DNA损伤诱导的长链非编码RNA(DINO)下调MMP2表达抑制人绒毛膜滋养层细胞侵袭与迁移

目的 探讨DNA损伤诱导的长链非编码RNA(DINO)通过调控迁移相关因子基质金属蛋白酶2(MMP2)的表达对人绒毛膜滋养层细胞侵袭与迁移的影响。方法 收集子痫前期(PE)患者和正常孕妇的胎盘组织(对照组)各20例,Masson染色观察胎盘组织结构改变,免疫荧光组织化学染色检测胎盘组织滋养层细胞MMP2的表达;实时荧光定量PCR检测DINO的表达;将HTR-8/SVneo人绒毛膜滋养层细胞转染DINO小干扰片段(si-DINO)后,实时荧光定量PCR验证DINO、MMP2 mRNA表达,Western blot法检测MMP2蛋白表达;Transwell^TM侵袭实验和细胞划痕实验检测细胞侵袭、迁移情况。结果 与对照组相比,PE患者血压、蛋白尿水平明显增高;胎盘组织可见纤维素样坏死,绒毛膜绒毛减少;免疫荧光显示胎盘滋养层细胞MMP2表达减弱、DINO表达增加;转染si-DINO后,DINO表达降低,而MMP2 mRNA及蛋白表达增加,HTR-8/SVneo细胞侵袭和迁移能力显著增加。结论 DINO下调MMP2表达,抑制胎盘滋养层细胞的侵袭与迁移。     

整合素β1对人体滋养层细胞黏附和迁移行为的影响

在体内,滋养层细胞是通过何种方式到达子宫螺旋动脉血管并对其基础结构进行重构的机制目前尚不清楚,但这一过程涉及到了细胞在血管内黏附和迁移的行为。采用微管吸吮技术和平行平板流动腔系统对整合素β1在滋养层细胞黏附和迁移中的作用进行检测。将滋养层细胞分别培养在I型鼠尾胶原和血管内皮细胞上,进行细胞黏附力测量和剪切应力加载,实验分为整合素β1抗体阻断组、非特异性抗体阻断组和正常对照组。结果显示:整合素β1抗体阻断组可显著降低细胞的黏附力和抵抗流体剪切应力引起位移的能力,而非特异性抗体组的结果与对照组差异不显著;内皮细胞可显著增强滋养层细胞的黏附力和抵抗流体剪切应力引起位移的能力。结果表明,整合素β1参与了滋养层细胞与血管内皮细胞间的黏附并调控了流体剪切应力作用下滋养层细胞的迁移行为。     

LPS介导滋养层细胞TLR4信号传导通路对HBD-2表达的影响

目的研究LPS体外刺激滋养层细胞是否诱导表达HBD-2,并进一步探讨该表达与TLR4信号传导通路的关系。方法建立不同孕周的滋养层细胞原代培养体系,应用TLR4阻断剂预处理滋养层细胞30 min前后,给予不同质量浓度的LPS(25、50、100、200、400 ng/ml)作用72 h,采用SYBR GreenⅠ实时荧光定量RT-PCR技术检测滋养层细胞HBD-2 mRNA的表达。结果 1)LPS能诱导滋养层细胞HBD-2 mRNA表达,且这种表达与其具有浓度和时间依赖性;当质量浓度为200 ng/ml,刺激24 h时,HBD-2 mRNA的相对表达量最高;2)TLR4阻断剂能抑制LPS对滋养层细胞表达HBD-2 mRNA的诱导作用,与未阻断之前相比差异具有统计学意义(P<0.01)。结论 LPS可能通过TLR4信号传导通路诱导滋养层细胞表达HBD-2 mRNA,将为宫内感染防治提供新靶点。     

