IVF-ET来源胎盘形态学、基因组学和胎盘屏障的研究：探讨辅助生育技术安全性
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摘要
辅助生殖技术（Assisted reproductive technology, ART）的安全性一直广受关注。早期调查表明，ART子代的出生缺陷率并未超过自然妊娠子代；但近年多项流行病学调查研究显示ART可能造成不良的妊娠结局，如：妊娠期合并症、早产、低出生体质量儿、围生儿发病率和死亡率升高、神经发育异常、表观遗传相关疾病等。ART增加孕母和子代不良健康风险的发生机制尚不清楚。学者们争论的焦点在于ART风险性增高到底是由于不孕患者不孕背景引起还是由ART技术引起。因此，对ART安全性的研究已成为临床生殖医学研究的热点。
围生期母婴的病理结局与胎盘功能有着密切的关联。胎盘作为妊娠期连接母胎的独特器官，具有重要的功能：（1）在母亲和胎儿异体移植物之间提供一个免疫界面；（2）在母胎间构建一个营养、代谢废物转运平台；（3）作为产生多种多肽和甾体类激素的来源，通过这些多肽和甾体激素来影响胎儿、胎盘以及母亲的相关代谢和发育。胎盘维系正常妊娠，和孕母和子代的围产期和远期安全密切相关。胎盘中的细胞间紧密连接参与形成胎盘屏障，后者具维持母胎稳态的重要功能，如转运水分、营养物质及代谢产物等。适宜氧气浓度对细胞屏障的形成和维持具有十分重要的作用，同时也是维持妊娠期正常滋养层细胞功能的必要条件。妊娠中晚期缺氧状态被认为是影响胎盘形态和功能的最关键因素。子宫胎盘血流量减少、宫内氧气供应和营养状态下降等因素，可能改变胎盘滋养层细胞的生物学特性，造成胎盘结构、功能改变。在这种情况下，胎盘通过改变相关蛋白表达或相关基因表观遗传修饰来适应这些改变，满足胎儿发育需求，发挥其代偿功能。一旦胎盘结构和功能出现异常超过自身代偿能力，就可能引起一系列不良结局，不仅会导致多种妊娠合并症的发生，如先兆子痫（Preeclampsia, PE）、妊娠期糖尿病（Gestaional diabetes mellitus, GDM）、胎儿宫内生长受限（Fetal growth restriction, FGR）等；还会造成宫内发育的胎儿发生程序性改变引起远期成年阶段各种代谢类疾病如高血压、糖尿病等发病率增高。
国内外已有少量关于动物和人来源ART胎盘的研究。动物研究显示，体外培养的动物胚胎经移植后妊娠，胎盘的大体结构、功能出现可疑异常，包括血管形成、氧化应激、激素代谢等方面；人ART来源胎盘的光镜研究显示，ART胎盘出现绒毛水肿、钙化的比率增高；蛋白质组学研究发现ART可能影响胎盘跨膜转运、代谢、核酸处理、应激反应和细胞骨架等功能；蛋白芯片研究显示ART来源胎盘血管形成因子表达存在异常。然而这些研究存在一定薄弱之处，也未见ART来源胎盘的超微结构研究、全基因组学的比较研究、差异表达基因的功能研究。
本研究以人类IVF-ET来源胎盘为研究对象，从超微结构和基因组学角度对ART来源胎盘进行研究，目的在于发现ART子代的胎盘结构和功能基因表达的差异。在此基础上，选择几个差异表达基因，以胎盘紧密连接为切入点，检测紧密连接相关因子表达差异；并进一步研究缺氧对胎盘滋养层细胞紧密连接功能影响，以此了解ART胎儿宫内发育环境潜在的改变，进一步评价ART安全性。
材料和方法
（1）收集543例ART来源胎盘和189例自然妊娠来源胎盘组织建立胎盘标本库，比较ART组383例单胎妊娠和自然妊娠组2981例单胎妊娠的临床结局，统计分析流行病学资料。以同期自然单胎妊娠胎盘为对照组，在标本库中选择因输卵管因素或轻度少弱精子症为不孕因素行IVF胚胎移植（Embryo transfer,ET）治疗后妊娠的胎盘标本进行后续实验，以降低不孕因素本身对实验结果的影响。受试者签署知情同意书，该研究经过本院伦理委员会审批。
（2）利用光镜和透射电镜的方法观察和比较8例IVF来源胎盘和15例自然妊娠来源胎盘形态学上的差异，观察指标主要包括胎盘屏障基本结构、胎盘绒毛形态、胎儿血管、绒毛间质等；
（3）提取总RNA，利用人类全基因组芯片Affymetrix HG U133Plus2.0基因芯片进行芯片杂交和分析，比较3例IVF来源胎盘和3例自然妊娠来源胎盘全基因表达谱差异，并通过Real time PCR和免疫组织化学方法验证，通过生物信息学分析将差异表达基因进行功能分类；
（4）根据形态学和芯片实验结果选择胎盘屏障转运、紧密连接功能作为后续ART胎盘功能研究的切入点，利用Real time PCR和Western Blot比较IVF来源胎盘和自然妊娠胎盘紧密连接相关因子CLDN4、CLDN8、OCLN等在mRNA和蛋白水平的表达差异，探讨两组之间是否存在紧密连接功能的改变；
（5）以滋养层细胞为研究模型，利用Millicell小室系统体外培养BeWo细胞，建立250μmol/L氯化钴（CoCl2）化学缺氧诱导模型，通过跨上皮电阻（Transepithelial electrical resistance, TER）和细胞旁渗透性（Paracellular permeability,CPP）的测定评估缺氧处理不同时间点（6h、12h、24h）后滋养层细胞紧密连接功能。并进一步通过检测缺氧处理后滋养层细胞紧密连接因子CLDN4、CLDN8、OCLN、ZO1表达水平，探讨缺氧导致滋养层细胞紧密连接功能障碍的可能机制，评估ART安全性。
结果
（1）与同期自然妊娠出生单胎新生儿相比，ART组早产、轻度子痫前期、GDM、边缘性前置胎盘以及羊水量异常的发生率明显升高，与国内外相关流行病学调查的结果一致。
（2）胎盘HE染色结果显示，两组胎盘大体结构正常，均存在典型的绒毛结构，绒毛屏障完整，无明显水肿、异常钙化等表现。利用透射电镜观察23例胎盘组织超微结构，其中IVF组8例，对照组15例。IVF组胎盘超微结构大体正常，绒毛结构典型，屏障完整。两组胎盘绒毛水肿、纤维素沉积以及核固缩等指标无明显差异(P>0.05)；和对照组胎盘相比，IVF组胎盘屏障厚度略有增加(P<0.05)， IVF来源胎盘合体滋养层细胞（Syncytiotrophoblast, ST）基底膜厚度略有增加(但P>0.05)；IVF组胎盘ST出现退行性改变，表现为ST细胞表面微绒毛密度降低，细胞内囊泡样结构增多(P<0.05)。
