疾病相关基因表达分析利用荧光定量RT-PCR检测ART老年小鼠脑组织Mapt, Bacel,Psen1,Psen2, App,Apoe,Igf-1,Igf-1r,Insr,Irs-1,Irs-2, miR-34与miR-101的表达变化。 (4)ART老年小鼠DNA损伤修复基因修饰分析应用焦磷酸测序技术检测ART老年小鼠脑组织相关损伤修复基因的CpG岛的甲基化状态。结果： (1)8-OHdG水平：对照组8-OHdG为4.26±1.54pg/ml, IVF组9.59±4.27pg/ml,ICSI组为8.57±3.90pg/ml,与体内组相比,差异均具有统计学意义。但IVF与ICSI组之间无显著性差异。 (2)DNA损伤修复相关基因mRNA水平：与体内组相比,IVF和ICSI组的Ogg1, Apex1和Pcna基因表达显著上调；IVF和ICSI组的Msh6,Msh2和Mlhl基因显著性低表达,差异均具有统计学意义。ICSI与IVF组相比,Pcna基因表达显著上调。 (3)神经退行性疾病相关基因mRNA和rmiRNA水平：与体内组相比,ICSI组Psen1,Igf-1基因表达水平升高,Igf-1r,Insr,Irs-1, miR-34和miR-101表达水平下降,差异具有统计学意义；IVF组Igf-1基因表达水平升高,Igf-1r,Insr,Irs-1和miR-34表达水平下降,差异具有统计学意义。IVF与ICSI组相比,Insr和miR-101在ICSI组表达显著性下降,差异具有统计学意义。 (4)DNA损伤修复相关基因DNA水平：Oggl和Mlhl的CpG岛的甲基化位点在IVF与ICSI组均与体内组存在甲基化率的差异,并有统计学意义。结论：1.IVF/ICSI出生小鼠老年时期脑组织可能存在较高氧化损伤风险。2.IVF/ICSI出生小鼠老年时期可能存在较高中枢神经系统退行性改变风险,但进一步机理尚需完善。
[Abstract]:The effect of assisted reproductive technology on the frequency of dynamic mutation in the first part
Objective:
Investigation of ART frequency and amplitude change was born with mutations in children compared with the dynamic difference between the natural pregnancy birth, to explore the influence of ART copy number mutation frequency and amplitude of dynamic mutation gene trinucleotide, provide evidence for the effect of ART on the stability of DNA.
Materials and methods:
Collected 2008-2012 years in women's Hospital School of medicine Zhejiang University completed the ART family delivery in 147 cases, including 75 cases of IVF families and 72 ICSI families were collected in the hospital. At the same time to complete the delivery of 99 cases of spontaneous pregnancy pedigrees, as normal control. The acquisition of neonatal umbilical cord blood and venous blood of the father mother extraction, DNA.
The dentatorubral pallidoluysian atrophy (dentatoubral and pallidoluysian atrophy, DRPLA), spinal bulbar muscular atrophy (spinal and BULAR muscular atrophy, SBMA), spinocerebellar ataxia type 1 (spinocerebellar ataxia SCA1), Magdo - Joseph's disease (Machado-Joseph, MJD), Huntington disease (Huntington's disease gene. HD), myotonic muscular atrophy (myotonic dystrophy, DM), fragile X syndrome (Fragile X, syndrome, FraX) gene 7 dynamic mutation disease: ATN1, AR, ATXN1, ATXN3, Huntington, DMPK, FMR-1 gene.
In 246 families collected (783 members), PCR amplified the 7 gene trinucleotide repeats, agarose gel electrophoresis, 8% non denaturing polyacrylamide gel electrophoresis, DNA sequencing analysis of complete families of the loci trinucleotide repeat copy number detection, statistics, comparisons between groups of trinucleotide repeat sequence dynamic mutation the frequency and amplitude of the copy number changes:
The ART offspring were detected 2466 alleles, 52 alleles mutation, the mutation rate was 2.11%, and the control group of offspring mutation rate was 0.77% (10/1300) compared to the difference was statistically significant. The IVF progeny mutation rate was 2.48% (32/1288), ICSI generation (the mutation rate was 1.70% 20/1178), compared with the control group, the difference was statistically significant, but no significant difference between IVF and ICSI group. In all these tested families, the dynamic mutation repeats has also decreased, but increased in the normal range.
