胎盘超微结构和呼吸链酶活性与子痫前期关系的研究

目的:研究胎盘组织超微结构的病理变化、呼吸链酶复合体活性变化与子痫前期的关系.方法:选择轻度子痫前期孕妇30例(轻度子痫前期组)、重度子痫前期孕妇15例(重度子痫前期组)和正常孕妇20例(正常妊娠组)行胎盘透射电镜观察,比较两组超微结构,同时采用紫外分光光度计法测定胎盘组织线粒体中两种呼吸链酶复合体(线粒体酶复合体Ⅰ、Ⅳ)的活性进行分析.结果:轻度子痫前期组与正常妊娠组胎盘超微结构比较,差异无统计学意义(P>0.05),重度子痫前期组与正常妊娠组和轻度子痫前期组胎盘超微结构比较,差异有统计学意义(P<0.05);重度子痫前期组胎盘组织中线粒体酶复合体Ⅳ的活性比正常妊娠组、轻度子痫前期组低,差异有高度统计学意义(P<0.01),轻度子痫前期组线粒体酶复合体Ⅳ的活性与正常妊娠组相比,差异无统计学意义(P>0.05).线粒体酶复合体Ⅰ在3组间差异无统计学意义(P>0.05).结论:子痫前期患者胎盘超微结构的变化、线粒体酶复合体Ⅳ活性降低,提示子痫前期发病机制中有能量代谢传递障碍,线粒体能量产生异常参与子痫前期的发病过程.     

脂联素在子痫前期患者胎盘组织中的表达与其发病的关系

目的 探讨脂联素在子痫前期患者胎盘组织中的表达与其发病的关系.方法 采用免疫组化链霉菌抗生物蛋白-过氧化物连接(SP)法及RT-PCR技术,检测20例正常足月妊娠孕妇(正常妊娠组)、12例轻度子痫前期(轻度子痫前期组)及22例重度子痫前期(重度子痫前期组)患者胎盘组织中脂联素蛋白及其mRNA的表达,并分析其与子痫前期发病的关系.结果 (1)3组孕妇胎盘绒毛合体滋养细胞及细胞滋养细胞胞质内脂联素蛋白均呈阳性表达,且各组内胎盘母面及子面脂联素蛋白的表达水平相互比较,差异均无统计学意义(P＞0.05).(2)重度子痫前期组胎盘组织中脂联素蛋白的表达水平(30 984±14 604)低于轻度子痫前期组(58 360±8910)及正常妊娠组(53 246±17 554),差异均有统计学意义(P＜0.01).重度子痫前期组中妊娠足月者胎盘组织中脂联素蛋白的表达水平(38 890±20 386)与未足月者(29 319±8997)比较,差异无统计学意义(P＞0.05);但与正常妊娠组比较,差异有统计学意义(P＜0.05).(3)3组孕妇胎盘组织中均有脂联素mRNA的表达.其中重度子痫前期组胎盘组织中脂联素mRNA表达水平(1.0±0.2)低于轻度子痫前期组(2.9±0.8)及正常妊娠组(3.3±1.1),差异有统计学意义(P＜0.05).结论 重度子痫前期患者胎盘组织中脂联素mRNA表达水平下降导致其蛋白表达水平也下降,提示脂联素的异常表达参与了子痫前期的发病.     

重度子痫前期孕妇脂氧素A4及11β羟基类固醇脱氢酶2的表达及意义

目的:检测重度子痫前期孕妇外周血中脂氧素A4(LXA4)水平以及胎盘组织中11β羟基类固醇脱氢酶2(11β-HSD2)表达,探讨LXA4和11β-HSD2参与重度子痫前期发生的机制。方法:选择在本院产前检查并分娩的妊娠妇女共45例,其中正常妊娠组20例,重度子痫前期组25例。ELISA法测定孕妇外周血血浆中LXA4水平;酶化学发光分析法检测孕妇血清中游离皮质醇浓度;免疫组织化学染色法检测胎盘组织11β-HSD2蛋白表达;实时荧光定量RT-PCR法测定胎盘组织11β-HSD2 mRNA的表达;分析LXA4与11β-HSD2蛋白或mRNA、皮质醇与平均动脉压之间的相关性。结果:重度子痫前期组与正常妊娠组相比,LXA4水平明显降低(P<0.05),皮质醇浓度虽有升高,但无显著性差异(P>0.05);胎盘组织中11β-HSD2蛋白及mRNA表达均显著低于正常妊娠组(P<0.05);两组孕妇的LXA4水平与11β-HSD2蛋白表达量、LXA4水平与11β-HSD2 mRNA表达量、皮质醇与平均动脉压均无相关性(P均>0.05)。结论:LXA4和11β-HSD2参与重度子痫前期的发生。     