（3）基因芯片结果显示，和对照组胎盘相比，IVF胎盘共有26个差异表达基因（差异倍数≥2倍），其中17个表达上调，9个表达下调。生物信息学分析显示这些差异表达基因所涉及的功能分为6类：免疫应答、跨膜转运、代谢、氧化应激、细胞分化以及其他功能。进一步利用Real time PCR对其中10个代表性差异表达基因进行验证，结果和芯片结果一致；用免疫组织化学技术对其中5个差异表达基因（REAP2、STAT4、MUC1、SCAP及VNN3）进行亚细胞定位研究显示这些基因产物主要定位在胎盘绒毛ST细胞膜和/或细胞浆中。
（4）根据形态学和芯片实验结果选择胎盘屏障转运、紧密连接功能作为后续ART胎盘功能研究的切入点。透射电镜对胎盘组织进行紧密连接结构观察，发现紧密连接定位于ST细胞间连接近游离面的顶端和血管内皮细胞细胞和细胞连接处；Real time PCR结果发现在mRNA水平上IVF组胎盘紧密连接因子CLDN4表达明显下调、CLDN8表达上调，差异具有统计学意义；其他因子CLDN1、CLDN3、CLDN5、OCLN和ZO1在两组胎盘组织中表达无明显差异；Western Blot结果显示CLDN4、CLDN8和OCLN蛋白表达水平在两组之间无明显差异。
（5）利用Millicell小室体外培养BeWo细胞，研究发现250μmol/L CoCl2作用不同时间点（6h、12h、24h）后，滋养层细胞紧密连接功能障碍，表现为TER降低，CPP升高。Real time PCR和Western Blot检测发现滋养层细胞紧密连接功能异常伴紧密连接功能因子CLDN4和ZO1表达下降、CLDN8表达升高。
结论
（1）本研究通过收集ART来源胎盘和自然妊娠来源胎盘组织，为探讨ART技术对胎盘结构、功能的影响提供了大量的样本基础。形态学实验发现，ART子代出现的某些胎盘超微结构，胎盘屏障厚度增加以及ST细胞出现部分退行性改变，主要是与胎盘屏障、转运功能相关的结构。但是，ART胎盘的HE组织学没有显著改变，反映ART胎儿发育的宫内环境、营养、屏障等，与自然妊娠胎儿大致相同。因此，从胎盘组织结构及重要功能分析，ART技术是相对安全的；但胎盘某些超微结构上的差异也应继续深入研究。
（2）本研究利用全基因组芯片对IVF胎盘基因表达谱进行研究，发现26个差异表达基因，涉及多种重要的胎盘功能，提示ART技术可能改变胎盘基因表达谱，影响胎盘发生的过程和胎盘功能，包括胎盘转运。
（3）本研究选择胎盘屏障转运相关基因作为ART安全性评估的切入点进行了基因功能研究，初步发现IVF胎盘中紧密连接因子CLDN4、CLDN8存在基因mRNA表达水平的差异，在蛋白质水平上差异不显著，可能为胎盘基因表达及其调节的代偿。这些结果，为ART安全性评估的研究提供了新的思路。
Assisted reproductive technology (ART) is defined as a series of treatments usedto achieve pregnancy, in which both oocytes and sperm are handled in vitro, such asconventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).Early data suggested that infants conceived with ART would develop normally.However in the past decade, increasing amount of well-designed studies have beenshowing that ART pregnancies could produce detrimental perinatal outcomes,compared to those of non-assisted pregnancies including: preterm birth; congenitalabnormality; low birth weight; perinatal morbidity; mortality; negativeneurodevelopmental outcome; or even cancer. However, the mechanism of theincreasing maternal and offspring health risks of ART remains unclear. Researchersare having debate on whether the increased risk of ART is due to infertilitybackground of these patients or caused by assisted reproductive technology. Resultsfrom a population-based cohort study indicated that adverse outcomes associated withassisted fertilization could be attributed not to the reproductive technology itself, butrather to certain other factors that lead either to infertility or subfertility. Recently,more molecular biology studies have proven that ARTs are the main causes byshowing ART were associated with anomalies in sperm chromatin packaging andabnormal placentation. Thus, this issue becomes even more controversial and itbecomes more important to conduct in depth investigations.
