【作者】 章瑜；

【导师】 黄荷凤； Peter Leung；

【作者基本信息】 浙江大学 ， 妇产科， 2010， 博士

【摘要】 辅助生殖技术(Assisted reproductive technology, ART)应用于临床30余年,在对各种不育症治疗作出巨大贡献的同时,也促使了人们对其操作安全性和子代健康的关注。近年来,有研究不断指出,ART可能会增加子代不良健康风险,包括出生缺陷、出生体重低下、染色体异常、肿瘤及由基因表观遗传修饰异常导致的罕见遗传病发病等。由于ART各项技术的直接临床应用均无实验室的基础研究背景,所以,ART增加子代不良健康风险的发生机制并不清楚。ART所涉及的各个环节,包括父母的不孕背景和大量非生理性干预,如超促排卵、配子和胚胎体外培养、胚胎移植、胚胎显微操作等步骤均有可能改变生命早期的发育,从而影响子代甚至再下一代的健康。面对目前人们对ART子代健康的担忧以及未来可能潜藏的更多危险,对ART的出生缺陷机制进行检测和评估已经刻不容缓。目前,多个国家的生殖协会或ART中心都开始对ART子代进行大型的流行病学调查。但是,大量关于ART安全性的评估,多集中于对新生儿结局的描述,而对妊娠并发症的描述较少,且结果不确定。关于ART新生儿不良结局发生的原因分析更少。相关国内资料尤其缺乏。妊娠并发症是妊娠期或分娩过程中出现的病理情况,一方面是异常妊娠过程的临床表现,另一方面,也是影响母儿健康的关键因素。因此,进一步对ART妊娠相关并发症的发生情况和妊娠结局的关系进行分析,有助于ART子代的全面安全性评估,从而更好的分析ART子代不良健康风险增加的可能原因和机制。胎盘是孕期连接胎儿和母体的重要器官,在妊娠过程中起物质交换和内分泌作用维持胎儿生长并保护胎儿免受外界损伤。胎盘微血管的生成及其网络构建是母儿物质交换的保证,对于正常妊娠的维持有着十分重要的意义。动物研究发现胚胎经体外培养后常伴有妊娠过程中的胎盘发育异常,人类ART子代也伴随胎盘发育异常几率增加,但发生机制不清。因此,对ART子代胎盘功能进行全面的检测,并对相关分子机制进行研究,有助于进一步评价ART操作对于胎盘发育和胎儿发育的影响,并探讨其因果关系。本研究以辅助生殖技术子代为研究对象,首先对ART妊娠并发症和妊娠结局进行临床分析；以临床发现ART妊娠和分娩并发症及新生儿不良结局发生率较自然妊娠显著增加为基础,应用高通量蛋白质组学研究技术,对ART子代胎盘差异蛋白表达谱进行全面检测和分析；以实验发现ART胎盘存在差异表达蛋白为基础,选取重点差异蛋白,分析目的蛋白在ART出生体重低下子代胎盘中的表达及相关病理机制,探讨ART胎盘发育与子代低出生体重发生的关系；总体评价ART技术安全性,分析ART不良妊娠结局发生的胎盘分子机制,并为临床改进ART操作、改善ART结局提供理论依据。第一部分辅助生殖技术晚期妊娠并发症和妊娠结局临床分析目的：评价ART获得妊娠与自然获得妊娠相比,是否存在妊娠晚期并发症和分娩并发症发生率及新生儿结局的差异；分析新生儿不良结局和ART妊娠并发症发生的关系。材料与方法：回顾性分析2003-2009年在我院分娩的ART妊娠(包括常规IVF和ICSI)1013例,以1：3配对、年龄匹配方式随机抽取同期在我院分娩的自然妊娠3069例作为对照组,比较两组之间多胎发生率、妊娠和分娩并发症发生率及新生儿结局是否存在差异。再将两组分为单胎妊娠和多胎妊娠后,分别统计以上指标是否存在差异。并进一步分析ART新生儿低出生体重和早产与妊娠并发症发生的关系。结果：ART组多胎发生率为37.8%,为自然妊娠组的20.9倍。ART组妊娠并发症及分娩并发症[包括妊娠期高血压、子痫前期、妊娠期糖尿病(GDM)、妊娠期肝内胆汁淤积症等(ICP)、前置胎盘、胎盘早剥、足月前胎膜早破(PPROM)、胎盘粘连、产后出血等]和新生儿早产、低出生体重、足月小样儿、新生儿窒息等发生率均显著高于自然妊娠组；相反,巨大儿发生率显著低于自然妊娠组。ART单胎妊娠和自然单胎妊娠相比,子痫前期、GDM、前置胎盘、PPROM、脐带附着异常、胎盘粘连、产后出血及新生儿早产、低出生体重、新生儿窒息等发生率仍显著增加。ART多胎妊娠和自然多胎妊娠相比,各妊娠并发症和分娩并发症发生率无明显差异,新生儿足月小样儿和双胎发育不均衡发生率显著低于对照组。ART新生儿低出生体重主要由早产引起,而在ART早产发生原因中,合并妊娠并发症者占73.2%,其中合并PPROM、ICP、妊娠期高血压疾病和前置胎盘的比率最高。结论：ART与妊娠并发症、分娩并发症发生率升高和新生儿不良结局相关,其原因与医源性的多胎妊娠密切相关。排除多胎因素后,ART单胎妊娠仍存在子痫前期、GDM等并发症发生率增高和新生儿结局不良,提示ART本身也与病理妊娠相关。ART新生儿低出生体重的发生主要由早产引起,并和妊娠并发症,特别是胎盘发育异常疾病的发生密切相关。因此,提倡单胚胎移植,严格控制ART适应症,改进临床ART操作,尽量减少非生理性干预,积极预防和治疗妊娠并发症有助于改善临床ART安全性。第二部分辅助生殖技术子代胎盘比较蛋白质组学研究目的：研究ART子代胎盘与自然妊娠子代胎盘相比总蛋白表达谱的改变,探讨ART增加子代不良健康风险的胎盘分子机制。材料与方法：6例常规IVF-ET妊娠,6例ICSI妊娠和6例同期自然妊娠胎盘提取细胞总蛋白,二维凝胶电泳分离蛋白组成分,银染染色后应用PDQuest软件分析数字化的凝胶图像,找出ART(包括常规IVF-ET和ICSI)胎盘组织表达有差异的蛋白质斑点；采用胶内酶解消化方式得到差异蛋白斑点多肽混合物,应用基质辅助激光解析串联飞行时间质谱(MALDI TOF-TOF)进行蛋白质鉴定；所有差异蛋白进行生物信息学检索、归类和功能分析；选取获得成功鉴定的代表性差异蛋白进行Western blotting验证。结果：1、通过分析二维电泳凝胶图谱,各胎盘组织检测到约1200个蛋白质斑点。常规IVF组和对照组比较,12个蛋白质点表达具有显著差异,其中7个蛋白质点表达上调,5个蛋白质点表达下调。ICSI组和对照组比较,18个蛋白质点表达具有明显差异,其中10个蛋白质点表达上调,8个蛋白质点表达下调。其中6个蛋白质点在常规IVF组和ICSI组中均具有表达改变,且变化趋势一致。2、通过胶内酶解消化、MALDI TOF-TOF分析得到差异表达蛋白斑点的肽质指纹图谱,通过MASCOT搜索引擎在NCBI蛋白质数据库中检索,其中20个蛋白斑点成功获得鉴定,其中5个蛋白Annexin A3, a-SNAP, hnRNP C1/C2, FTL和ATP5A在常规IVF组和ICSI组中均有表达差异。3、应用生物信息学方法对蛋白进行归类和功能分析,差异表达蛋白功能涉及胞膜转运、代谢、应激反应、细胞骨架和核酸代谢等多个方面。4、应用Western blotting方法对代表性蛋白Annexin A3, a-SNAP, hnRNP C1/C2和FTL进行验证,结果与二维电泳一致。结论：ART妊娠子代胎盘组织中存在与胞膜转运、代谢、应激反应、细胞骨架和核酸代谢相关的蛋白质表达变化,提示ART操作可能导致胎盘发育的异常,而机体也存在相应的保护性措施和代偿机制进行纠正。该结果初步筛选了ART相关胎盘差异表达蛋白,为深入探讨ART妊娠过程中的胎盘异常发育机制,进一步明确ART与不良妊娠结局和子代健康之间的关系提供了可靠的资料。第三部分Annexin A3和a-SNAP在ART低出生体重子代胎盘中的表达及意义目的：检测ART低出生体重子代胎盘中annexin A3和a-SNAP的表达水平,分析其与胎盘微血管生成的关系,并探讨annexin A3和a-SNAP表达在ART子代低出生体重发生中的胎盘分子病理机制。材料和方法：以12例ART妊娠合并低出生体重(LBW),12例ART妊娠正常出生体重(NBW)和12例同期自然妊娠子代胎盘为研究对象,采用Western blotting方法检测ART/LBW组、ART/NBW组和对照组中annexin A3和α-SNAP表达量的差异。采用免疫组织化学方法进一步检测annexin A3和a-SNAP在ART/LBW组、ART/NBW组和对照组胎盘组织中的定位和表达差异,用CD34标记代表微血管密度(MVD)方法比较三组间胎盘绒毛微血管分布及密度,并分析胎盘annexin A3和a-SNAP表达与MVD的相关性。结果：Western blotting结果显示,与对照组相比,ART/LBW组胎盘中annexinA3和a-SNAP的表达显著下降,ART/NBW组annexin A3和a-SNAP的表达显著上调。免疫组织化学结果显示annexin A3和a-SNAP在胎盘绒毛滋养细胞,血管内皮细胞及绒毛间质细胞均有表达,表达水平与western blotting结果一致。ART/LBW组胎盘组织中MVD较对照组显著下降,ART/NBW组MVD较对照组无明显改变。在对照组和ART/LBW组中,胎盘annexin A3的表达、α-SNAP的表达均与MVD成显著正相关。结论：Annexin A3和a-SNAP表达水平的显著下降在ART子代低出生体重发生机制中发挥重要的作用,可能与胎盘微血管生成障碍相关。更多还原