EGCG通过下调COX-2和MMP-2表达抑制人脑胶质瘤细胞的迁移和侵袭

目的:探讨表没食子儿茶素没食子酸酯(EGCG)下调COX-2及MMP-2的表达、抑制人脑胶质瘤细胞迁移及侵袭的作用机制。方法:MTT法检测EGCG处理24h后SWO-38细胞的活性,确定药物作用浓度;细胞侵袭与迁移实验检测EGCG处理24h后SWO-38细胞的迁移与侵袭能力;Western blotting对比分析EGCG处理24h后SWO-38细胞COX-2和MMP-2的表达。通过肿瘤坏死因子α(TNF-α)促进COX-2的表达,观察EGCG是否通过COX-2/MMP-2通路抑制肿瘤的迁移侵袭。结果:细胞侵袭与迁移实验发现,EGCG对SWO-38细胞的迁移和侵袭能力有抑制作用,与对照组SWO-38细胞相比较,有显著差异(P0.01)。Western blotting发现经过EGCG处理24h后,SWO-38细胞COX-2和MMP-2蛋白的表达水平降低,提示EGCG抑制SWO-38迁移的机制可能与该药物抑制COX-2的表达而降低SWO-38细胞酶解细胞外基质的能力相关。结论:EGCG抑制了人脑胶质瘤SWO-38细胞的迁移与侵袭,其机制与EGCG抑制COX-2表达后,调节了MMP-2诱导的酶解细胞外基质的能力相关。     

柔毛水杨梅甲醇提取物通过调控miR-758表达对滋养层细胞增殖、迁移和侵袭的影响

目的 探讨柔毛水杨梅甲醇提取物对滋养层细胞增殖、迁移侵袭的影响。方法 不同浓度的柔毛水杨梅甲醇提取物处理人正常滋养细胞HTR8/SVneo后，四甲基偶氮唑蓝（MTT）实验检测细胞增殖，Transwell实验检测细胞迁移和侵袭。实时荧光定量PCR(RT-qPCR)检测HTR8/SVneo、正常胎盘滋养细胞、子痫前期胎盘滋养细胞中miR-758表达水平。将miR-758抑制物转染HTR8/SVneo，检测抑制miR-758表达对HTR8/SVneo细胞增殖、迁移和侵袭的影响。将miR-758模拟物转染HTR8/SVneo，检测过表达miR-758对柔毛水杨梅作用的HTR8/SVneo细胞增殖、迁移和侵袭的影响。结果 柔毛水杨梅甲醇提取物处理后HTR8/SVneo细胞的增殖、迁移和侵袭能力显著升高，miR-758表达显著降低，并呈一定浓度依赖性（P<0.05）。在子痫前期胎盘滋养细胞中miR-758表达升高，抑制miR-758表达后HTR8/SVneo细胞的增殖、迁移和侵袭能力显著升高（P<0.05）。过表达miR-758能够逆转柔毛水杨梅甲醇提取物对HTR8/SVneo增殖、迁...     

藤黄酸对人结肠癌HT-29细胞生物学行为及JNK信号通路的影响

目的探讨藤黄酸对人结肠癌HT-29细胞增殖、凋亡、迁移及ASK-1、p-ASK-1、JNK、p-JNK表达的影响。方法将人结肠癌HT-29细胞分为空白对照组与藤黄酸组(0.5、1.0、2.5、5.0μmol/ml)。不同浓度藤黄酸干预24、48、72h后,MTT方法检测藤黄酸对HT-29细胞增殖的抑制作用。2.5μmol/ml藤黄酸干预48 h后,流式细胞术检测HT-29细胞的凋亡率;划痕实验检测藤黄酸对HT-29细胞迁移能力的影响;Western blot实验检测藤黄酸对HT-29细胞JNK、p-JNK、ASK-1、p-ASK-1的表达。结果不同浓度藤黄酸均能显著抑制HT-29细胞增殖,且呈时间和浓度依赖性。2.5μmol/ml藤黄酸干预48 h后,HT-29细胞凋亡率升高,迁移能力降低(P0.01);JNK、p-JNK、ASK-1、p-ASK-1蛋白表达水平均显著升高(P0.01)。结论藤黄酸能够抑制人结肠癌HT-29细胞增殖与迁移,并促进凋亡,其作用机制可能与调控JNK信号通路相关。     

脂多糖改变牙髓干细胞的生物学特性

背景:龋病发生时,包括细菌在内的各种刺激沿牙本质小管传导至牙髓,牙髓组织中的牙髓干细胞受到刺激后增殖迁移至受损的牙本质下方,形成修复性牙本质以补充和修复受损组织。在此过程中,脂多糖作为细菌的主要毒力因子,是否可以影响牙髓干细胞增殖、迁移及成牙本质向分化等生物学行为,还有待研究。目的:观察脂多糖对牙髓干细胞增殖能力、迁移能力和成牙本质向分化能力的影响。方法:组织块酶消化法培养牙髓干细胞,给予0,0.1,1,10 mg/L脂多糖刺激后,采用MTT法检测牙髓干细胞增殖情况,划痕实验和Transwell实验检测牙髓干细胞的迁移能力;给予1 mg/L脂多糖和矿化诱导液刺激21 d后,茜素红染色检测细胞矿化结节形成能力,RT-PCR检测成牙本质相关基因的表达。结果与结论:①0.1,1,10 mg/L脂多糖组的吸光度值在第1,3,5,7天4个时间点均低于0 mg/L脂多糖组(P<0.01),迁移能力均高于0 mg/L脂多糖组;②牙髓干细胞矿化诱导21 d后,1 mg/L脂多糖刺激组所形成的矿化结节数量和面积明显小于0 mg/L脂多糖组(P<0.01),成牙本质相关基因OCN、BSP、ALP表达量显著低于0 mg/L脂多糖组(P<0.01);③结果表明,脂多糖刺激牙髓干细胞后其增殖能力和成牙本质向分化能力降低,迁移能力明显增强。     