The placenta is a temporary organ with many physiological functions, which isclosely related to pregnancy outcomes. It maintains fetal homoeostasis by providingan immune interface between the maternal and fetal allograft, transports nutrients andwaste products between mother and fetus, and acts as source of many peptide andsteroid hormones that influence fetal, placental, and maternal metabolism anddevelopment. Disturbances of placental development, including the reducedutero-placental blood flow, decreased oxygen supply in the uterus or abnormal maternal nutrient status can alter the biological characteristics of trophoblast cells andbring about changes in normal placental function. Under such circumstances, theplacenta may adapt by altering its functional protein expression or changingepigenetic modification of placental gene expression in order to meet the requirementof fetal development. Once the placental structure and function is abnormal, it willnot only lead to a variety of pregnancy complications, such as Preeclampsia (PE),Gestational diabetes mellitus (GDM) and Fetal growth restriction (FGR) and so on,but also 'programmed' the developing fetus for increased risk of developing variousendocrine diseases in later adult life.
Variations in ART procedures such as: gonadotropins for superovulation;medication taken for pregnancy sustenance; intracytoplasmic sperm injection;blastocyst culture; assisted hatching; or preimplantation genetic diagnosis couldpossibly disturb placental development which is assumed might alter placentation andpossibly cause the harmful outcome in offspring derived from ART treatment.Therefore, it is essential to study ART derived placentae to ensure the maternal-fetalsafety. Animal studies have demonstrated that abnormal placentation includingincreasing inflammations, oxidative stress and apoptosis were investigated afterembryo in vitro culture and transfer. Researches of human placentae have also provedthe incidence of villous edema and microcalcifications were increased in ART derivedplacentae; proteomics study have revealed that abnormal protein profiles related toanomalous placental functions are involved in ART placentae. Although the abovestudies have been done, systematic studies on ART derived placentae are still required.Researches of placental morphology, gene expression profiling and the underlyingmolecular mechanisms will help further evaluation the effects of the ARTmanipulation on placental development and fetal development, and to explore thepotential causal relationship to pregnancy outcomes.
Placental barrier transport is a vital function to maintain the maternal-fetalhomeostasis by transporting water, nutrients and metabolites. The tightness and theselectivity of paracellular transport is one of the important factors for the transportfunction of the placental barrier system, which is mainly dependent on the tightjunction structure between epithelial cells. Dysfunction of placental tight junctions would accompany by the alteration of transepithelial resistance (TER) andparacellular permeability (CPP), leading to dysfunction of transplacental transportand other functions, which involved in the occurrence and development of a varietyof placenta-related pregnancy complications (such as PE, hydatidiform moles, etc).However, mechanisms of tight junction in the placentae haven’t been deeplyinvestigated. Researchers conducted in blood-brain barrier, air blood barrier systemsuggest that tight junction were disturbed in oxygen depletion, estrogen and insulinstimulation and inflammatory environment, accompanied by the alteration ofexpression and location of tight junction proteins, leading to epithelial barrierdysfunction and changed paracellular transport capacity. Oxygen is the basiccondition for cell growth and differentiation. During early pregnancy thedevelopment of both placenta and fetus are established in a relatively hypoxicenvironment. Concentration of oxygen in various stages of pregnancy will affectplacental functions, and is believed as the key to a successful pregnancy. Placentalischemia and hypoxia have been proven to be the causal factors of a variety ofpregnancy complications. Therefore, hypoxia has been considered to be the mostcritical factors to affect placental morphology and function during the late pregnancy.The effects of hypoxia on tight junctions and tansplacental transport capacities havenot been carried out in human placentae and trophoblasts. The in-depth study of theplacental functions in tight junction will be of great significance for us to clarify theoccurrence of pregnancy related complications.
Herein, by using transmission electron microscopy (TEM) and microarrayanalysis, we compared morphological appearance and global gene expression patternsof placentae from mothers in ART group with those from control group. The objectiveof this study is to discover the potential effects of ART treatment on themorphological appearance and gene expression in placentae and to possible causalrelationship between ART procedures and offspring health. Further, we chosetransplacental barrier transport as our main target based on the basis of morphologicaland microarray experiments, especially focused on placental tight junctions, toinvestigate whether there are differentiated expressed factors in the ART group.Moreover, we studied the effects and mechanisms of hypoxia on tight junction functions in human trophoblast cells, providing new clues to clarify the pathogenesisof some pregnancy related complications.