【Abstract】 Assisted reproductive technology (ART) has helped thousands of couples conceive children in the past 30 years. Early studies suggested ART might be safe and children born from ART developped normally. However, in recent years, more and more well-designed studies have consistently documented associations of ART with an increased risk of birth defects, low birth weight, chromosome abnormalities, childhood cancer and epigenetic defects. As IVF was introduced into practice without formal evaluation of its effects on the health of children conceived with this procedure, there is little information about the mechanisms underlying the increased health defects. It is also unclear whether the infertility background of the parents or which stage(s) from the controlled ovarian hyperstimulation, gametes and embryo manipulation, in vitro culture to embryo transfer to the baby’s birth may raise the risk of health defects. Although the precise significance and origin of ART and offspring safety still require confirmation and clarification, available evidence has suggested the concerns for the long-term safety of ART procedures.In rencent years, more and more epidemiological investigations have been carried out by the reproductive associations or ART centres for the ART offsprings. However, most researches focused on the overall pregnancy outcomes such as birth weight and number of multiple gestations when evaluating the safety of ART. Limited data are available to describe patterns of adverse obstetric and delivery complications and their relationship with prenatal outcome, both in China and abroad. Pregnancy complications are the diseases raised during the pregnancy or delivery process. They are not only the clinical manifestations of abnormal pregnancy, but also the main reasons of adverse outcome of mother and newborn. Therefore, assessment of the influence of ART on the pregnancy complications and newborn outcome will benefit the knowledge of existed information on safety evaluation of ART offspring, in order to well analyze the mechanisms of adverse health of ART offspring.The placenta is a temporary organ that consists of a fetal component and a maternal component and it is a structure unique to pregnancy functioning to sustain and protect the fetus until birth. Placenta vascular formation is important for fetal growth and development. Proper development of placenta ensures the exchange of oxygen/nutrients and blood flow necessary for fetal growth. Abnormal placenta vasculogenesis has been consistently associated with different pregnancy complications and worse perinatal outcome. Abnormal placentation has been observed following in vitro embryo production in animals. It was also reported that human placenta derived from ART were associated with more frequent pathological findings. These results of abnormal placentation in