【作者】 黄玉晶；

【导师】 舒为群；

【作者基本信息】 第三军医大学 ， 劳动卫生与环境卫生学， 2014， 博士

【摘要】 1.研究背景我们前期研究发现重庆市孕产妇体内存在邻苯二甲酸酯（PAEs）污染。国外有流行病学研究及我们前期小样本量流行病学研究认为其可能与早产及胎儿发育相关，但是这种关联还具有争议，具体机制也不清楚。过氧化物酶体增殖物激活受体（peroxisome proliferator-activatived receptors，PPARs）是由配体激活的转录因子，属于核激素受体超家族，与能量代谢及炎症密切相关。有研究提示，PPARs同样与妊娠相关，胎盘中PPARs的表达可能通过调节性激素水平、调控炎症相关蛋白（例如基质金属蛋白酶（MMPs））及能量代谢影响早产及胎儿发育。我们前期研究发现，MEHP（邻苯二甲酸二异辛酯代谢产物）可诱导颗粒细胞过氧化物酶体增殖物激活受体（PPARs）PPARγ亚型表达增加。其他研究还发现，多种PAEs可作为配体激活PPARs。因此，我们推测PAEs可能通过PPARs引起早产及影响胎儿发育。本实验通过流行病学调查，验证PAEs含量与早产和胎儿发育的关系及PPARs在其中的作用；同时构建DEHP染毒动物模型，以PPARs的亚型PPARγ为核心，围绕性激素调节和炎症相关蛋白表达，研究PPAR在PAEs诱发早产和与影响胎儿发育的作用和机制。2.研究内容2.1孕妇脐血PAEs含量与早产及胎儿发育关联的流行病学研究。2.1.1研究对象的选择及样本采集：以我校西南医院妇产科住院孕产妇为研究对象，根据《病例对照研究样本量表》确定纳入人数207人以上。通过调查表了解孕妇一般情况。纳入标准：至少最近1年居住在三峡库区或重庆。排除标准：吸烟、职业性PAEs暴露、未进行过产检、患有与早产或胎儿发育相关的遗传性疾病或其他严重疾病者。促凝管采集每名孕产妇脐血5mL用于血清激素检测；抗凝管采集每名孕产妇脐血5mL用于检测PAEs含量；采集胎盘组织样本用于检测PPARγ蛋白表达。2.1.2人群资料分析：以孕龄＜37周作为早产标准将孕产妇分为早产组与对照组。根据病历了解孕产妇输液情况及妊娠并发症（包括：胎膜早破、绒毛膜羊膜炎、妊娠期肝内胆汁淤积症、前置胎盘、妊娠期高血压综合征、妊娠期糖尿病、胎盘早剥，由资深妇产科医生确诊）发病情况。根据产科记录收集胎儿发育指标（包括：胎儿身长、胎儿体重、腹围、股骨长、双顶径、头围）及胎儿发育影响因素指标（胎盘重量、孕期）。应用病例对照研究分析早产及妊娠并发症与脐血PAEs含量、胎盘PPARγ表达及血清激素水平的关联；应用横断面研究分析脐血PAEs含量、胎盘PPARγ表达、血清激素水平及胎儿发育的关联。2.1.3脐血PAEs浓度检测：正己烷与TBME液相萃取脐血PAEs，通过GC/MS检测15种环境中常见的PAEs浓度（Accustandard PAEs混标：邻苯二甲酸二甲酯（DMP）、邻苯二甲酸二乙酯（DEP）、邻苯二甲酸二异丁酯（DIBP）、邻苯二甲酸二丁酯（DBP）、邻苯二甲酸二(2-甲氧基乙)酯（DMEP）、邻苯二甲酸二-4-甲基-2-戊酯（BMPP）、邻苯二甲酸二-2-乙氧基乙酯（DEEP）、邻苯二甲酸二戊酯（DPP）、邻苯二甲酸二正己酯（DNHP）、邻苯二甲酸丁苄酯（BBP）、邻苯二甲酸二丁氧基乙酯（DBEP）、邻苯二甲酸二环己酯（DCHP）、邻苯二甲酸二异辛酯（DEHP）、邻苯二甲酸二正辛酯（DNOP）、邻苯二甲酸二壬酯（DNP），色谱纯）。2.1.4血清激素水平检测：分离血清，采用ELISA方法检测基质金属蛋白酶和前列腺素E2水平，采用放射免疫法测定孕酮、雌二醇、雌三醇水平。2.1.5产妇胎盘PPARγ蛋白表达：IP细胞裂解液提取胎盘组织总蛋白，BCA法检测蛋白浓度；Western Blot检测PPARγ蛋白表达：SDS-PAGE电泳分离蛋白；Bio-rad标准半干式转PVDF膜；ECL Kit（碧云天）化学发光检测蛋白表达。2.1.6数据处理与分析：采用SPSS13.0软件包管理和分析数据。采用独立样本T检验、二元logistic回归分析早产、妊娠并发症与脐血PAEs含量及胎盘PPARγ表达及血清激素水平含量的相关性。采用独立样本T检验、线性回归分析脐血PAEs含量、胎盘PPARγ表达、血清激素水平及胎儿发育的相关性。2.2胎盘PPARγ表达在DEHP染毒影响雌鼠早产及胎鼠发育中的作用的动物实验2.2.1实验动物分组及处理：6-7周龄健康雌性SPF级SD大鼠60只，按随机数字法随机分为2组：对照组（玉米油2ml/kg）20只、染毒组（DEHP（作为PAEs代表）2ml/kg），连续灌胃27天。将染毒组进一步分成染毒组（DEHP2ml/kg+2%DMSO灌胃，20只）和干预组（DEHP2ml/kg+2%DMSO+0.5mg/kg GW9662（PPARγ特异性拮抗剂）灌胃,20只），在灌胃3天后合笼；以看见阴栓作为妊娠第0天，以妊娠第20天前分娩作为早产，无早产者于妊娠第20天处死，称量去除子宫的孕鼠体重，与妊娠第0天体重差值作为妊娠期体重增加值。2.2.2孕鼠激素水平检测：促凝管采集心脏血，分离血清；采用ELISA方法检测雌酮、睾酮、前列腺素E2、前列腺素F2a、硫酸脱氢表雄酮、雄烯二酮水平；采用放射免疫法测定孕酮、雌二醇水平。2.2.3胎鼠质量及胎盘重量观察：纵形切开子宫，暴露植入体，取出胎鼠，分离胎鼠和胎盘，分别称重胎鼠和胎盘，观察吸收胎、死胎和活胎。2.2.4母鼠胎盘PPARγ蛋白表达：方法同2.1.5。2.2.5实时定量qPCR检测胎盘PPARγ、PPARα、CYP191（P450芳香化酶）、MMP2、MMP9基因表达：使用Trizol提取胎盘总RNA；Nano Drop分光光度计定量；以OligodT为引物，反转录cDNA；以cDNA为模板进行实时荧光定量PCR（SYBR Green I嵌合荧光法），然后对所得Ct值进行分析，以β-Actin的Ct值为内参比较基因转录水平的变化。2.2.6统计学处理：通过SPSS13.0软件进行单因素方差分析，如存在组间差异再根据方差齐性检验结果进行两两比较。繁殖实验结果使用卡方检验。3.结果与讨论3.1孕妇脐血PAEs含量与早产及妊娠并发症的关联性除DCHP外，其余14种PAEs在早产孕妇脐血中含量显著高于对照组且均呈正相关，提示脐血PAEs含量可能与早产相关。除DNHP、DMEP、DBEP、DCHP外，其余9种PAEs在胎膜早破孕妇脐血中含量显著高于对照组，回归分析也显示10种PAEs（除DNHP、DMEP、DBP、DIBP、DCHP外）与胎膜早破正相关，提示PAEs含量与胎膜早破有关。