孕早期高雌激素暴露出生低体重/小于胎龄儿的表遗传调节机制研究
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摘要
第一部分低出生体重/小于胎龄儿与妊娠早期血清雌激素水平
目的：评估多胎妊娠早期减为单胎或双胎妊娠后的出生体重情况,并且比较分析早期多胎妊娠与单胎妊娠母亲外周血雌激素水平及妊娠早期母亲外周血雌激素水平与新生儿出生体重的关系。
材料和方法：对2006年1月至2010年7月在浙江大学医学院附属妇产科医院进行辅助生殖技术(Assisted reproductive technology, ART)治疗的病例进行回顾性分析,以自然或人为减为单胎妊娠或双胎妊娠的减胎组为实验组,以同期相应的未进行减胎的原始双胎或单胎作为对照组,比较分析实验组单胎和双胎与各自相应对照组间新生儿出生体重和小于胎龄儿发生率之间的差异。同时收集195例ART后妊娠7-8周时母亲的外周血(包括单胎、双胎、三胎和四胎妊娠),利用化学发光法对外周血血清雌二醇水平进行检测,比较各组间雌二醇水平,分析妊娠早期母亲外周血雌激素水平与减胎术后出生子代体重的相关性。
结果：1.多胎妊娠无论是减为单胎妊娠还是双胎妊娠,减胎组新生儿平均出生体重均低于相应的未进行减胎的原始单胎妊娠和双胎妊娠组,单胎组分别为3217.05±546.88g和3377.85±477.05g,P<0.01；双胎组分别为2428.80±485.64g和2495.31±520.06g,P<0.05。小于胎龄儿发生率前者均显著高于后者,单胎组分别为2.9%(6/210)和11.4%(12/105),P<0.01；双胎组分别为27.3%(472/1728)和38.5%(167/434),P<0.01,早产的发生率均无显著性差异(P>0.05)。
2.多胎妊娠组母亲外周血雌二醇水平显著高于单胎妊娠组,并且随着妊娠胎数的增加,外周血雌二醇水平显著升高(P<0.01)。
3.妊娠早期母亲外周血雌二醇水平与多胎妊娠减胎术后胎儿出生体重存在负性相关性(r=-0.32,P=0.018)。
结论：处于高雌激素环境的早期多胎妊娠能增加子代低体重和小于胎龄儿发生率,并且这种影响不能利用早期减胎术进行消除,而妊娠早期母亲外周血高雌激素水平可能在低出生体重和小于胎龄儿的发生中起着重要作用。
第二部分早期妊娠胎儿组织及子代出生后脐血和胎盘组织中印记基因的表达及调控机制
目的：评估多胎妊娠早期胎儿组织及子代出生后脐血和胎盘组织表观遗传学状态。
材料和方法：对多胎妊娠行早期减胎术后获得的胎儿组织与同期同孕周自然单胎妊娠行人工流产获得的胎儿组织,以及减胎术后出生的双胎脐血及胎盘组织与相应的未进行减胎的双胎脐血及胎盘组织进行对比研究,利用荧光定量PCR对印记基因IGF2、H19、PHLDA2和CDKN1C,及甲基化转移酶DNMT1的表达水平进行检测、利用甲基化特异性PCR及亚硫氢酸盐测序的方法对上述印记基因相应的差异甲基化区域H19DMR和KvDMRl的甲基化水平进行检测。
结果：CDKN1C和DNMT1不仅在多胎妊娠早期的胎儿组织中高表达,而且同时在减胎术后出生的双胎脐血和胎盘组织中的表达均显著上升(P<0.05),同时,CDKN1C的甲基化调控区域KvDMR1的甲基化水平均显著上升(P<0.05)。IGF2在多胎妊娠早期的胎儿组织中表达上升,但其甲基化调控区H19DMR的甲基化水平未发生显著改变,H19和PHLDA2的表达在在多胎妊娠早期的胎儿组织中表达未发生显著变化(P>0.05)。
结论：妊娠早期高水平的雌激素可能通过上调胎儿组织中DNA甲基化转移酶DNMT1的表达使KvDMR1的甲基化升高,使生长抑制性印记基因CDKNIC的表达上调,从而对胎儿的生长发育产生持续性影响,导致低出生体重和小于胎龄儿的发生,这可能是早期高雌激素水平使低出生体重和小于胎龄儿的发生率增加的重要调控机制之一。
第三部分雌激素对CDKN1C表达的表遗传调控机制
目的：探讨雌激素是否直接参与了CDKNIC的表达及其表遗传调控机制。
材料和方法：1.体外实验以人类滋养细胞系HTR-8为研究对象,通过不同浓度的雌激素和(或)同时加入雌激素受体阻断剂、DNMT1小干扰RNA处理细胞,采用荧光定量PCR技术检测细胞中CDKN1C和DNMT1的表达。
2.通过高浓度的雌激素处理HTR-8细胞,采用甲基化特异性PCR技术及亚硫氢酸盐测序技术检测高浓度雌激素刺激后细胞KvDMR1区域甲基化水平。
3. DNMT1promotor-pGL-3重组质粒构建,质粒转染到HTR-8细胞,培养后裂解细胞进行荧光素酶活性检测。
4.利用染色质免疫共沉淀技术,检测DNMT1启动子区域雌激素反应元件与雌激素受体的结合。
结果：1.雌激素能够明显上调DNMT1和CDKNIC的表达,使KvDMR1区甲基化水平上调；雌激素受体阻断剂ICI182780及DNMT1小干扰RNA能够阻断E2对CDKN1C的上调作用。
2. DNMT1转录起始位点-659/-647bp处存在雌激素反应元件结构,在HTR-8细胞中,E2可能通过该雌激素反应元件调节DNMT1的表达,从而对CDKNIC的表达起到间接调控作用。
结论
雌激素通过作用于DNMT1转录起始位点-659/-647bp处的雌激素反应元件使KvDMR1的甲基化升高,从而使生长抑制性印记基因CDKNIC的表达上调,这可能是孕早期高水平的雌激素对胎儿的生长发育产生持续性影响,导致低出生体重和小于胎龄儿的发生增加的重要调控机制之一。
Part I:Low birth weight/small for gestational age and the maternal serum estradiol levels in first trimester pregnancies
Objective:To evaluate the birth weights of multifetal pregnancies reduced to singletons or twins, compare the maternal serum estradiol (E2) levels of early singleton pregnancies with those of multiple pregnancies and analyze the correlation between maternal serum E2levels and the birth weights of their offsprings.