assisted reproduction related procedures in animals and human being reveal partial evidence for the possibility of worse outcome of ART offspring. To overall evaluate the specific gene products in placenta following ART, global screening of protein expression in placenta is needed.In this study, we first analysis the pregnancy complications and petinatal outcomes associated with ART. Then comparative proteomic analysis of ART placenta was carried out using two-dimensional electrophorosis and mass spectrometry. At last, role of two differentially expressed proteins, annexin A3 and a-SNAP, which were detected by proteomics as differentially expressed in ART placenta, was further studied in ART placenta of newborn with low birth weight. PartⅠLate pregnancy complications and perinatal outcomes analyses of assisted reproductive technologyObjective:To determine whether the use of ART is associated with an increase incidence of maternal late pregnancy complications, labor and delivery complications or adverse perinatal outcomes.Patients and methods:A retrospective cohort study was conducted on the 1013 cases of pregnancy conceived through ART (containing standard IVF and ICSI) during 2003-2009 in Zhejiang Women’s Hospital.3069 cases of natural conceived pregnancy with matched maternal age were considered as control group. Maternal late pregnancy complications, labor and delivery complications and perinatal outcomes were evaluated. Then, these indicators were evaluated respectively after ART group and control group were separated into singleton pregnancy and multiple pregnancy.Results:The multiple pregnancy rate was 37.8% in ART group, significantly higher than control group. ART was associated with higher incidence of maternal pregnancy and delivery complications [containing gestational hypertension, pre-eclampsia, gestational diabetes mellitus (GDM), intrahepatic cholestasis of pregnancy (ICP), placenta previa, placental abruption, preterm premature rupture of memberane (PPROM), placental conglutination and postpartum hemorrage] and worse perinatal outcomes [containing preterm labor, low birth weight (LBW), small for term labor and newborn asphyxia]. ART singleton pregnancy was associated with higher incidence of pre-eclampsia, GDM, placenta previa, PPROM, abnormal placental umbilical cord insertion (UCI), placental conglutination, postpartum hemorrhage, preterm labor, LBW and newborn asphyxia. ART multiple pregnancy was associated with lower incidence of small for term labor and twin discordance. Low birth weight newborn is mainly associated with preterm labor.73.2% of preterm labor was induced by different pregnancy complications.Conclusion:Patients who undergo ART are at increased risk for pregnancy complications and worse perinatal outcomes. Multiple pregnancy is closely related to these outcomes. ART singleton pregnancy was also associated with an increased risk for some pregnancy complications, which indicated that ART itself might contribute to the worse outcome of offspring. Low birth weight newborn is mainly associated with preterm labor and prenancy complications, especially for the placenta-related diseases. Therefore, promote single embryos transfer, strict control ART indication, improve ART manipulation, minimize the non-physiological interference, prevent and treat the pregnancy complications, will benefit to improve