COX-1,COX-2在子痫前期患者胎盘和脐带组织的表达

目的:研究环氧合酶-1(COX-1),环氧合酶-2(COX-2)在子痫前期患者胎盘和脐带组织的表达,探讨它们在子痫前期发病机制中的作用。方法:用免疫组化SP法检测50例子痫前期患者(轻度30例,重度20例)胎盘和脐带组织中COX-1,COX-2表达,20例正常孕妇为对照组。结果:(1)COX-1、COX-2在轻、重度子痫前期组的表达水平均明显高于对照组,两两比较,差异有统计学意义(P<0.05)。COX-1在重度子痫前期组和轻度子痫前期组中表达均无统计学差异(P>0.05),而COX-2重度子痫前期组与轻度子痫前期组的差异有统计学意义(P<0.05)。结论:COX-1,COX-2参与了子痫前期胎盘螺旋小动脉和脐带血管动脉粥样硬化的发展过程。     

长链脂肪酸氧化酶mRNA在正常妊娠及重度子痫前期胎盘中的表达

目的研究长链羟酰基辅酶A脱氢酶(long-chain3-hydroxyacyl-CoAdehydrogenase,LCHAD)在正常妊娠不同孕期绒毛或胎盘组织的表达情况以及在伴有肝脏损害和不伴有肝脏损害重度子痫前期胎盘的表达差异。方法应用原位杂交和RT-PCR方法对早孕绒毛组织(10例)、妊娠中期胎盘组织(10例)、正常妊娠晚期胎盘组织(10例)及32例重度子痫前期胎盘组织进行LCHAD基因的定位表达及半定量测定。结果原位杂交实验显示正常妊娠早、中、晚期绒毛或胎盘组织及重度子痫前期胎盘组织滋养细胞中存在LCHAD阳性表达。RT-PCR实验显示①妊娠早期绒毛LCHAD表达与妊娠中期比较,P=0.844;妊娠早期绒毛LCHAD表达高于晚期胎盘,P=0.020;妊娠中期胎盘LCHAD表达也高于晚期胎盘P=0.026;②发病孕周≤34周早发型重度子痫前期伴肝损害胎盘组织中LCHAD表达均值为(0.449±0.038),不伴肝损害LCHAD表达均值为(0.482±0.042),伴肝损害较不伴肝损害者表达有减弱,但两组比较,P=0.084;发病孕周≤34周早发型重度子痫前期伴肝损害LCHAD表达量与正常晚期比较,P=0.05,而不伴肝损害重度子痫前期LCHAD表达量与正常晚期比较,P=0.775。结论本研究显示在妊娠的早中晚期滋养细胞中均存在长链脂肪酸氧化代谢,妊娠早中期LCHAD的mRNA表达高于妊娠晚期;早发型重度子痫前期伴肝损害胎盘组织中LCHAD表达均值与不伴有肝损害者比较虽无统计学差异,但是有明显降低趋势。提示长链脂肪酸氧化代谢对子痫前期伴发肝脏损害的影响还有待酶活性和蛋白水平以及代谢调节方面的深入研究。     