Materials and Methods:A retrospective review was performed on data obtained from patients between January2006and June2010undergoing assisted reproductive technology (ART) at the Assisted Reproductive Unit of the Women's Hospital, School of Medicine, Zhejiang University. Spontaneous or selective reduction of one or more gestational sacs and/or embryos occurred to singletons or twins before the12th week of gestation were included. The control group included assisted reproduction patients with non-reduced singletons or twins performed over the same period. Non-reduced singleton was matched by a stratified random selection. Compare the perinatal outcomes of reduced groups with that of their own controls. Peripheral blood samples of195women with ART conceptions on7-8weeks' gestation were collected and measured serum E2levels with Roche cobas immunoassay.
Results:1.The birth weights of singletons and twins after reduction were significantly lower than non-reduced pregnancies (3217.05±546.88g vs3377.85±477.05g, P=0.009and2428.80±485.64g vs2495.31±520.06g, p=0.016, respectively). The rates of small for gestational age (SGA) after reduction were significantly higher than their own control (P<0.05). No significant difference was observed in duration of gestation (P>0.05).
2. The serum E2levels for multiple pregnancies were significantly higher than those for singleton pregnancies (P<0.05), the serum E2levels elevated with the increased number of fetuses.
3. Multivariate correlation analyses showed that maternal serum E2level shows a inverse correlation with the birth weight of offspring (r=-0.32, P=0.018).
Conclusion:Early multiple pregnancies with high maternal serum E2level can lower the birth weight and increase the risk of SGA, and the adverse effect could not be eliminatec by early multifetal reduction. The high maternal serum E2levels in first trimeste pregnancies may play an important role in low birth weight and SGA.
Part II:Expression and methylation status of imprinting genes in fetal tissues, umbilical cord blood and placenta tissues
Objective:To investigate epigenetic status of early multiple pregnancies.
Materials and Methods:Fetal tissues of multiple pregnancies were collected from multifetal pregnancy reduction (MFPR), and fetal tissues of singleton pregnancies were collected from elective terminations of normal pregnancies as control group. Umbilical cord blood (UCB) and placenta tissues from reduced twins and primary twin pairs were collected. The expression of IGF2, H19, CDKN1C, PHLDA2and DNMT1was determined by real-time quantitative PCR. The methylation status of H19DMR and KvDMR1was determined by methlylation specific PCR (MSP) and bisulfite sequencing PCR (BSP).