但是无胎膜早破孕妇中早产组脐血PAEs除DCHP、DNHP外，其余13种浓度仍然显著高于对照组，且除DCHP外均与早产发病正相关（包括DNHP），提示胎膜早破可能部分参与PAEs与早产的关联，但不是引起PAEs含量与早产关联的主要原因。有产前输液行为的孕妇3种PAEs浓度（DMP、DEEP、BMPP）显著增高（与无产前输液行为者比）；无产前输液行为的孕妇除BMPP、DNHP、DCHP外，其余12种PAEs在早产组含量仍然显著高于对照组，且15种PAEs均与早产呈正相关（包括BMPP、DNHP、DCHP），提示产前输液行为同样不是引起PAEs含量与早产关联的主要原因。3.2脐血PAEs含量与胎儿发育的关联性孕妇脐血15种PAEs高暴露组（浓度大于中位数者（检出率大于50%）或有检出者（检出率小于50%））孕期显著降低，且PAEs水平与孕期负相关。但是将早产组和非早产组孕妇分开比较，PAEs与孕期的相关性消失，提示PAEs含量与孕期缩短相关性主要来源于早产的影响。在全部女性婴儿中，大多数PAEs与胎儿发育指标相关；通过孕期校正后，相关性大量下降；单独评价非早产孕妇PAEs与女婴发育指标的关联，同样只有少量存在关联。在全部男性婴儿中，大多数PAEsPAEs与胎儿发育指标相关；通过孕期校正后，或者单独评价非早产孕妇PAEs与发育指标的关联，相关性同样显著下降。PAEs与出生指标的关联，提示PAEs含量可能与胎儿发育不良有关，但是这种关联可能主要来源于早产和孕期缩短的影响。使用孕期校正后或单独观察非早产孕妇组时PAEs对男婴和女婴出生发育指标的不良影响存在差异，包括对PAEs的敏感种类（女婴：DMEP、DEP、DNHP、BBP、DNP、DBP、DCHP和DEHP；男婴：DIBP、DPP和DBEP）和敏感指标（女婴：胎儿出生体重、胎儿腹围、胎儿股骨长、胎儿头围、胎儿双顶径；男婴：胎儿出生身长、胎儿头围），提示PAEs对胎儿发育的影响存在性别差异。3.3孕妇脐血PAEs含量、早产及妊娠并发症、胎儿发育与胎盘PPARγ表达的关联3.3.1脐血PAEs含量与胎盘PPARγ蛋白表达的关系全部孕妇中，15种PAEs高暴露组PPARγ表达显著增加，且15种PAEs含量与PPARγ表达正相关。非早产组中，DPP、DMP、DCHP、DBP、DEHP高暴露组PPARγ表达增加，除这5种外，还有DNP、DIBP、BMPP与PPARγ表达正相关。早产组中，BMPP、DIBP、DBP、DEHP高暴露组PPARγ表达增加，在该组中，除DMEP、DEEP，其余13种PAEs脐血浓度与胎盘PPARγ表达正相关。提示PAEs含量可能与PPARγ表达增加相关。3.3.2胎盘PPARγ蛋白表达与早产、胎膜早破的关系早产组胎盘PPARγ表达均显著高于对照组，且PPARγ表达与早产发病呈正相关。胎膜早破组胎盘PPARγ表达显著高于对照组，且PPARγ表达与胎膜早破发病呈正相关。非胎膜早破孕妇群体中，早产组胎盘PPARγ表达均显著高于对照组，且PPARγ表达与早产发病呈正相关。提示PPARγ与胎膜早破的关联可能来源于早产的影响。3.3.3胎盘PPARγ蛋白表达与胎儿发育的关系全部孕妇，胎盘PPARγ高表达组孕期显著缩短且两者负相关，但是将孕妇分成非早产组和早产组分别观察时，胎盘PPARγ表达与孕期缩短的相关性消失。在女性婴儿中，不管是全部女性婴儿还是非早产组女性婴儿，均未见胎盘PPARγ高表达组胎儿发育指标显著改变。全部女性婴儿胎盘PPARγ表达与胎儿身长、胎儿体重、双顶径、头围、腹围、股骨长负相关；单独比较非早产组女性婴儿胎盘PPARγ表达与出生指标的关联，相关性消失。在男性婴儿中，全部男性婴儿胎盘PPARγ高表达组胎儿身长、胎儿体重、双顶径、头围、显著降低，胎盘PPARγ表达与胎儿身长、胎儿体重、双顶径、头围、腹围负相关；单独比较非早产组男性婴儿，胎盘PPARγ高表达组只剩头围显著降低，胎盘PPARγ表达与胎儿发育指标的相关性消失。我们推测，胎盘PPARγ表达与胎儿发育的关联与脐血PAEs类似，主要来源于其与早产的关联。3.4孕妇脐血PAEs含量、胎盘PPAGγ蛋白表达、早产及妊娠并发症、胎儿发育与血清性激素水平及炎症相关蛋白的关联。3.4.1脐血PAEs与血清性激素水平及炎症相关蛋白的关系全部孕妇中，5种PAEs（DEP、DCHP、DIBP、DBP、DEHP）高浓度组雌三醇水平显著下降；DEHP高浓度组雌二醇水平下调；DEEP、DMEP高暴露组孕酮水平显著上调。非早产孕妇中，DNOP高暴露组雌三醇水平显著上调，DBP高暴露组雌三醇水平显著下调；DEHP高暴露组雌二醇水平显著下调；DEEP、BMPP、DMEP高暴露组孕酮水平显著上调。早产组中，DNHP高暴露组雌三醇水平显著上调；DEEP、DMEP、DBEP高暴露组雌二醇水平显著上调。推测脐血PAEs含量可能与血清雌三醇、雌二醇、孕酮水平正相关。全部孕妇中，DEP、BBP高暴露组基质金属蛋白酶水平显著下调；DEHP高暴露组前列腺素E2水平显著下调。非早产孕妇中，DNHP、BBP、DEEP高暴露组基质金属蛋白酶水平显著下调；DCHP、DEHP高暴露组前列腺素E2水平显著下调。早产组中，DBP高暴露组基质金属蛋白酶水平显著下调。推测脐血PAEs含量可能与基质金属蛋白酶、前列腺素E2水平负相关。3.4.2胎盘PPARγ表达与血清性激素水平及炎症相关蛋白的关系全部孕妇中，胎盘PPARγ表达与雌三醇水平负相关；非早产孕妇中，胎盘PPARγ表达与前列腺素E2水平负相关，与性激素水平无关。3.4.3血清性激素水平及炎症相关蛋白与早产、胎膜早破的关系早产组孕妇血清雌三醇水平显著下降，胎膜早破组孕妇血清性激素水平未出现显著改变。推测胎膜早破与激素水平可能无关或相关性被其他因素干扰。3.4.4血清性激素水平及炎症相关蛋白与胎儿发育的关系非早产组女性婴儿中，脐血血清雌二醇高浓度组头围、腹围显著降低，且雌二醇浓度与双顶径、头围、腹围负相关；孕酮高浓度组腹围显著降低，且孕酮浓度与腹围负相关。非早产组男性婴儿中，脐血血清雌二醇高浓度组胎儿体重显著降低，且雌二醇浓度与胎儿体重负相关。我们推测，脐血雌激素水平可能与胎儿发育负相关。非早产组女性婴儿中，脐血血清前列腺素E2高浓度组胎儿身长显著增加。非早产组男性婴儿中，脐血血清基质金属蛋白酶高浓度组胎儿体重显著增加，且基质金属蛋白酶浓度与胎儿体重正相关。我们推测，脐血基质金属蛋白酶、前列腺素水平可能与胎儿发育正相关。3.5胎盘PPARγ表达在DEHP染毒影响雌鼠早产及胎鼠发育中的作用3.5.1雌鼠及胎鼠基本情况染毒期间，各组雌鼠体重变化、受孕率无显著差异。染毒组和干预组孕鼠正常妊娠率（有活胎）、活胎率、平均胎儿数、胎鼠均重均显著低于对照组。但染毒组与干预组均未观察到早产。