低分子量肝素对滋养层细胞MMP-2、TIMP-2表达及侵袭力的影响

目的:探讨低分子量肝素对体外培养人早孕绒毛滋养层细胞MMP-2、TIMP-2表达及侵袭力的影响。方法:将经胰蛋白酶/DNA酶Ⅰ联合消化,通过Percoll细胞分离液纯化得到的绒毛滋养层细胞进行体外培养。采用不同浓度低分子量肝素干预培养24h后,ELISA法测定细胞上清液中MMP-2、TIMP-2的浓度;采用Tr-answell小室观察滋养层细胞的侵袭能力。结果:不同浓度的低分子量肝素(1.0×102IU/L,1.0×103IU/L,1.0×104IU/L)干预人早孕绒毛滋养层细胞后,与对照组相比,MMP-2的表达上调,滋养层细胞侵袭力增强,在1.0×103IU/L时MMP-2表达最高,滋养层细胞侵袭力最强(P0.05)。TIMP-2的表达随着低分子量肝素浓度的增加而逐渐下降,与对照组比较,在1.0×103IU/L、1.0×104IU/L组明显降低(P0.05),但1.0×103IU/L组与1.0×104IU/L组之间TIMP-2的表达无差异(P0.05)。结论:低分子量肝素可能直接通过影响绒毛滋养层细胞MMP-2、TIMP-2的表达进而影响绒毛滋养层细胞的侵袭能力。     

HBV感染人胎盘滋养层细胞机制的初步探讨

目的 采用透射电镜观察HBV体外感染人胎盘滋养层细胞的超微结构变化.方法 HBV体外感染人胎盘滋养层细胞.ELISA检测培养上清中HBsAg,PCR检测细胞培养上清和滋养层细胞中的HBV DNA.HBV荧光定量PCR检测细胞培养上清中HBV DNA量(拷贝/ml).透射电镜观察滋养层细胞的超微结构.结果 感染组滋养层细胞培养上清中HBsAg在PBS清洗后12 h时A值为0.942,96 h时上升为1.264.PCR检测感染组细胞培养上清和感染组细胞HBV DNA均为阳性.PBS彻底清洗后0、12、36、60、84 h感染组细胞培养上清的HBV DNA分别为:＜103,3×104,6×105,5×105,3×105拷贝/ml.透射电镜观察到感染组滋养层细胞膜附近存在包涵素(Clathrin)形式的内吞小体形成,并且发现内吞小体内存在病毒颗粒样结构.在感染组细胞粗面内质网内发现HBsAg特异性的纤维丝状结构.结论 HBV可能经包涵素依赖的细胞内吞形式进入滋养层细胞,进而实现感染细胞或通过胞释作用将病毒排至细胞的对侧而实现穿越滋养层细胞屏障.     

普鲁卡因抑制脂多糖诱导的结肠癌细胞肿瘤侵袭性的作用机制

目的探讨普鲁卡因抑制脂多糖诱导结肠癌细胞肿瘤侵袭性行为的调控及其机制。方法 CCK-8细胞活性实验检测不同浓度的普鲁卡因对结肠癌细胞活性的影响,Western blotting分析普鲁卡因对脂多糖结合蛋白表达水平的影响,划痕愈合实验检测普鲁卡因对结肠癌细胞迁移能力的影响,Transwell侵袭实验检测普鲁卡因对结肠癌细胞侵袭能力的影响。结果浓度为0.08 mg/m L的普鲁卡因对结肠癌细胞的活性影响情况最明显,差异有统计学意义(P0.05);浓度为0.08 mg/m L的普鲁卡因对脂多糖结合蛋白的表达水平抑制最明显,差异有统计学意义(P0.05);划痕愈合测定显示普鲁卡因可以直接抑制结肠癌细胞的运动能力;Transwell侵袭实验测定显示普鲁卡因可以直接抑制结肠癌细胞的侵袭能力,差异有统计学意义(P0.05)。结论普鲁卡因可以通过影响脂多糖诱导结肠癌细胞的迁移和侵袭,可作为化疗辅助药物,为探索结肠癌诊疗的分子机制和新的潜在治疗策略提供相应理论帮助,为结肠癌的临床治疗方式提供有效支持。     