胎盘生长因子与子痫前期发病及一氧化氮关系的研究

目的:探讨胎盘生长因子(PLGF)在子痫前期发病中的作用及其与一氧化氮的关系。方法:选择妊娠期高血压疾病患者45例,其中妊娠期高血压10例,轻度子痫前期12例,重度23例;选择同期正常妊娠妇女20例作为对照组。采用免疫组织化学染色法和逆转录-聚合酶链式反应(RT-PCR)检测两组患者胎盘PLGF蛋白及mRNA的表达。采用硝酸盐还原酶法测定两组胎盘组织NO浓度的变化。结果:(1)免疫组化结果显示,轻度和重度子痫前期的胎盘绒毛合体滋养细胞、绒毛间质PLGF表达均显著低于正常妊娠组(P<0.05),妊娠期高血压组与正常组无差别;PLGF在妊娠期高血压、子痫前期组及正常妊娠组分布范围基本一致,主要分布在绒毛合体滋养细胞和间质细胞胞浆,部分血管合体膜上也有表达;(2)轻、重度子痫前期胎盘组织PLGF mRNA平均灰度分别为3.33±0.39、1.97±0.29,显著低于正常妊娠组的平均灰度4.87±0.60(P<0.01);(3)轻、重度子痫前期胎盘组织中NO浓度分别为8.20±5.56μmol/g、6.46±2.25μmol/g,显著低于对照组18.10±7.12μmol/g(P<0.05);妊娠期高血压组胎盘组织NO浓度与对照组差异无显著性;(4)胎盘组织中胎盘生长因子表达水平与胎盘组织NO浓度呈显著正相关(r=0.54,P<0.05)。结论:子痫前期胎盘组织中胎盘生长因子水平降低,NO浓度下降,可能在子痫前期的发病中起一定作用。     

重度子痫前期患者可溶性人类白细胞抗原G在血浆和胎盘组织中的表达

目的:研究可溶性人类白细胞抗原G(sHLA-G)在孕晚期重度子痫前期患者血浆的含量水平及其亚型mRNA在胎盘组织中的表达,探讨sHLA-G的来源及其在子痫前期发病中的意义.方法:选择2008年10月至2009年4月第四军医大学唐都医院重度子痫前期患者20例(重度子痫前期组),20例正常足月妊娠者(正常足月妊娠组)及20例正常未妊娠者(正常未妊娠组).采用酶联免疫吸附法(ELISA)检测血浆sHLA-G的含量;采用实时荧光定量PCR法比较sHLA-G亚型(HLA-G5、G6)mRNA在重度子痫前期组和正常足月妊娠组胎盘组织表达的差异.结果:①重度子痫前期组血浆sHLA-G含量水平明显低于正常足月妊娠组(P<0.05),正常未妊娠组与重度子痫前期组和正常足月妊娠组比较,差异有统计学意义(P<0.05).②重度子痫前期组胎盘HLA-G5、G6 mRNA相对表达量(0.32)明显低于正常足月妊娠组相对表达量(1.0).结论:重度子痫前期患者血浆sHLA-G及其胎盘组织中亚型mRNA含量水平均降低,可能与子痫前期发病有关,胎盘可能是sHLA-G的来源之一.     

人类白细胞抗原G的表达与子痫前期发病的关系

目的 通过检测子痫前期孕妇相关组织中人类白细胞抗原G (HLA-G)的表达,探讨其与子痫前期发病的关系.方法 选择2009年3月至12月在陕西省妇幼保健院产科住院分娩的子痫前期孕妇30例,根据病情分为轻度子痫前期组8例,重度子痫前期组22例.选择同期健康孕妇30例为对照组.3组孕妇均以剖宫产方式分娩.采用ELISA法检测孕妇外周血、脐血及羊水中的可溶性HLA-G(sHLA-G)水平;采用蛋白印迹法检测胎盘、胎膜及脐带组织中HLA-G蛋白的表达.结果 (1)各组孕妇外周血、脐血、羊水中sHLA-G水平:轻、重度子痫前期组孕妇外周血sHLA-G水平分别为(50±14)、(30±6) μg/L,新生儿脐血中sHLA-G水平分别为(34±10)、( 26±8)μg/L,均明显低于对照组的(100±16)、(70±9) μg/L,分别与对照组比较,差异均有统计学意义(P＜0.01);重度子痫前期组与轻度子痫前期组比较,差异也有统计学意义(P＜0.01).重度子痫前期组新生儿脐血中sHLA-G水平虽低于轻度子痫前期组,但差异无统计学意义(P＞0.05).轻、重度子痫前期组孕妇羊水中sHLA-G水平分别为(26±7)、( 25±5) μg/L,均低于对照组的(27±6)μg/L,但分别与对照组比较,差异无统计学意义(P＞0.05);轻度子痫前期组与重度子痫前期组比较,差异也无统计学意义(P＞0.05).(2)各组孕妇胎盘、胎膜及脐带组织中的HLA-G蛋白表达:对照组胎盘组织中HLA-G蛋白表达水平为1.59±0.36,胎膜组织中表达水平为0.42±0.09,胎盘组织中的HLA-G蛋白表达水平明显高于胎膜组织,两者比较,差异有统计学意义(P＜0.05);对照组脐带组织中HLA-G蛋白表达水平为0.24±0.17,分别与胎盘及胎膜组织中的HLA-G蛋白表达水平比较,差异均有统计学意义(P＜0.01).轻度子痫前期组胎盘组织中HLA-G蛋白表达水平为0.78±0.21,重度子痫前期组为0.29±0.17,分别与对照组比较,差异均有统计学意义(P＜0.01).轻、重度子痫前期组孕妇胎膜及脐带组织中均未检测出HLA-G蛋白的表达.结论 与健康孕妇相比,HLA-G在子痫前期孕妇外周血、脐血及胎盘组织中呈明显低表达,HLA-G表达水平的异常可能与子痫前期的发病有关.     