Materials and Methods
(1) Five hundreds and forty-three placental samples were collected from thewomen who had undergone ART treatment in our study to establish sample bank ofplacenta. Pregnancy outcomes have been compared between383ART singletonpregnancies and control group consisted of2981natural conceived pregnant womenin Jiangsu Province People Hospital in the same period.
(2) In order to minimize the ambiguity of our results, placental samplessubjected to IVF-ET due to tubal factor or mild male factors were selected for furtherstudy. Morphological appearance has been compared between ART group and controlgroups using light microscope and TEM focused on the placental blood barrier,villous stroma, and the cytotrophoblasts (CT) and syncytiotrophoblasts (ST) withtheir substructures in terminal villi.
(3) A GeneChip Affymetrix HG U133Plus2.0Array was utilized to analyze theglobal gene expression patterns between goups, which were further certified usingReal time PCR. Immunohistochemistry (IHC) was conducted to investigate thelocation of dysregulated genes in the placentae. The differentially expressed geneswere classified according to their biological processes.
(4) Transplacental barrier transport was chosen as our main target based on themorphological and microarray results, especially focused on placental tight junctions.Real time PCR and Western Blot were utilized to analyze the expression of tightjunctions factors CLDN4, CLDN8and OCLN between ART and control groups bothat mRNA and protein level, respectively.
(5) Millicell chambers were utilized for human trophoblast cell line BeWo cell invitro culture and250uM cobalt chloride was used to establish the chemical anoxiamodel of hypoxia. TER and CPP were detected at different time points (6h,12h,24h)after exposed to hypoxia, thus evaluating the alteration of tight junctions oftrophoblast cells under hypoxic conditions. Real time PCR and Western Blot wereemployed for the detection of both mRNA and protein expression of tight junctionrelated factors CLDN4, CLDN8, OCLN and ZO1of trophoblast cells at different time points (6h,12h,24h) after exposed to hypoxia, trying to elucidate the mechanism ofdisturbance of tight junction in hypoxia.
Results
(1) Our results showed ART singleton pregnancy was associated with higherincidence of preterm labor, PE, GDM, placenta pervia, pretern premature rupture ofmemberane and abnormal amniotic fluid volume than control group.
(2) Light microscopy results showed that both ART derived and controlplacentae had normal microscopic histological features, indicating that the ARTprocedure did not affect the gross structure of the placentae, and also demonstratedthat the samples were of sufficient quality to be analyzed using TEM. Moreover,twenty-three placental samples including8in ART group and15in control groupwere underwent TEM examination to compare their ultrastructure. The present studyrevealed a mild alteration of the placental barrier in ART derived placentae, includinga thickening of the placental barrier, decreased density of syncytiotrophoblastic apicalmicrovilli, and increased vacuoles observed in ST, all of which are potentiallyinvolved in the downregulation of transplacental transports and exchanges.
(3) After the microarray analysis, twenty-six differentially expressed genes wereidentified in the ART treated placentae:17up-regulated;9down-regulated. Eighteenof these were classified into six groups according to critical placental function:immune response; transmembrane transport; metabolism; oxidative stress; celldifferentiation; and other functions. Ten differentially expressed genes were furthercertified by Real time PCR and IHC showed some of these gene products wereexpressed in the placental villus tissues, either in the cytoplasm or in the membrane ofST.
(4) Tight junctions in human placentae were detailed investigated in our furtherstudy and the structure of tight junction were conformed to be localized in the apicalpart of the syncytium and also between the cell-cell contacts of fetal blood vesselendothelial after TEM examination. After Real time PCR and Western Blot analysis,the mRNA level of CLDN4and CLDN8were significantly differentially expressed inART derived placentae when compared with control groups. However, the proteinlevels of these tight junctions did not differ between groups.
(5) The in vitro monolayer culture model of trophoblast cells has been successfulestablished using Millicell chamber. Tight junction dysfunction of trophoblast cellswas found at different time points after exposed to250uM cobalt chloride whichmimic chemical hypoxia, with the observation of decreased TER and increased CPP.The results of Real time PCR and Western Blot showed both mRNA and proteinexpressions of ZO1, CLDN4of trophoblast cells were decreased, and CLDN8oftrophoblast cells were increased.
Conclusions
(1) The collection of large amount of placental samples both from ART andnatural conceived pregnancy in our study has provided a sample basis for furtherresearches on placental morphology and functions. Ultrastructural examinationsrevealed a mild alteration of the placental barrier in ART derived placentae, includinga thickening of the placental barrier, decreased density of syncytiotrophoblastic apicalmicrovilli, and increased vacuoles observed in ST. However major morphologicalstructure has not significantly changed, reflecting that the intrauterine environment ofART pregnancy is roughly the same as naturally conceived pregnancy. Therefore,ART technology is relatively safe based on the structure studies while theultrastructural alteration in ART placentae should also be continually investigated.
(3) Microarray analysis have investigated26differentially expressed genes wereidentified in the ART treated placentae related to different placental functions. Thechanged gene expression files have been speculated to be associated with thealteration of some placental functions attributed to ART manipulations and thepossibly changed pregnancy outcomes.