Trophoblast-macrophage interactions: a regulatory network for the protection of pregnancy

PROBLEM: Macrophages are one of the first immune cells observed at the implantation site. Their presence has been explained as the result of an immune response toward paternal antigens. The mechanisms regulating monocyte migration and differentiation at the implantation site are largely unknown. In the present study, we demonstrate that trophoblast cells regulate monocyte migration and differentiation. We propose that trophoblast cells 'educate' monocytes/macrophages to create an adequate environment that promote trophoblast survival. METHOD OF STUDY: CD14(+) monocytes were isolated from peripheral blood using magnetic beads. Co-culture experiments were conducted using a two-chamber system. Monocytes were stimulated with lipopolysaccharide (LPS) and cytokine levels were determined using multiplex cytokine detecting assay. RESULTS: Trophoblast cells increase monocyte migration and induce a significant increase in the secretion and production of the pro-inflammatory cytokines [interleukin-6 (IL-6), IL-8, tumor necrosis factor-alpha] and chemokines (growth-related oncogen-alpha, monocyte chemoattractant protein-1, macrophage inflammatory protein-1beta, RANTES). Furthermore, the response of monocytes to LPS was different in monocytes pre-exposed to trophoblast cells. CONCLUSION: The results of this study suggest that trophoblast cells are able to recruit and successfully educate monocytes to produce and secrete a pro-inflammatory cytokine and chemokine profile supporting its growth and survival. Furthermore we demonstrate that trophoblast cells can modulate monocytes response to bacterial stimuli.     

Impaired migration of trophoblast cells caused by simvastatin is associated with decreased membrane IGF-I receptor, MMP2 activity and HSP27 expression

BACKGROUND: Statins inhibit 3-hydroxy-3-methylglutaryl coenzyme-A reductase, the rate-limiting enzyme of the mevalonate pathway, and are used successfully in the treatment of hypercholesterolaemia. Statins are contraindicated during pregnancy. Lately, we have shown that simvastatin has adverse affects on human first trimester placental explants' proliferation and migration. The objective of the present study was to investigate the molecules involved in mediating simvastatin's effect on trophoblast cell migration. We hypothesized that simvastatin attenuates insuline-like growth factor-I (IGF-I) receptor expression (involved in trophoblast motility), matrix metalloproteinase (MMP) activities, and heat shock protein 27 (HSP27) levels (whose mRNA is actively transcribed during trophoblast differentiation) in trophoblast cells thus consequently effecting their migration. METHODS: Human placental explants were cultured above a matrigel with/without simvastatin (10 microM) for 5 days. In this model, trophoblast migrates from the villi into the matrigel. Western-blot and immunohistochemistry served for analysing HSP27 expression. Immunohistochemistry was used for assessing IGF-I receptor localization. MMPs activity was assayed by gel zymography. RESULTS: Simvastatin reduced IGF-I receptor membranal expression, MMP2 activity and HSP27 expression in trophoblast cells (P < 0.05). CONCLUSIONS: The inhibitory effect of simvastatin on trophoblast cell migration is associated with a significant decrease in the tested molecules, which probably contributes to the impaired migration.     

The effect of 6-mercaptopurine on early human placental explants

BACKGROUND: 6-mercaptopurine (6-MP) is an antineoplastic and immunosuppressive drug. Recently, more women have received this drug during pregnancy. Animal studies have shown that 6-MP has deleterious effects on the fetus, while human data include prematurity, intrauterine growth restriction, low birth weight and malformations that occur especially when the drug is administered in the first trimester of pregnancy. OBJECTIVES: To study the effects of 6-MP on cellular functions of human trophoblast explants. METHODS: Human placental explants (5.5-9 weeks gestational age), that were grown on matrigel, were exposed to medium containing 6-MP for 5 days. Medium alone served as control. Extravillous trophoblast (EVT) cell migration assessment was performed by visual observation. Analysis of proliferating events of the trophoblast cells was assessed by immunohistochemical examination. Apoptosis was analyzed by Tunnel procedure and by anti-caspase 3 staining and hormone level by enzyme-linked immunosorbent assay. RESULTS: 6-MP inhibited migration of EVT cells from the villi to the matrigel with a lower proliferation rate and increased apoptosis of cytotrophoblast cells compared to controls. However, no significant effect of 6-MP on hormone levels was observed. CONCLUSIONS: 6-MP inhibited migration and proliferation of trophoblast cells in first-trimester human placental explant culture.     