HtrA1在子痫前期孕妇血清、胎盘组织中的表达及意义

目的:探讨HtrA1(high temperature requirement A1)在子痫前期孕妇血清、胎盘组织中的表达及意义。方法:HtrA1属分泌型蛋白,可采用酶联免疫吸附法(ELISA)测定同期住院孕妇的空腹血清HtrA1浓度,其中正常晚期妊娠30例、轻度子痫前期25例、重度子痫前期20例;同时应用免疫组化SABC法检测胎盘组织中HtrA1的表达。结果:正常晚期妊娠、轻度子痫前期及重度子痫前期孕妇血清HtrA1浓度分别为(160.63±32.52)pg/ml、(210.82±32.52)pg/ml及(233.68±38.61)pg/ml,组间差异显著(P<0.05);正常晚期妊娠、轻度子痫前期及重度子痫前期孕妇胎盘组织中HtrA的平均灰度值分别为156.03±5.07、149.89±2.69及140.05±4.96,组间差异显著(P<0.05)。结论:随着病情加重,孕妇血清及胎盘组织中HtrA1的表达显著增高。HtrA1与子痫前期的发生、发展有关。     

子痫前期孕妇血清和胎盘组织中干扰素-γ诱导蛋白10的表达及意义

目的:检测子痫前期孕妇胎盘组织及血清中干扰素-γ诱导蛋白10(IP-10)的表达情况,探讨其与子痫前期发病的关系。方法:收集正常妊娠、轻度子痫前期、重度子痫前期孕妇的胎盘组织及血清,采用ELISA、实时荧光定量PCR及Western blot方法检测IP-10在3组孕妇胎盘组织及血清中的表达变化。结果:ELISA结果显示,与正常对照组相比,轻度子痫前期组、重度子痫前期组孕妇血清中IP-10表达均明显增加(P均<0.05);实时荧光定量PCR及Western blot结果显示,与正常对照组相比,轻度子痫前期组、重度子痫前期组孕妇胎盘组织中IP-10 mRNA及蛋白表达水平均有不同程度增高(P均<0.05)。结论:子痫前期孕妇胎盘及血清中IP-10表达升高,可能与子痫前期的发病有关。     

Analysis of the metabolic footprint and tissue metabolome of placental villous explants cultured at different oxygen tensions reveals novel redox biomarkers

Pre-eclampsia (PE) is a multi-system disorder of pregnancy hypothesised to arise from circulating factors derived from an unhealthy placenta. Some changes in placental phenotype seen in PE can be reproduced by culture in altered oxygen (O(2)) tension. Currently, these circulating factors are unidentified, partly due to the complexity of maternal plasma. Investigation of factors released from placental tissue provides a potential method to identify bioactive compounds. Experimental strategies to study compounds present in a biological system have expanded greatly in recent years. Metabolomics can detect and identify endogenous and secreted metabolites. We aimed to determine whether metabolites could be identified in placental cultures with acceptable experimental variability and to determine whether altered O(2) tension affects the composition of the placental metabolome. In this study we used gas-chromatography-mass spectroscopy to determine the presence of metabolites in conditioned culture medium (CCM) and tissue lysates of placental villous explants cultured in 1, 6 and 20% atmospheric O(2) for 96h. This experimental strategy had an intra-assay variation of 6.1-11.6%. Intra and inter-placental variability were 15.7-35.8% and 44.8-46.2% respectively. Metabolic differences were identified between samples cultured in 1, 6 and 20% O(2) in both CCM and tissue lysate. Differentially expressed metabolites included: 2-deoxyribose, threitol or erythritol and hexadecanoic acid. We conclude that metabolomic strategies offer a novel approach to investigate placental function. When conducted under carefully controlled conditions, with appropriate statistical analysis, metabolic differences can be identified in placental explants in response to altered O(2) tension. Metabolomics could be used to identify changes in conditions associated with placental pathology.     