Results:CDKN1C and DNMT1mRNA levels were significantly higher in early fetal tissues of multiple pregnancies than singleton pregnancies. Consistent with the results in fetal tissues, the expression levels of CDKN1C and DNMT1in UCB and placenta tissues of reduced twin pairs were significantly increased when compared to their matched twins. KvDMRl was hypermethylated in fetal tissues of MFPR, UCB and placenta tissues of reduced twins. Significant upregulation level of IGF2was also observed in fetal tissues of multiple pregnancies, but the methylated level of H19DMR was normal. The expression levels of H19and PHLDA2in fetal tissues of multiple pregnancies were unchanged when compared with their controls.
Conclusion:Estradiol may induce DNMT1overexpression and thus, promote hypermethylation of KvDMRl and overexpression of CDKN1C, which is a critical mechanism for low birth weight and SGA of early multiple pregnancies.
Part Ⅲ:The mechanism of estradiol regulating the expression of CDKN1C
Objective:To investigate the mechanism of estradiol (E2) regulating the expression of CDKN1C.
Materials and Methods:HTR8/SVneo (HTR8) cells were exposed to E2and/or estrogen receptors antagonist (ICI182780), DNMT1small interfering RNAs (siRNAs). The expression of CDKN1C and DNMT1was determined by real-time quantitative PCR. The methylation status of KvDMR1in10-5M E2treated HTR8cells was determined by methlylation specific PCR (MSP) and bisulfite sequencing PCR (BSP). DNMT1promotor-pGL-3plasmid was constructed and luciferase activity was measured using the dual luciferase reporter assay system. Chromatin immunoprecipitation (ChIP) experiments were performed to detect the binding of ERa on the ERE-like site of DNMT1promoter.
Results:E2increased the expression of CDKN1C, DNMT1and the methylation level of KvDMR1in HTR8cells. The effects were inhibited by ERs antagonists and DNMT1siRNA. ERa activated DNMT1transcription through an estrogen responsive elements (ERE) located at-659/-647bp upstream of the transcriptional start site.
Conclusion:E2can promote DNMT1transcription through an ERE located at the transcriptional start site. High expression of DNMT1is associated with the hypermethylation of KvDMRl and the upregulation of CDKN1C. This is may be one of the important reasons for the increase of low birth weight and SGA.
目的：评估多胎妊娠早期减为单胎或双胎妊娠后的出生体重情况,并且比较分析早期多胎妊娠与单胎妊娠母亲外周血雌激素水平及妊娠早期母亲外周血雌激素水平与新生儿出生体重的关系。