the ART outcome.Part II Comparative proteomic analysis of human placenta derived from assisted reproductive technologyObjective:To examine differences in protein expression in placenta derived from assisted reproductive technology (ART) and normal pregnancy using global proteomics-based approach.Methods:We applied a proteomic approach on six placenta tissues from patients underwent standard IVF, six from ICSI and six from natural conceived pregnancy. Two-dimensional gel electrophoresis (2-DE) and MALDI-TOF/TOF Mass Spectrometry (MS) were performed to describe differential protein expression profiles in ART placenta and control placenta. Western blot analysis was used to validate the result of 2-DE.Results:Using 2-DE we found that, compared with the control group,12 spots in standard in vitro fertilization (IVF) group and 18 spots in intracytoplasmic sperm injection (ICSI) group were significantly differentially expressed. Among them,6 spots were differentially expressed in both standard IVF and ICSI groups with the same change tendency. Totally,20 proteins were successfully identified by MALDI TOF/TOF MS, including proteins involved in the membrane traffic, metabolism, nucleic acid processing, stress response and cytoskeleton. Notably, five proteins detected to be differentially expressed in both ART groups were identified as annexin A3, hnRNP C1/C2, a-SNAP, FTL and ATP5A. Some of the proteins were confirmed by Western blot analysis. Conclusion:Our study allowed for the initial identification of these proteins related to various functions in placentation with significantly altered abundance in ART groups. The present results reveal that abnormal protein profiles are involved in ART placenta and these differentially expressed proteins may be valuable for the evaluation of potential association between ART treatment and offspring outcome.Part III Roles of annexin A3 and a-SNAP in placenta derived from ART offspring with low birth weightObjective:To investigate the expression of annexin A3 and a-SNAP, the density of microvessels in placenta derived from ART offspring with low birth weight (LBW), discuss the relationship of abnormal expression of annexin A3, a-SNAP and microvascular density (MVD) in ART/LBW offspring placenta.Methods:12 placentas derived from ART/LBW offspring,12 from ART normal birth weight (NBW) offspring and 12 placentas from normal pregnancy were examined. Western blot analysis was performed to analyze the expression level of annexin A3 and a-SNAP. Immunohistochemistry (IHC) for annexin A3 andα-SNAP together with quantitative CD34 IHC used for detecting MVD was carried out to analyze the relativity between expression levels of annexin A3 or a-SNAP and MVD.Results:Western blot results showed that expression levels of annexin A3 and a-SNAP in ART/LBW group were significantly lower than control group, which is opposite with result in ART/NBW group. IHC results also showed significantly down-regulated expression of annexin A3 and a-SNAP in ART/LBW group, together with significantly lower density of microvessels. There were significant correlation between the expression of annexin A3 or a-SNAP and MVD.Conclusion:The significantly lower expression of annexin A3 andα-SNAP in placenta may play an important role in the pathology of low birth weight in ART derived pregnancy by affecting placenta angiogenesis.更多还原