Heme Oxygenase Expression in Human Placental Villous Tissue in Response to Exposure to in vitro Ischemia-Reperfusion Injury

Heme oxygenases (HO-1 and HO-2) are responsible for the production of carbon monoxide, a vasodilator. The products of heme oxygenase are also anti-oxidants. HO is expressed within the placenta and is important in controlling placental blood flow. HO can be sensitive to oxygen, with responses differing depending on the cell type. Recent studies have suggested that in preeclampsia, the placenta would be subjected to fluctuations in oxygen tension analogous to an ischemia-reperfusion injury. Thus the present study tested the hypothesis that HO-1 and or HO-2 expression in placental villous explants would be altered by an ischemic-reperfusion insult. Human term placental explants were exposed to hypoxia then re-oxygenation in 5% or 20% O₂ or repeated cycles of hypoxia-re-oxygenation. HO protein concentrations were assessed by Western blotting. No changes in HO-1 or HO-2 were found with any treatment protocol. Chemical induction of HO-1 was possible in explants showing that HO-1 induction in explants is possible. The results suggest that cells in term placental villous tissue do not respond to hypoxia-re-oxygenation by altering the amount of HO-1 or HO-2 protein.     

Review: The effects of oxygen on normal and pre-eclamptic placental tissue--insights from metabolomics

Placental dysfunction is central to many complications of human pregnancy including pre-eclampsia (PE), intra-uterine growth restriction (IUGR) and stillbirth. The precise molecular pathophysiology of placental dysfunction in these conditions is not known, although oxidative and nitrative stresses have been implicated. Metabolites are low molecular weight chemicals which play an important role in biological function, primarily through metabolism and regulation of biological processes. The holistic study of metabolites, defined as metabolomics or metabolic profiling, has the objective to detect and identify all, or a large complement of all metabolites. Metabolomics is applied to discover new knowledge regarding biological processes and systems. We hypothesised that a metabolomic strategy could (1) provide a reproducible technique to investigate the intracellular metabolism of placental tissue and also metabolites consumed from or secreted in to the extracellular 'metabolic footprint' of in vitro culture systems (2) identify metabolic related differences in placental tissue culture systems subjected to perturbations in oxygen tension and from pregnancies complicated by PE. We review our early studies which demonstrate that a reproducible experimental protocol is required, including the preparation of culture medium and the site of the placenta applied for sampling tissue. We have detected changes in the intracellular metabolome and metabolic footprint of placental tissue in response to altered oxygen tension and PE. We have demonstrated that placental tissue from uncomplicated pregnancies cultured in 1% oxygen (hypoxia) had metabolic similarities to explants from PE pregnancies cultured at 6% oxygen (normoxia). Metabolites requiring further study include lipids, glutamate and glutamine and metabolites related to tryptophan, leukotriene and prostaglandin metabolism. Metabolomics has the potential to identify changes in clinical conditions, such as PE, that are associated with placental molecular pathophysiology.     

Decreased tryptophan catabolism by placental indoleamine 2,3-dioxygenase in preeclampsia

OBJECTIVE: Tryptophan degradation and depletion resulting from activation of indoleamine 2,3-dioxygenase is characteristic of inflammatory reactions and may control their intensity. Normal third-trimester pregnancy is characterized by a maternal systemic inflammatory response, which is more intense in preeclampsia. Therefore, we studied tryptophan metabolism in pregnant women, with or without preeclampsia, as well as expression and function of placental indoleamine 2,3-dioxygenase. STUDY DESIGN: Plasma concentrations of tryptophan and kynurenine in women with preeclampsia, appropriately matched women with normal pregnancy, and healthy nonpregnant women were measured. Placental enzymatic activity and messenger RNA (mRNA) expression level of indoleamine 2,3-dioxygenase were determined from the same placental material. Peripheral blood mononuclear cell proliferation was determined in medium conditioned by prior culture with villous tissue. RESULTS: The plasma ratio of kynurenine to tryptophan, an in vivo index of enzyme activity, was significantly increased compared with nonpregnant controls in normal pregnancy but not in preeclampsia. The activity and mRNA expression level of indoleamine 2,3-dioxygenase in term placentas were significantly lower in preeclampsia. Medium conditioned by culture of villous tissue explants of preeclampsia was less effective in inhibiting peripheral blood mononuclear cell proliferation compared with that of normal pregnancy. CONCLUSION: These observations suggest that in preeclampsia, reduced placental indoleamine 2,3-dioxygenase activity (and relatively elevated plasma tryptophan) could cause dysregulation of the inflammatory response that is intrinsic to normal pregnancy. This may contribute to the pathogenesis of the maternal syndrome of preeclampsia.     