材料和方法：对2006年1月至2010年7月在浙江大学医学院附属妇产科医院进行辅助生殖技术(Assisted reproductive technology, ART)治疗的病例进行回顾性分析,以自然或人为减为单胎妊娠或双胎妊娠的减胎组为实验组,以同期相应的未进行减胎的原始双胎或单胎作为对照组,比较分析实验组单胎和双胎与各自相应对照组间新生儿出生体重和小于胎龄儿发生率之间的差异。同时收集195例ART后妊娠7-8周时母亲的外周血(包括单胎、双胎、三胎和四胎妊娠),利用化学发光法对外周血血清雌二醇水平进行检测,比较各组间雌二醇水平,分析妊娠早期母亲外周血雌激素水平与减胎术后出生子代体重的相关性。
结果：1.多胎妊娠无论是减为单胎妊娠还是双胎妊娠,减胎组新生儿平均出生体重均低于相应的未进行减胎的原始单胎妊娠和双胎妊娠组,单胎组分别为3217.05±546.88g和3377.85±477.05g,P<0.01；双胎组分别为2428.80±485.64g和2495.31±520.06g,P<0.05。小于胎龄儿发生率前者均显著高于后者,单胎组分别为2.9%(6/210)和11.4%(12/105),P<0.01；双胎组分别为27.3%(472/1728)和38.5%(167/434),P<0.01,早产的发生率均无显著性差异(P>0.05)。
2.多胎妊娠组母亲外周血雌二醇水平显著高于单胎妊娠组,并且随着妊娠胎数的增加,外周血雌二醇水平显著升高(P<0.01)。
3.妊娠早期母亲外周血雌二醇水平与多胎妊娠减胎术后胎儿出生体重存在负性相关性(r=-0.32,P=0.018)。
结论：处于高雌激素环境的早期多胎妊娠能增加子代低体重和小于胎龄儿发生率,并且这种影响不能利用早期减胎术进行消除,而妊娠早期母亲外周血高雌激素水平可能在低出生体重和小于胎龄儿的发生中起着重要作用。
第二部分早期妊娠胎儿组织及子代出生后脐血和胎盘组织中印记基因的表达及调控机制
目的：评估多胎妊娠早期胎儿组织及子代出生后脐血和胎盘组织表观遗传学状态。
材料和方法：对多胎妊娠行早期减胎术后获得的胎儿组织与同期同孕周自然单胎妊娠行人工流产获得的胎儿组织,以及减胎术后出生的双胎脐血及胎盘组织与相应的未进行减胎的双胎脐血及胎盘组织进行对比研究,利用荧光定量PCR对印记基因IGF2、H19、PHLDA2和CDKN1C,及甲基化转移酶DNMT1的表达水平进行检测、利用甲基化特异性PCR及亚硫氢酸盐测序的方法对上述印记基因相应的差异甲基化区域H19DMR和KvDMRl的甲基化水平进行检测。
结果：CDKN1C和DNMT1不仅在多胎妊娠早期的胎儿组织中高表达,而且同时在减胎术后出生的双胎脐血和胎盘组织中的表达均显著上升(P<0.05),同时,CDKN1C的甲基化调控区域KvDMR1的甲基化水平均显著上升(P<0.05)。IGF2在多胎妊娠早期的胎儿组织中表达上升,但其甲基化调控区H19DMR的甲基化水平未发生显著改变,H19和PHLDA2的表达在在多胎妊娠早期的胎儿组织中表达未发生显著变化(P>0.05)。
结论：妊娠早期高水平的雌激素可能通过上调胎儿组织中DNA甲基化转移酶DNMT1的表达使KvDMR1的甲基化升高,使生长抑制性印记基因CDKNIC的表达上调,从而对胎儿的生长发育产生持续性影响,导致低出生体重和小于胎龄儿的发生,这可能是早期高雌激素水平使低出生体重和小于胎龄儿的发生率增加的重要调控机制之一。
第三部分雌激素对CDKN1C表达的表遗传调控机制
目的：探讨雌激素是否直接参与了CDKNIC的表达及其表遗传调控机制。
材料和方法：1.体外实验以人类滋养细胞系HTR-8为研究对象,通过不同浓度的雌激素和(或)同时加入雌激素受体阻断剂、DNMT1小干扰RNA处理细胞,采用荧光定量PCR技术检测细胞中CDKN1C和DNMT1的表达。
2.通过高浓度的雌激素处理HTR-8细胞,采用甲基化特异性PCR技术及亚硫氢酸盐测序技术检测高浓度雌激素刺激后细胞KvDMR1区域甲基化水平。
3. DNMT1promotor-pGL-3重组质粒构建,质粒转染到HTR-8细胞,培养后裂解细胞进行荧光素酶活性检测。
4.利用染色质免疫共沉淀技术,检测DNMT1启动子区域雌激素反应元件与雌激素受体的结合。
结果：1.雌激素能够明显上调DNMT1和CDKNIC的表达,使KvDMR1区甲基化水平上调；雌激素受体阻断剂ICI182780及DNMT1小干扰RNA能够阻断E2对CDKN1C的上调作用。
2. DNMT1转录起始位点-659/-647bp处存在雌激素反应元件结构,在HTR-8细胞中,E2可能通过该雌激素反应元件调节DNMT1的表达,从而对CDKNIC的表达起到间接调控作用。
结论
雌激素通过作用于DNMT1转录起始位点-659/-647bp处的雌激素反应元件使KvDMR1的甲基化升高,从而使生长抑制性印记基因CDKNIC的表达上调,这可能是孕早期高水平的雌激素对胎儿的生长发育产生持续性影响,导致低出生体重和小于胎龄儿的发生增加的重要调控机制之一。
Part I:Low birth weight/small for gestational age and the maternal serum estradiol levels in first trimester pregnancies
Objective:To evaluate the birth weights of multifetal pregnancies reduced to singletons or twins, compare the maternal serum estradiol (E2) levels of early singleton pregnancies with those of multiple pregnancies and analyze the correlation between maternal serum E2levels and the birth weights of their offsprings.