提示DEHP染毒在未对雌鼠生长造成显著影响的剂量下已经具备严重的生殖毒性，但这种生殖毒性并不表现为早产。染毒组和干预组PPARγ蛋白表达均显著高于对照组，PPARα、PPARγ基因表达均显著低于对照组，提示DEHP可能是从转录后水平提高PPARγ的表达且与PPARγ活性抑制无关，其诱导的PPARγ蛋白水平提高可能反过来抑制了PPARs基因表达（包括PPARα和PPARγ）。人群研究也显示PPARγ与早产及脐血PAEs均正相关，我们推测其可能参与了脐血PAEs与早关的关联。3.5.2性激素水平及激素调节基因的变化及作用与对照组相比，染毒组雌二醇水平显著升高，但反映P450芳香化酶活性的睾酮与雌二醇比值显著下降，雌激素合成限速酶P450芳香化酶（CYP191a1）基因表达显著下降，反映17β-HSD家族活性的雌二醇与雌酮比值显著升高。拮抗PPARγ后，雌二醇与雌酮比值显著增加，且CYP191a1表达有所增加。提示我们前期发现的PPARγ介导的P450芳香化酶表达抑制可能参与妊娠期DEHP调节雌二醇水平，但并不是主要途径，17β-HSD1/7活性增加或17β-HSD4/8活性抑制介导的雌酮向雌二醇转换可能参与PAEs调节雌二醇水平。PAEs诱导雌激素水平升高，可能产生两方面作用：①雌激素过度增加导致雌激素与孕酮水平失衡，引起胎膜早破和早产。②人群研究发现雌二醇与胎儿发育负相关，其他人的研究也证实雌激素受体基因多态性与胎儿发育密切相关且呈性别差异，雌二醇水平增加还可能参与PAEs与胎儿发育的负相关。3.5.3炎症相关蛋白的改变及作用染毒组和干预组MMP2、MMP9基因表达显著低于对照组，干预组MMP9基因表达略高于染毒组，提示DEHP可能从转录水平抑制MMPs表达，这种抑制有PPARγ参与，人群研究同样显示血清MMPs水平与脐血PAEs暴露负相关；前列腺素E2在动物试验中未出现明显改变，但人群研究中同样与脐血PAEs暴露负相关。有研究认为，MMPs与前列腺素E2与母胎循环密切相关，其缺少可能导致母胎循环不足，影响胎儿发育。我们的人群研究也显示MMPs和前列腺素E2水平可能与胎儿发育正相关，而PPARγ可抑制基质金属蛋白酶表达且与前列腺素E2水平负相关，提示PPARγ可能通过抑制前列腺素E2和基质金属蛋白酶介导脐血PAEs浓度与胎儿生长发育的负相关。4.结论4.1病例对照研究显示脐血邻苯二甲酸酯水平与早产及胎膜早破存在关联。4.2横断面研究显示脐血邻苯二甲酸酯水平与胎儿发育负相关，动物实验也证实邻苯二甲酸二异辛酯孕期暴露致胎鼠体重下降。4.3横断面研究显示脐血邻苯二甲酸酯水平与血清雌激素（雌二醇、雌三醇）水平正相关，动物实验证实邻苯二甲酸二异辛酯孕期暴露上调雌二醇水平。4.4横断面研究显示脐血邻苯二甲酸酯水平与胎盘PPARγ蛋白表达正相关，动物实验证实邻苯二甲酸二异辛酯孕期暴露可从转录后水平上调胎盘PPARγ蛋白表达。横断面研究显示脐血邻苯二甲酸酯水平和胎盘PPARγ蛋白表达均与血清前列腺素E2、基质金属蛋白酶水平负相关，动物实验证实胎盘PPARγ蛋白表达介导邻苯二甲酸二异辛酯孕期暴露致基质金属蛋白酶表达下调。更多还原

【Abstract】 1. Back groundPhthalates Acid Ester (PAEs) are a class of synthetic chemicals which are produced inlarge volumes and used in a wide variety of industrial and common household products andare commonly found in the environment because of its widespread use in the world. Somephthalates can cross the placental barrier and be detected in cord blood. In our previousstudies, phthalates were found in water samples from the Yangtze and Jialing rivers. And9phthalates were detected in cord blood in pregnant women. Nevertheless, the effects ofprenatal exposure to phthalates on preterm delivery and fetal growth are still unknown.Other studies have suggested an association between phthalate exposure and shortergestational age and lower birth weight. but other studies found no significant relationshipbetween prenatal exposure to phthalates and birth outcomes.Our previous studies shown that the MEHP, one metabolite of DEHP, could increasethe expression of peroxisome proliferator activated receptor gamma (PPARγ) in ovariangranulosa cell. Others found phthalates can active PPARs as its ligands. PPARs in placentacan affect the hormone production, expression of protein associated with inflammation likematrix metalloproteinases (MMPs), fat and glucose metabolism. Both of them areassociated with preterm delivery and development. so we presume that the expression ofPPARs in placenta were play a role in the association of phthalates with preterm deliveryand fetal development.In