Interleukin-11 up-regulates endoplasmic reticulum stress induced target,PDIA4 in human first trimester placenta and in vivo in mice

Interleukin (IL)11 is a crucial factor for human trophoblast function and placentation. Elevated levels are associated with pregnancy complications including preeclampsia, intrauterine growth restriction (IUGR) and preterm birth. However, the regulation of IL11 in the placenta has not been investigated. We examined the effect of pro-inflammatory cytokines IL1β and TNFα, as well as low oxygen tension (2%) on IL11 levels in first trimester placental villous explants. IL1β upregulated IL11 mRNA and protein, while TNFα and low oxygen had no effect. Using mass spectrometry, we identified protein disulfide isomerase 4 (PDIA4) in IL11-treated first trimester human placental explants (100 ng/ml, 24 h, n = 3), but not PBS control tissues. PDIA4 is a member of the PDI family, also known as endoplasmic reticulum (ER) stress protein (ERP)72. We previously identified GRP78 (a master regulator for ER stress) in human placenta for the first time and demonstrated that IL11 up-regulates GRP78 in the placenta. In this report, we demonstrated that IL11 upregulates PDIA4 protein in human placental villous tissue, HTR8-SVneo trophoblasts (cell line) and in vivo in IL11-treated mouse placenta. We aimed to determine whether IL11 upregulates other ER stress proteins in human first trimester placental villous. IL11 stimulated ERP44, but not GRP94, or PDI. Placental endoplasmic reticulum stress has been postulated in the pathophysiology of preeclampsia and IUGR, but its activation remains elusive. Together, these data suggest that IL11 could trigger an ER stress response in the placenta, which may contribute to obstetric complications such as preeclampsia.     

Hypoxia Upregulates GCM1 in Human Placenta Explants

Studies in mice have shown that a variety of genes, including GCM1, regulate the differentiation of trophoblast cells. GCM1 is also expressed in the human placenta. Placental GCM1 protein has been reported to be reduced in preeclampsia. In view of the close link between hypoxia, hypoxia-reoxygenation, preeclampsia, placental development and the reported reduction in GCM1, we hypothesised that GCM1 expression would be affected by hypoxia. The aim was to determine the effects of hypoxia on GCM1 expression in the human placenta. Two model systems were used; villous explants and cultured primary cytotrophoblast cells. GCM1 protein was detectable at low levels in explants maintained for 7 h in 8 or 20% O₂. A striking increase in GCM1 was observed when villous explants were incubated for 1h in 1% O₂ (p < 0.002). Incubation of explants for 1 h in 1% O₂ followed by re-oxygenation for 6 h in 8 or 20% O₂ resulted in a decline in GCM1 protein. Expression of GCM1 was also analysed in primary cytotrophoblast and syncytiotrophoblast cultured in 8 or 20% O₂ or reduced oxygen (1–2% O₂) conditions. GCM1 protein was not detected in any of the experimental conditions used. This study has shown that acute hypoxia increases GCM-1 protein in villous explants. The experiments with purified trophoblast do not support a role for hypoxia increasing GCM-1 in these cells under the conditions used. The present findings are in keeping with the complex effects of oxygen depending on the conditions used. The hypoxic effects on GCM1 warrant further investigation as they may provide further information on the pathogenesis of preeclampsia.     