(4) The changed mRNA level of CLDN4and CLDN8have been demonstrated inART derived placentae, however their protein level has no alteration between groups,indicating the regulation of placental gene expression and its ability of compensation.Tight junction factors might have their possible roles in the regulation oftransplacental barrier transport after ART manipulation, which have provided the newaspect for the ART security studies.
围生期母婴的病理结局与胎盘功能有着密切的关联。胎盘作为妊娠期连接母胎的独特器官，具有重要的功能：（1）在母亲和胎儿异体移植物之间提供一个免疫界面；（2）在母胎间构建一个营养、代谢废物转运平台；（3）作为产生多种多肽和甾体类激素的来源，通过这些多肽和甾体激素来影响胎儿、胎盘以及母亲的相关代谢和发育。胎盘维系正常妊娠，和孕母和子代的围产期和远期安全密切相关。胎盘中的细胞间紧密连接参与形成胎盘屏障，后者具维持母胎稳态的重要功能，如转运水分、营养物质及代谢产物等。适宜氧气浓度对细胞屏障的形成和维持具有十分重要的作用，同时也是维持妊娠期正常滋养层细胞功能的必要条件。妊娠中晚期缺氧状态被认为是影响胎盘形态和功能的最关键因素。子宫胎盘血流量减少、宫内氧气供应和营养状态下降等因素，可能改变胎盘滋养层细胞的生物学特性，造成胎盘结构、功能改变。在这种情况下，胎盘通过改变相关蛋白表达或相关基因表观遗传修饰来适应这些改变，满足胎儿发育需求，发挥其代偿功能。一旦胎盘结构和功能出现异常超过自身代偿能力，就可能引起一系列不良结局，不仅会导致多种妊娠合并症的发生，如先兆子痫（Preeclampsia, PE）、妊娠期糖尿病（Gestaional diabetes mellitus, GDM）、胎儿宫内生长受限（Fetal growth restriction, FGR）等；还会造成宫内发育的胎儿发生程序性改变引起远期成年阶段各种代谢类疾病如高血压、糖尿病等发病率增高。
国内外已有少量关于动物和人来源ART胎盘的研究。动物研究显示，体外培养的动物胚胎经移植后妊娠，胎盘的大体结构、功能出现可疑异常，包括血管形成、氧化应激、激素代谢等方面；人ART来源胎盘的光镜研究显示，ART胎盘出现绒毛水肿、钙化的比率增高；蛋白质组学研究发现ART可能影响胎盘跨膜转运、代谢、核酸处理、应激反应和细胞骨架等功能；蛋白芯片研究显示ART来源胎盘血管形成因子表达存在异常。然而这些研究存在一定薄弱之处，也未见ART来源胎盘的超微结构研究、全基因组学的比较研究、差异表达基因的功能研究。
本研究以人类IVF-ET来源胎盘为研究对象，从超微结构和基因组学角度对ART来源胎盘进行研究，目的在于发现ART子代的胎盘结构和功能基因表达的差异。在此基础上，选择几个差异表达基因，以胎盘紧密连接为切入点，检测紧密连接相关因子表达差异；并进一步研究缺氧对胎盘滋养层细胞紧密连接功能影响，以此了解ART胎儿宫内发育环境潜在的改变，进一步评价ART安全性。
材料和方法
（1）收集543例ART来源胎盘和189例自然妊娠来源胎盘组织建立胎盘标本库，比较ART组383例单胎妊娠和自然妊娠组2981例单胎妊娠的临床结局，统计分析流行病学资料。以同期自然单胎妊娠胎盘为对照组，在标本库中选择因输卵管因素或轻度少弱精子症为不孕因素行IVF胚胎移植（Embryo transfer,ET）治疗后妊娠的胎盘标本进行后续实验，以降低不孕因素本身对实验结果的影响。受试者签署知情同意书，该研究经过本院伦理委员会审批。
（2）利用光镜和透射电镜的方法观察和比较8例IVF来源胎盘和15例自然妊娠来源胎盘形态学上的差异，观察指标主要包括胎盘屏障基本结构、胎盘绒毛形态、胎儿血管、绒毛间质等；
（3）提取总RNA，利用人类全基因组芯片Affymetrix HG U133Plus2.0基因芯片进行芯片杂交和分析，比较3例IVF来源胎盘和3例自然妊娠来源胎盘全基因表达谱差异，并通过Real time PCR和免疫组织化学方法验证，通过生物信息学分析将差异表达基因进行功能分类；
（4）根据形态学和芯片实验结果选择胎盘屏障转运、紧密连接功能作为后续ART胎盘功能研究的切入点，利用Real time PCR和Western Blot比较IVF来源胎盘和自然妊娠胎盘紧密连接相关因子CLDN4、CLDN8、OCLN等在mRNA和蛋白水平的表达差异，探讨两组之间是否存在紧密连接功能的改变；
（5）以滋养层细胞为研究模型，利用Millicell小室系统体外培养BeWo细胞，建立250μmol/L氯化钴（CoCl2）化学缺氧诱导模型，通过跨上皮电阻（Transepithelial electrical resistance, TER）和细胞旁渗透性（Paracellular permeability,CPP）的测定评估缺氧处理不同时间点（6h、12h、24h）后滋养层细胞紧密连接功能。并进一步通过检测缺氧处理后滋养层细胞紧密连接因子CLDN4、CLDN8、OCLN、ZO1表达水平，探讨缺氧导致滋养层细胞紧密连接功能障碍的可能机制，评估ART安全性。
结果
（1）与同期自然妊娠出生单胎新生儿相比，ART组早产、轻度子痫前期、GDM、边缘性前置胎盘以及羊水量异常的发生率明显升高，与国内外相关流行病学调查的结果一致。
（2）胎盘HE染色结果显示，两组胎盘大体结构正常，均存在典型的绒毛结构，绒毛屏障完整，无明显水肿、异常钙化等表现。利用透射电镜观察23例胎盘组织超微结构，其中IVF组8例，对照组15例。IVF组胎盘超微结构大体正常，绒毛结构典型，屏障完整。两组胎盘绒毛水肿、纤维素沉积以及核固缩等指标无明显差异(P>0.05)；和对照组胎盘相比，IVF组胎盘屏障厚度略有增加(P<0.05)， IVF来源胎盘合体滋养层细胞（Syncytiotrophoblast, ST）基底膜厚度略有增加(但P>0.05)；IVF组胎盘ST出现退行性改变，表现为ST细胞表面微绒毛密度降低，细胞内囊泡样结构增多(P<0.05)。