this study we used both epidemiology and experiments research to investigate theassociations of phthalate levels in cord blood with fetal growth and preterm delivery and themechanism of them.2. Material and methods2.1epidemiology experiments research for the relationship of phthalate levels in cord blood with fetal growth and preterm delivery.2.1.1Study population. Two-hundred and seven volunteers were recruited atSouthwest Hospital in Chongqing, Southwest China. None of the participants had a familyor personal history of occupational exposure to phthalates. We administered a questionnaireto the participants after labor to obtain information on socio-demographic characteristics,medical history and lifestyle factors. Delivery characteristics and birth outcomes wereobtained from the perinatal database of Southwest Hospital, including preterm delivery(defined as gestational age less than37weeks), premature rupture of membranes (PROM),chorioamnionitis, intrahepatic cholestasis of pregnancy (ICP), placenta previa,pregnancy-induced hypertension syndrome (PIH, including preeclampsia), gestationaldiabetes mellitus (GDM), abruption placentae, intravenous transfusion history(defined asbeen put on a drip within the last week before labor), gestational age, birth weight, birthlength, head circumference (HC), biparietal diameter (BPD), abdominal circumference (AC)and femur length (FL). Ten millilitre of cord blood were obtained from subjects within10min of delivery,5ml was stored in a heparinized glass container for phthalatesmeasurements and5ml was separated the serum for hormonal assess. The placental samplefrom each pregnant were collected to detect the protein expression of PPARγ.2.1.2Phthalates measurements. Phthalates in cord blood were extracted by liquidextraction and analyzed by GC/MS. Fifteen phthalates were identified and quantitated bytheir characteristic retention time, including Dimethyl phthalate (DMP), Diethyl phthalate(DEP), Diisobutyl phthalate (DIBP), Dibutyl phthalate (DBP), bis (2-methoxyethyl)phthalate (DMEP), bis (4-methyl-2-pentyl) phthalate (BMPP), bis (2-ethoxyethyl) phthalate(DEEP), Diamyl phthalate (DPP), Dihexyl phthalate (DNHP), Benzyl butyl phthalate(BBP), bis (2-n-butoxyethyl) phthalate (DBEP), Dicyclohexyl phthalate (DCHP), bis(2-ethyl hexyl) phthalate (DEHP), Di-n-octyl phthalate (DNOP) and Dinonyl phthalate(DNP).2.1.3Hormonal assess. The matrix metalloproteinases (MMPs) and prostaglandin E2(PGE2) in serum were assessed by ELISA method and the progesterone (P), estradiol (E2)and estriol (E3) were assessed by radioimmunoassay.2.1.4Protein assess. The protein was extract from placenta by RIPA Lysis Buffer andthe expression of PPARγ assessed by Western Blot and BeyoECL Plus. 2.1.5Statistical analyses. All statistical analyses were performed with SPSS