Materials and Methods:A retrospective review was performed on data obtained from patients between January2006and June2010undergoing assisted reproductive technology (ART) at the Assisted Reproductive Unit of the Women's Hospital, School of Medicine, Zhejiang University. Spontaneous or selective reduction of one or more gestational sacs and/or embryos occurred to singletons or twins before the12th week of gestation were included. The control group included assisted reproduction patients with non-reduced singletons or twins performed over the same period. Non-reduced singleton was matched by a stratified random selection. Compare the perinatal outcomes of reduced groups with that of their own controls. Peripheral blood samples of195women with ART conceptions on7-8weeks' gestation were collected and measured serum E2levels with Roche cobas immunoassay.
Results:1.The birth weights of singletons and twins after reduction were significantly lower than non-reduced pregnancies (3217.05±546.88g vs3377.85±477.05g, P=0.009and2428.80±485.64g vs2495.31±520.06g, p=0.016, respectively). The rates of small for gestational age (SGA) after reduction were significantly higher than their own control (P<0.05). No significant difference was observed in duration of gestation (P>0.05).
2. The serum E2levels for multiple pregnancies were significantly higher than those for singleton pregnancies (P<0.05), the serum E2levels elevated with the increased number of fetuses.
3. Multivariate correlation analyses showed that maternal serum E2level shows a inverse correlation with the birth weight of offspring (r=-0.32, P=0.018).
Conclusion:Early multiple pregnancies with high maternal serum E2level can lower the birth weight and increase the risk of SGA, and the adverse effect could not be eliminatec by early multifetal reduction. The high maternal serum E2levels in first trimeste pregnancies may play an important role in low birth weight and SGA.
Part II:Expression and methylation status of imprinting genes in fetal tissues, umbilical cord blood and placenta tissues
Objective:To investigate epigenetic status of early multiple pregnancies.
Materials and Methods:Fetal tissues of multiple pregnancies were collected from multifetal pregnancy reduction (MFPR), and fetal tissues of singleton pregnancies were collected from elective terminations of normal pregnancies as control group. Umbilical cord blood (UCB) and placenta tissues from reduced twins and primary twin pairs were collected. The expression of IGF2, H19, CDKN1C, PHLDA2and DNMT1was determined by real-time quantitative PCR. The methylation status of H19DMR and KvDMR1was determined by methlylation specific PCR (MSP) and bisulfite sequencing PCR (BSP).
Results:CDKN1C and DNMT1mRNA levels were significantly higher in early fetal tissues of multiple pregnancies than singleton pregnancies. Consistent with the results in fetal tissues, the expression levels of CDKN1C and DNMT1in UCB and placenta tissues of reduced twin pairs were significantly increased when compared to their matched twins. KvDMRl was hypermethylated in fetal tissues of MFPR, UCB and placenta tissues of reduced twins. Significant upregulation level of IGF2was also observed in fetal tissues of multiple pregnancies, but the methylated level of H19DMR was normal. The expression levels of H19and PHLDA2in fetal tissues of multiple pregnancies were unchanged when compared with their controls.
Conclusion:Estradiol may induce DNMT1overexpression and thus, promote hypermethylation of KvDMRl and overexpression of CDKN1C, which is a critical mechanism for low birth weight and SGA of early multiple pregnancies.
Part Ⅲ:The mechanism of estradiol regulating the expression of CDKN1C
Objective:To investigate the mechanism of estradiol (E2) regulating the expression of CDKN1C.
Materials and Methods:HTR8/SVneo (HTR8) cells were exposed to E2and/or estrogen receptors antagonist (ICI182780), DNMT1small interfering RNAs (siRNAs). The expression of CDKN1C and DNMT1was determined by real-time quantitative PCR. The methylation status of KvDMR1in10-5M E2treated HTR8cells was determined by methlylation specific PCR (MSP) and bisulfite sequencing PCR (BSP). DNMT1promotor-pGL-3plasmid was constructed and luciferase activity was measured using the dual luciferase reporter assay system. Chromatin immunoprecipitation (ChIP) experiments were performed to detect the binding of ERa on the ERE-like site of DNMT1promoter.
Results:E2increased the expression of CDKN1C, DNMT1and the methylation level of KvDMR1in HTR8cells. The effects were inhibited by ERs antagonists and DNMT1siRNA. ERa activated DNMT1transcription through an estrogen responsive elements (ERE) located at-659/-647bp upstream of the transcriptional start site.
Conclusion:E2can promote DNMT1transcription through an ERE located at the transcriptional start site. High expression of DNMT1is associated with the hypermethylation of KvDMRl and the upregulation of CDKN1C. This is may be one of the important reasons for the increase of low birth weight and SGA.
引文
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