According to the infertility background, then we will use IVF and ICSI of each group is divided into the following: female factors including tubal infertility, endometriosis, ovulation disorder; male factors including oligospermia, asthenospermia, abnormal sperm and obstructive azoospermia; both male and female factors; unexplained infertility and other factors. We found in ICSI in the group, for the woman's infertility factors (3.90%) and both factors (2.59%) the occurrence of dynamic mutation is higher than that of the pure male factors (0.76%), the difference was statistically significant.
conclusion
The frequency of dynamic mutation of the 1.ART progeny is higher than that of the natural pregnancy offspring.
The 2.ART technique and its infertility background may affect the frequency of the dynamic mutation of the offspring.
The study of the second part ART on the repair genes of DNA injury in the placenta of the offspring
Objective:
Objective to study the expression of DNA damage repair related genes in the placenta of ART offspring, reveal the cause of genomic instability in ART progeny, and explore the relationship between the alteration of DNA methylation rate and the abnormal expression of genes in promoter regions.
Materials and methods:
From 2009 -2013 years in 52 cases of term affiliated obstetrics and Gynecology Hospital of Zhejiang University School of medicine in cesarean delivery, fetal ART single offspring placenta tissue, including 32 cases of IVF and 20 cases of normal offspring ICSI offspring. The control group for the same period in our hospital 32 cases of cesarean section pregnancy offspring. Parent records Pro age, birth weight, gestational age and other general information.
On the basis of the first part of this paper shows that ART generation dynamic mutation frequency increased this result, dynamic mutation of DNA repair gene as target gene: OGG1:8-oxoguanine DNA glycosylase; MSH6:mutS homolog6; MSH2:mutS homolog2; MSH3:mutS homolog3; MLH1: MutL homolog1; MLH3:MutL homolog3; PMS2:postmeiotic segregation increased2; PMS1:postmeiotic segregation increased1; XPA:xeroderma pigmentosum, complementation group A APEXI:apurinic/apyrimidinic; endonuclease1; RPA1: replication protein Al, select the placenta as the target tissue, study on the mechanism of occurrence of instability mutation of ART progeny dynamic.
(1) analysis of gene expression related to DNA injury and repair in ART subplacental placenta
The expression changes of DNA damage repair related genes OGG1, MSH6, MSH2, MSH3, MLH1, MLH3, PMS2, PMS1, MLH3, and ART were detected by fluorescent quantitative RT-PCR.
(2) analysis of gene modification related to DNA injury and repair in ART subplacental placenta
The methylation status of the five gene promoter regions of OGG1, RPA1, PMS2, MSH6 and XPA was detected by pyrosequencing.
(3) analysis of gene protein expression related to DNA injury and repair in ART subplacental placenta
The expression of MLH1, OGG1, RPA1, PMS2, MSH2, MSH6 and XPA proteins in the placental tissue was detected by Western-blot.
Result:
(1): IVF and mRNA levels in group ICSI, PMS2, RPA1, XPA, MSH2, MSH6 gene has high expression compared with the control group, the difference was statistically significant.IVF in the MLH1 group compared with control group and ICSI group expressed significantly; in addition, compared with the IVF group, ICSI group, MSH2. XPA, OGG1 and RPA1 gene has high expression significantly, the difference was statistically significant.
(2) DNA level: the methylation sites of CpG island of OGG1, RPA1, PMS2, MSH6 and XPA in the IVF and ICSI groups are different from those in the body group, and the methylation rate is statistically significant, which is consistent with the change of gene mRNA level.
(3) protein level: the expression level of PMS2, MSH6 and MLH1 protein in ICSI group was significantly higher than that in the IVF group and the control group, and the difference was statistically significant.