forwindows version18.0. Independent sample T-test and Chi-square test were used to analyzethe characteristics of pregnant women in preterm and term delivery group and theiroffspring. We used linear regressions and Independent sample T-test to estimate theassociation of phthalates with expression of PPARγ, hormonal, gestational age and birthoutcomes, and used binary logistic regression and Independent sample T-test to estimate theassociation of preterm delivery with phthalates, expression of PPARγ in placenta and serumhormonal.2.2The role of PPARγ in the adverse effects of DEHP in pregnant rats.2.2.1A total60female Sprague-Dawley rats (6-7weeks old) were randomized into2groups,20in control group which received by gavage corn oil,40in exposure group whichreceived by gavage2g/kg of DEHP for27days. Then the exposure group were equallyrandomized into2group (20per group) and respectively received by gavage2g/kg ofDEHP (exposure group), DEHP2ml/kg+0.5mg/kg GW9662（antagonist of PPARγ）(intervention group) for3days and mate with health male rats. After one week for mating,the pregnant rats were still gavaged corn oil (the control group),2g/kg of DEHP (theexposure group), DEHP2ml/kg+0.5mg/kg GW9662(the intervention group). Themorning in which a vaginal plug was found was designated day1of gestation. Treated ratswere sacrificed between at day20of gestation. The uteri were excised immediately toevaluate the effect of DEHP and GW9662on fetal development. Blood samples werecollected to assess the effect of Bap and DBP on plasma hormonal concentrations. Placentawere isolated for the detection of protein and mRNA expression.2.2.2Hormonal assess. The estrone (E1), testesterone (T), dehydroepi androsteronesulfate (DHEAS),5-androstenedione (ASD) prostaglandin E2(PGE2) and prostaglandinF2a (PGF2a) in serum were assessed by ELISA method and the progesterone (P), estradiol(E2) were assessed by radioimmunoassay.2.2.3Protein and mRNA assess. The protein was extract from placenta by RIPA LysisBuffer and the expression of PPARγ was assessed by Western Blot and BeyoECL Plus. ThemRNA was extract from placenta by Trizol and the expression of PPARγ, PPARα, CYP191,MMP2and MMP9were assessed by Realtime RT-PCR (SYBR Green I).2.2.4Statistical analyses. All statistical analyses were performed with SPSS for windows version18.0. Independent sample T-test and Chi-square test were used to analyzethe effect of DEHP and GW9662.3. Results and discussion3.1Cord blood phthalate levels and preterm delivery.Cord blood levels of14phthalates (except DCHP) were increased in preterm group.Cord blood levels of phthalates (except DNHP, DMEP, DBEP and DCHP) were increased inPROM group which was associated with preterm. But the cord blood levels of13phthalates(except DCHP and DNHP) were also increased in preterm group in non-PROM