Placental and trophoblastic in vitro models to study preventive and therapeutic agents for preeclampsia

In the field of preeclampsia, enormous efforts are ongoing to identify biomarkers predicting the syndrome already in the first trimester of pregnancy. At the same time, there is the need for in vitro models to test such biomarkers prior to their use in clinical trials. In addition, in vitro models may accelerate the development and evaluation of the benefit of any putative therapeutics. Therefore, in vitro systems have been established to evaluate the release of biomarkers and measure the effect of putative therapeutics using placental villous explants as well as the choriocarcinoma cell line BeWo. For explants, a cryogenic method to freeze, transport and thaw villous explants was developed to use such tissues for a multi-site tissue culture evaluation.Here we focus on three out of many in vitro models that have been established for human placental trophoblast. (1) Choriocarcinoma cell lines such as BeWo, Jeg-3 and Jar cells (2) isolated primary trophoblast cells, and (2) villous explants from normal placentas delivered at term. Cell lines were used to assess the effect of differentiation and fusion on the expression and release of a preeclampsia marker (placental protein 13; PP13) and beta-hCG. Moreover, cell lines were used to study the effect of putative preeclampsia therapeutics such as vitamins C and E, heparin and aspirin on marker release and viability. Cryopreservation of villous explants enabled shipment to a remote laboratory and testing of parameters in different countries using explants from one and the same placenta.Recently published data make it tempting to speculate that the choriocarcinoma cell line BeWo as well as fresh and cryogenically stored placental villous explants may well serve as in vitro models to study preventive and therapeutic agents in the field of preeclampsia.     

Does oxygen concentration affect shedding of trophoblastic debris or production of inflammatory mediators from first trimester human placenta?

Preeclampsia is a major cause of pregnancy morbidity and mortality. It is hypothesised that necrotic syncytial knots and/or inflammatory factors released from the placenta into the maternal circulation are responsible for inducing the widespread endothelial cell activation that is seen in preeclampsia. Poor placental perfusion has been associated with preeclampsia, this had led to the hypothesis that placental hypoxia has an important role in the pathogenesis of preeclampsia. In this study, using a placental explant model, we investigated whether different oxygen environments induced abnormal shedding of trophoblastic debris or secretion of cytokines from the placenta. There was no significant difference in the numbers of trophoblasts shed from explants cultured in 1% or 8% oxygen containing environments. There was also no difference in the levels of activated caspases in trophoblasts shed from explants cultured in these two oxygen environments nor was there a significant difference in the endothelial cell responses to trophoblasts shed from explants cultured in 1% or 8% oxygen. Similarly, there was no significant change in the secretion of nine cytokines into the conditioned medium from explants cultured in 1% or 8% oxygen. This study supports the growing evidence that levels of oxygen in the placental environment during the first trimester of pregnancy may not in itself be the essential component contributing to the pathogenesis of preeclampsia.     

Matrix metalloproteinase release from placental explants of pregnancies complicated by intrauterine growth restriction

OBJECTIVE: There is evidence of impaired placental development in intrauterine growth restriction (IUGR). Matrix metalloproteinases (MMPs) are extracellular matrix-degrading enzymes that are released by placental cells during tissue remodeling processes. We hypothesized 1) that release of MMP-2 and -9 is decreased and/or release of tissue inhibitors of metalloproteinases (TIMPs) is increased from placental explants in pregnancies complicated by IUGR and 2) that oxygen levels affect such release. METHODS: Placental villous explants from normal (n = 7) and IUGR (n = 7) pregnancies were cultured at high (20%) and low (3%) oxygen levels for 24 hours. Supernatants were analyzed for MMP-2 and MMP-9 by zymography and for TIMP-1 and -2 by western blot analysis. RESULTS:: At 20% oxygen there was significantly reduced MMP-2 (P < .05) and TIMP-1 (P < .01) release and a trend for decreased MMP-9 release (P = .07) in explants from IUGR pregnancies compared with normal pregnancies; however, there were no differences at 3% oxygen. TIMP-2 was below detectable levels in all samples. Although MMP-2 and TIMP-1 release was significantly reduced at 3% compared with 20% oxygen in explants from both normal (P < .001; P < .05) and IUGR (P < .05) pregnancies, MMP-2 release changed less in IUGR compared with normal explant cultures. There were no significant effects of oxygen on MMP-9 release. CONCLUSION: Placental explants from IUGR pregnancies demonstrated reduced MMP-2, MMP-9, and TIMP-1 release compared with explants from normal pregnancies at high (20%) but not low (3%) oxygen.