（3）基因芯片结果显示，和对照组胎盘相比，IVF胎盘共有26个差异表达基因（差异倍数≥2倍），其中17个表达上调，9个表达下调。生物信息学分析显示这些差异表达基因所涉及的功能分为6类：免疫应答、跨膜转运、代谢、氧化应激、细胞分化以及其他功能。进一步利用Real time PCR对其中10个代表性差异表达基因进行验证，结果和芯片结果一致；用免疫组织化学技术对其中5个差异表达基因（REAP2、STAT4、MUC1、SCAP及VNN3）进行亚细胞定位研究显示这些基因产物主要定位在胎盘绒毛ST细胞膜和/或细胞浆中。
（4）根据形态学和芯片实验结果选择胎盘屏障转运、紧密连接功能作为后续ART胎盘功能研究的切入点。透射电镜对胎盘组织进行紧密连接结构观察，发现紧密连接定位于ST细胞间连接近游离面的顶端和血管内皮细胞细胞和细胞连接处；Real time PCR结果发现在mRNA水平上IVF组胎盘紧密连接因子CLDN4表达明显下调、CLDN8表达上调，差异具有统计学意义；其他因子CLDN1、CLDN3、CLDN5、OCLN和ZO1在两组胎盘组织中表达无明显差异；Western Blot结果显示CLDN4、CLDN8和OCLN蛋白表达水平在两组之间无明显差异。
（5）利用Millicell小室体外培养BeWo细胞，研究发现250μmol/L CoCl2作用不同时间点（6h、12h、24h）后，滋养层细胞紧密连接功能障碍，表现为TER降低，CPP升高。Real time PCR和Western Blot检测发现滋养层细胞紧密连接功能异常伴紧密连接功能因子CLDN4和ZO1表达下降、CLDN8表达升高。
结论
（1）本研究通过收集ART来源胎盘和自然妊娠来源胎盘组织，为探讨ART技术对胎盘结构、功能的影响提供了大量的样本基础。形态学实验发现，ART子代出现的某些胎盘超微结构，胎盘屏障厚度增加以及ST细胞出现部分退行性改变，主要是与胎盘屏障、转运功能相关的结构。但是，ART胎盘的HE组织学没有显著改变，反映ART胎儿发育的宫内环境、营养、屏障等，与自然妊娠胎儿大致相同。因此，从胎盘组织结构及重要功能分析，ART技术是相对安全的；但胎盘某些超微结构上的差异也应继续深入研究。
（2）本研究利用全基因组芯片对IVF胎盘基因表达谱进行研究，发现26个差异表达基因，涉及多种重要的胎盘功能，提示ART技术可能改变胎盘基因表达谱，影响胎盘发生的过程和胎盘功能，包括胎盘转运。
（3）本研究选择胎盘屏障转运相关基因作为ART安全性评估的切入点进行了基因功能研究，初步发现IVF胎盘中紧密连接因子CLDN4、CLDN8存在基因mRNA表达水平的差异，在蛋白质水平上差异不显著，可能为胎盘基因表达及其调节的代偿。这些结果，为ART安全性评估的研究提供了新的思路。
Assisted reproductive technology (ART) is defined as a series of treatments usedto achieve pregnancy, in which both oocytes and sperm are handled in vitro, such asconventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).Early data suggested that infants conceived with ART would develop normally.However in the past decade, increasing amount of well-designed studies have beenshowing that ART pregnancies could produce detrimental perinatal outcomes,compared to those of non-assisted pregnancies including: preterm birth; congenitalabnormality; low birth weight; perinatal morbidity; mortality; negativeneurodevelopmental outcome; or even cancer. However, the mechanism of theincreasing maternal and offspring health risks of ART remains unclear. Researchersare having debate on whether the increased risk of ART is due to infertilitybackground of these patients or caused by assisted reproductive technology. Resultsfrom a population-based cohort study indicated that adverse outcomes associated withassisted fertilization could be attributed not to the reproductive technology itself, butrather to certain other factors that lead either to infertility or subfertility. Recently,more molecular biology studies have proven that ARTs are the main causes byshowing ART were associated with anomalies in sperm chromatin packaging andabnormal placentation. Thus, this issue becomes even more controversial and itbecomes more important to conduct in depth investigations.