pregnants.The intravenous transfution history was associated with preterm delivery and3phthalates(DMP, DEEP and BMPP). And14phthalates (except BMPP, DNHP and DCHP) were alsoassociated with preterm delivery in the group which hadn t has the intravenous transfutionhistory. Those indicates that the PROM and the intravenous transfution history may play arole in the between phthalates in cord blood and preterm, but not the key one.3.2Cord blood phthalate levels and birth outcomes.Higher phthalate concentrations in cord blood had decreased birth weight, birth length,abdominal circumference, femur length, biparietal diameter, head circumference, both inmale and female infants, respectively. Majority of these associations disappeared afteradjusting for gestational age or analysis in term group. And the preterm delivery andgestational age reduction were also associated with birth outcomes. These suggested thatthe phthalates in cord blood may have adverse effects on fetal development, which may betotally dependent on the gestational duration reduction and preterm delivery. After adjustingfor gestational age, the sensitive to phthalates and sensitive indicator were differencesbetween the genders. These indicate the gender differencesmay exist in the associationsbetween phthalates exposure and birth outcomes.3.3The associations of protein expression of PPARγ in placenta with phthalates incord blood, preterm, and birth outcomes.3.3.1The associations of protein expression of PPARγ in placenta with phthalates incord blood.Higher phthalate concentrations in cord blood had significantly higher proteinexpression of PPARγ in placenta. And the phthalate levels were positive correlation withprotein expression of PPARγ in placenta. These associations were also existed in term and preterm group, respectively. These indicated that the phthalate levels were directly positivecorrelation with protein expression of PPARγ in placenta.3.3.2The associations of protein expression of PPARγ in placenta with preterm.The protein expression of PPARγ were significantly increased in preterm and PROMgroup. And in non-PROM pregnant, the protein expression of PPARγ were alsosignificantly increased in preterm. These indicated that the protein expression of PPARγmay associated with preterm delivery which may partially dependent on PROM and theprotein expression of PPARγ in placenta may play a role in the associations betweenphthalates in cord blood and preterm delivery.3.3.3The associations of protein expression of PPARγ in placenta with birth outcomes.The protein expression of PPARγ was negative correlated with birth weight, birthlength, abdominal circumference, femur length, biparietal diameter, head circumference,both in male and female infants, respectively. Majority of these associations disappearedwhen analysis