Conclusion:
1.ART operation and infertility background may be related to the abnormal expression level of DNA damage repair related genes in the placenta tissue.
The 2.ART operation and the infertility background may affect the DNA methylation of DNA damage repair related genes.
The mRNA level of 3.DNA damage repair related genes is not consistent with the protein level, which may be related to post transcriptional regulation.
The effect of the third part ART on the oxidative damage and degenerative changes of DNA in the central nervous system of the aged mice
Objective:
Objective to study the oxidative damage, DNA damage repair genes and neurodegenerative diseases related genes in ART born mice in the elderly, and to evaluate the effect of ART on the oxidative damage and degenerative changes of DNA in the central nervous system of the aging mice.
Materials and methods:
Including the ART old C57BL/6J mouse model application has been built: 8 IVF mice and 8 ICSI mice, 8 mice (male and female mice in vivo fertilization rate, similar) 8-OHdG as markers of oxidative DNA damage, DNA damage repair genes Msh2, Apex1, Msh6, Ogg1, Pcna, Xpa and Mlh1; nerve degeneration disease related Mapt, Bacel, Psen1, Psen2, App, Apoe and microRNA and Insulin/IGF-1 pathway genes as target gene. Selection of brain tissue as the target tissue of ART on the impact of birth old age mouse central nervous system DNA oxidative damage and degenerative changes in risk.
(1) analysis of 8-OHdG level in ART aged mice
The level of 8-OHdG in brain tissue of ART aged mice was detected by ELISA.
(2) analysis of gene expression related to DNA injury and repair in ART
The changes in the expression of Msh2, Apex1, Msh6, Oggl, Pcna, Xpa and Mlhl in the brain tissues of ART aged mice were detected by fluorescence quantitative RT-PCR.
(3) analysis of gene expression related to senile neurodegenerative diseases in ART
The expression changes of Mapt, Bacel, Psen1, Psen2, App, Apoe, Igf-1, Igf-1r, Insr, Igf-1, Igf-1r, RT-PCR, ART in brain tissue of aged mice were detected by fluorescent quantitative RT-PCR.
(4) analysis of DNA damage repair gene modification in ART aged mice
The methylation status of CpG island in the brain tissue related damage repair gene of ART aged mice was detected by pyrosequencing.
Result:
(1) 8-OHdG level: 8-OHdG in the control group was 4.26 + 1.54pg/ml, IVF group was 9.59 + 4.27pg/ml, ICSI group was 8.57 + 3.90pg/ml, the difference was statistically significant compared with the in vivo group, but there was no significant difference between IVF and ICSI group.
(2) the level of DNA damage repair related gene mRNA: compared with group IVF and group ICSI in vivo, Ogg1, Apex1 and Pcna gene expression was significantly up-regulated in IVF and ICSI group; Msh6, low expression of Msh2 and Mlhl gene significantly, the differences were statistically significant.ICSI compared with IVF group, Pcna gene expression increased significantly.
(3) gene related neurodegenerative diseases, mRNA and rmiRNA levels: compared with the in vivo group, ICSI Psen1 group, the expression level of Igf-1 increased, Igf-1r, Insr, Irs-1, the expression level of miR-34 and miR-101 decreased, the difference was statistically significant; increasing the expression level of IVF Igf-1 gene Igf-1r, Insr, and Irs-1 levels miR-34 decreased, the difference was statistically significant.IVF compared with ICSI group, the expression of Insr and miR-101 in ICSI group was significantly decreased, the difference was statistically significant.
(4) DNA damage repair related gene DNA level: Oggl and Mlhl CpG island methylation sites in IVF and ICSI groups were different from the body group methylation rate, and had statistical significance.
Conclusion: there may be a high risk of oxidative damage in the brain tissue of 1.IVF/ICSI born mice in the old age. There may be a higher risk of central nervous system degenerative changes in.2.IVF/ICSI born mice in the old age, but the mechanism needs further improvement.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R714.8

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