The placenta is a temporary organ with many physiological functions, which isclosely related to pregnancy outcomes. It maintains fetal homoeostasis by providingan immune interface between the maternal and fetal allograft, transports nutrients andwaste products between mother and fetus, and acts as source of many peptide andsteroid hormones that influence fetal, placental, and maternal metabolism anddevelopment. Disturbances of placental development, including the reducedutero-placental blood flow, decreased oxygen supply in the uterus or abnormal maternal nutrient status can alter the biological characteristics of trophoblast cells andbring about changes in normal placental function. Under such circumstances, theplacenta may adapt by altering its functional protein expression or changingepigenetic modification of placental gene expression in order to meet the requirementof fetal development. Once the placental structure and function is abnormal, it willnot only lead to a variety of pregnancy complications, such as Preeclampsia (PE),Gestational diabetes mellitus (GDM) and Fetal growth restriction (FGR) and so on,but also 'programmed' the developing fetus for increased risk of developing variousendocrine diseases in later adult life.
Variations in ART procedures such as: gonadotropins for superovulation;medication taken for pregnancy sustenance; intracytoplasmic sperm injection;blastocyst culture; assisted hatching; or preimplantation genetic diagnosis couldpossibly disturb placental development which is assumed might alter placentation andpossibly cause the harmful outcome in offspring derived from ART treatment.Therefore, it is essential to study ART derived placentae to ensure the maternal-fetalsafety. Animal studies have demonstrated that abnormal placentation includingincreasing inflammations, oxidative stress and apoptosis were investigated afterembryo in vitro culture and transfer. Researches of human placentae have also provedthe incidence of villous edema and microcalcifications were increased in ART derivedplacentae; proteomics study have revealed that abnormal protein profiles related toanomalous placental functions are involved in ART placentae. Although the abovestudies have been done, systematic studies on ART derived placentae are still required.Researches of placental morphology, gene expression profiling and the underlyingmolecular mechanisms will help further evaluation the effects of the ARTmanipulation on placental development and fetal development, and to explore thepotential causal relationship to pregnancy outcomes.
Placental barrier transport is a vital function to maintain the maternal-fetalhomeostasis by transporting water, nutrients and metabolites. The tightness and theselectivity of paracellular transport is one of the important factors for the transportfunction of the placental barrier system, which is mainly dependent on the tightjunction structure between epithelial cells. Dysfunction of placental tight junctions would accompany by the alteration of transepithelial resistance (TER) andparacellular permeability (CPP), leading to dysfunction of transplacental transportand other functions, which involved in the occurrence and development of a varietyof placenta-related pregnancy complications (such as PE, hydatidiform moles, etc).However, mechanisms of tight junction in the placentae haven’t been deeplyinvestigated. Researchers conducted in blood-brain barrier, air blood barrier systemsuggest that tight junction were disturbed in oxygen depletion, estrogen and insulinstimulation and inflammatory environment, accompanied by the alteration ofexpression and location of tight junction proteins, leading to epithelial barrierdysfunction and changed paracellular transport capacity. Oxygen is the basiccondition for cell growth and differentiation. During early pregnancy thedevelopment of both placenta and fetus are established in a relatively hypoxicenvironment. Concentration of oxygen in various stages of pregnancy will affectplacental functions, and is believed as the key to a successful pregnancy. Placentalischemia and hypoxia have been proven to be the causal factors of a variety ofpregnancy complications. Therefore, hypoxia has been considered to be the mostcritical factors to affect placental morphology and function during the late pregnancy.The effects of hypoxia on tight junctions and tansplacental transport capacities havenot been carried out in human placentae and trophoblasts. The in-depth study of theplacental functions in tight junction will be of great significance for us to clarify theoccurrence of pregnancy related complications.
Herein, by using transmission electron microscopy (TEM) and microarrayanalysis, we compared morphological appearance and global gene expression patternsof placentae from mothers in ART group with those from control group. The objectiveof this study is to discover the potential effects of ART treatment on themorphological appearance and gene expression in placentae and to possible causalrelationship between ART procedures and offspring health. Further, we chosetransplacental barrier transport as our main target based on the basis of morphologicaland microarray experiments, especially focused on placental tight junctions, toinvestigate whether there are differentiated expressed factors in the ART group.Moreover, we studied the effects and mechanisms of hypoxia on tight junction functions in human trophoblast cells, providing new clues to clarify the pathogenesisof some pregnancy related complications.
Materials and Methods
(1) Five hundreds and forty-three placental samples were collected from thewomen who had undergone ART treatment in our study to establish sample bank ofplacenta. Pregnancy outcomes have been compared between383ART singletonpregnancies and control group consisted of2981natural conceived pregnant womenin Jiangsu Province People Hospital in the same period.