in term group. These suggested that, like the phthalates in cord blood, theprotein expression of PPARγ may have adverse effects on fetal development, which may bepartially to dependent on the preterm delivery.3.4The associations of serum hormonal and inflammation-associated protein withphthalates in cord blood, protein expression of PPARγ in placenta, preterm, and birthoutcomes3.4.1The associations of phthalates in cord blood with serum hormonal andinflammation-associated protein.In total pregnant, higher phthalate concentrations in cord blood had significantlyhigher progesterone levels, and lower estriol, estradiol, matrix metalloproteinases andprostaglandin E2levels. But in term and preterm group, respectively, the phthalateconcentrations in cord blood was positive correlated with estriol and estradiol level. Theseindicates that the phthalate concentrations in cord blood was positive correlated with estrins(include estriol and estradiol) and progesterone level, and negative correlated withinflammation-associated protein (include matrix metalloproteinases and prostaglandin E2).3.4.2The associations of protein expression of PPARγ in placenta with serumhormonal and inflammation-associated protein.The protein expression of PPARγ in placenta was negatively associated with prostaglandin E2in term group.3.4.3The associations of serum hormonal and inflammation-associated protein withpreterm delivery and PROM.The preterm group had significantly lower estriol level. And the PROM group had notany significantly change in serum hormonal and inflammation-associated protein.3.4.3The associations of serum hormonal and inflammation-associated protein withbirth outcomes.The estradiol level was negatively related with abdominal circumference, biparietaldiameter and head circumference; and the progesterone level was negatively related withabdominal circumference; and prostaglandin E2was positively related with birth length infemale infants in term group. The estradiol level was negatively related with birth weight;and matrix metalloproteinases was positively related with birth length in male infants interm group. Because the phthalates in cord blood are positive correlated with estriol andnegative correlated with matrix metalloproteinases and prostaglandin E2, and the proteinexpression of PPARγ in placenta was negatively associated with prostaglandin E2. wesupposed that serum hormonal and inflammation-associated protein a role in theassociations of phthalates in cord blood and the protein expression of PPARγ in placentawith preterm.3.5The role of PPARγ in the reproductive toxicity of DEHP in pregnant rats.3.5.1The effect of DEHP and PPARγ on rat growth and fetuses development.The exposure and intervention group had significangt changes in body gains ofpregnant rats versus control group before and after pregnancy. And the pregnancy Rate,viable fetuses rate, fetuses number per pregnant rat and fetal weight were significantlydecreased in both exposure and intervention group. Indicates the exposure dose used in thestudy had serious reproductive toxicity but didn’t affacted the growth of pregnent rats.3.5.2The expression of PPARγ in placenta in DEHP exposed pregnant rats.The protein expression of PPARγ in placenta were significantly increased and themRNA expression of PPARγ and PPARα, another isoform of PPARs, in placenta weresignificantly decreased in both exposure and intervention group. These indicates the DEHPmay up-regulate the protain expression of PPARγ after Transcription, not Transcription level.3.5.3The role of PPARγ in hormonal regulation of DEHP in pregnent rats. The estradiol level was significantly increased and the ratio of testesterone to estradiol,which reflected the active of P450Arom was significantly decreased when exposoed toDEHP. But the mRNA expression of P450Arom was significantly decreased in the exposuregroup. And the ratio of estradiol to estrone, which reflected the active of17β-HSDs wassignificantly increased. When inhibiting the activity of PPARγ by GW9662, the mRNAexpression of P450Arom increased and ratio of estradiol to estrone increased moresignificantly. So wo supposed that the inhibition of P450Arom expression by PPARγ mayplay a role in hormonal regulation of DEHP, but not the key one. The change of17β-HSDsactivity may be more important in hormonal regulation of DEHP. And the DEHP canincrease the estrins level, which may play a role in the association of phthalates in cordblood with preterm and birth outcomes.3.5.4The role of PPARγ in regulation of inflammation-associated protein in DEHPexposed pregnent rats.The mRNA expression of MMP2, MMP9were significantly decreased when exposedto DEHP, and the prostaglandin E2and prostaglandin F2a levels in serum were not changed.When inhibiting the activity of PPARγ by GW9662, the mRNA expression of MMP9wereincreased. These indicated that the PPARγ play a role in regulating the mRNA expression ofMMP9by DEHP.The MMPs and prostaglandin E2were important to fetal development, which werepositively correlated to birth outcomes in our epidemiologic study. Our study suggested thatthe phthalates exposure may down regulate the MMPs and the prostagland in E2partiallythrough PPARγ pathway, which may play a role in the association of phthalates exposureand birth outcomes.4. ConclusionIn our study, there is a strong evidence of the association between phthalate levels incord blood and preterm delivery and birth outcomes. The expression of PPARγ in placentamay play a role in there association through alterating the serum sexual hormone andinflammation-associated protein like MMPs and prostaglandin E2.更多还原