(2) In order to minimize the ambiguity of our results, placental samplessubjected to IVF-ET due to tubal factor or mild male factors were selected for furtherstudy. Morphological appearance has been compared between ART group and controlgroups using light microscope and TEM focused on the placental blood barrier,villous stroma, and the cytotrophoblasts (CT) and syncytiotrophoblasts (ST) withtheir substructures in terminal villi.
(3) A GeneChip Affymetrix HG U133Plus2.0Array was utilized to analyze theglobal gene expression patterns between goups, which were further certified usingReal time PCR. Immunohistochemistry (IHC) was conducted to investigate thelocation of dysregulated genes in the placentae. The differentially expressed geneswere classified according to their biological processes.
(4) Transplacental barrier transport was chosen as our main target based on themorphological and microarray results, especially focused on placental tight junctions.Real time PCR and Western Blot were utilized to analyze the expression of tightjunctions factors CLDN4, CLDN8and OCLN between ART and control groups bothat mRNA and protein level, respectively.
(5) Millicell chambers were utilized for human trophoblast cell line BeWo cell invitro culture and250uM cobalt chloride was used to establish the chemical anoxiamodel of hypoxia. TER and CPP were detected at different time points (6h,12h,24h)after exposed to hypoxia, thus evaluating the alteration of tight junctions oftrophoblast cells under hypoxic conditions. Real time PCR and Western Blot wereemployed for the detection of both mRNA and protein expression of tight junctionrelated factors CLDN4, CLDN8, OCLN and ZO1of trophoblast cells at different time points (6h,12h,24h) after exposed to hypoxia, trying to elucidate the mechanism ofdisturbance of tight junction in hypoxia.
Results
(1) Our results showed ART singleton pregnancy was associated with higherincidence of preterm labor, PE, GDM, placenta pervia, pretern premature rupture ofmemberane and abnormal amniotic fluid volume than control group.
(2) Light microscopy results showed that both ART derived and controlplacentae had normal microscopic histological features, indicating that the ARTprocedure did not affect the gross structure of the placentae, and also demonstratedthat the samples were of sufficient quality to be analyzed using TEM. Moreover,twenty-three placental samples including8in ART group and15in control groupwere underwent TEM examination to compare their ultrastructure. The present studyrevealed a mild alteration of the placental barrier in ART derived placentae, includinga thickening of the placental barrier, decreased density of syncytiotrophoblastic apicalmicrovilli, and increased vacuoles observed in ST, all of which are potentiallyinvolved in the downregulation of transplacental transports and exchanges.
(3) After the microarray analysis, twenty-six differentially expressed genes wereidentified in the ART treated placentae:17up-regulated;9down-regulated. Eighteenof these were classified into six groups according to critical placental function:immune response; transmembrane transport; metabolism; oxidative stress; celldifferentiation; and other functions. Ten differentially expressed genes were furthercertified by Real time PCR and IHC showed some of these gene products wereexpressed in the placental villus tissues, either in the cytoplasm or in the membrane ofST.
(4) Tight junctions in human placentae were detailed investigated in our furtherstudy and the structure of tight junction were conformed to be localized in the apicalpart of the syncytium and also between the cell-cell contacts of fetal blood vesselendothelial after TEM examination. After Real time PCR and Western Blot analysis,the mRNA level of CLDN4and CLDN8were significantly differentially expressed inART derived placentae when compared with control groups. However, the proteinlevels of these tight junctions did not differ between groups.
(5) The in vitro monolayer culture model of trophoblast cells has been successfulestablished using Millicell chamber. Tight junction dysfunction of trophoblast cellswas found at different time points after exposed to250uM cobalt chloride whichmimic chemical hypoxia, with the observation of decreased TER and increased CPP.The results of Real time PCR and Western Blot showed both mRNA and proteinexpressions of ZO1, CLDN4of trophoblast cells were decreased, and CLDN8oftrophoblast cells were increased.
Conclusions
(1) The collection of large amount of placental samples both from ART andnatural conceived pregnancy in our study has provided a sample basis for furtherresearches on placental morphology and functions. Ultrastructural examinationsrevealed a mild alteration of the placental barrier in ART derived placentae, includinga thickening of the placental barrier, decreased density of syncytiotrophoblastic apicalmicrovilli, and increased vacuoles observed in ST. However major morphologicalstructure has not significantly changed, reflecting that the intrauterine environment ofART pregnancy is roughly the same as naturally conceived pregnancy. Therefore,ART technology is relatively safe based on the structure studies while theultrastructural alteration in ART placentae should also be continually investigated.
(3) Microarray analysis have investigated26differentially expressed genes wereidentified in the ART treated placentae related to different placental functions. Thechanged gene expression files have been speculated to be associated with thealteration of some placental functions attributed to ART manipulations and thepossibly changed pregnancy outcomes.
(4) The changed mRNA level of CLDN4and CLDN8have been demonstrated inART derived placentae, however their protein level has no alteration between groups,indicating the regulation of placental gene expression and its ability of compensation.Tight junction factors might have their possible roles in the regulation oftransplacental barrier transport after ART manipulation, which have provided the newaspect for the ART security studies.
引文
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