Uterine NK Cells and Trophoblast HLA Class I Molecules

PROBLEM: To investigate the proposal that NK cells in decidua may control trophoblast migration during implantation of the human placenta. METHOD: Use Mab specific for HLA-G and for HLA-C in association with flow cytometry and immunoprecipitation to determine the expression of these HLA molecules by trophoblast. Expression of Killer inhibitory/activatory receptors (KIR/KAR) and the CD94 receptor by decidual NK cells was also studied. RESULTS: Extravillous trophoblast expressed HLA-G and HLA-C in both β₂m-associated form and as free heavy chains. KIR and KAR are expressed by decidual NK cells. The repertoire of receptors varied between different women and also between blood and decidual NK cells from the same women. The expression of CD94 was also different between blood and decidual NK cells. CONCLUSION: The recognition of HLA-G/HLA-C by KIR/KAR and CD94 could provide a mechansm by which decidual NK cells control trophoblast migration.     

MicroRNA-155 通过调节CXCR4 / PI3K / AKT 途径影响滋养细胞的侵袭与迁移

目的:以人绒毛膜滋养层细胞系JEG-3 细胞为研究对象,结合该细胞侵袭和迁移能力的变化情况,研究转染miR-155 mimics 和miR-155 inhibitor 之后CXCR4 的表达变化及其对下游PI3K/ AKT 信号通路的影响作用,从而探讨miR-155参与子痫前期发生发展的分子机制。方法:设计miR-155mimics 和miR-155 inhibitor,对JEG-3 进行转染,通过Transwell 侵袭实验、划痕实验,观察转染后细胞的侵袭和迁移能力的变化;利用Real-time PCR 检测CXCR4 mRNA 的表达;利用Western blot检测CXCR4 及下游p鄄AKT 蛋白的表达水平。结果:Real-time PCR 结果显示,miR-155 mimics 转染组CXCR4 mRNA 相对表达量(0.589依0.096)明显低于空白对照组(1.503依0.090)和阴性对照组(1.146依0.153),差异有统计学意义(P<0.05);miR-155 inhibitor 转染组CXCR4 mRNA 相对表达量(1.739依0.083)与两组对照组相比差异也有统计学意义(P<0.05)。Western blot 结果显示miR-155 mimics 转染组CXCR4 蛋白和下游p-AKT 蛋白表达水平均明显降低,而miR-155 inhibitor 转染组CXCR4 和p-AKT 蛋白水平则升高,与空白对照组和阴性对照组相比差异均有统计学意义(P<0.05)。Transwell 侵袭实验结果显示,与空白对照组(63郾46依2郾37)和阴性对照组(49.29依5.81)侵袭细胞数相比,miR-155 mimics 转染组侵袭细胞数(22.89依9.42)明显减少,差异有统计学意义(P<0.05);同时,miR-155 inhibitor 转染组侵袭细胞数(81.50依11.25)明显升高,差异有统计学意义(P<0.05)。划痕实验结果显示,转染miR-155 mimics 后,JEG-3 细胞相对迁移距离(0.159依0.058)低于空白对照组(1.080依0.045)和阴性对照组(0.823依0.201),差异有统计学意义(P<0.05);而miR-155 inhibitor 转染组,JEG-3 细胞相对迁移距离(1.640依0.078)明显增加,差异有统计学意义(P<0.05)。结论:miR-155 可能通过抑制CXCR4 的表达进而抑制其下游PI3K/AKT 信号通路的活化,从而影响滋养细胞的侵袭及迁移能力,最终导致子痫前期的发生发展。     

The use of Alamar Blue assay for quantitative analysis of viability, migration and invasion of choriocarcinoma cells

BACKGROUND: The current techniques for quantifying trophoblast viability, migration and invasion are mainly limited by the need to sacrifice the cells during the test procedure. In this study, the vital dye AB (AB) was used to quantify cell number and viability of BeWo and JEG-3 choriocarcinoma cells, as well as their migration and invasion through fibronectin-coated filters. METHODS :AB was directly added to culture medium of incubated test and control cells. At various time intervals, the redox reaction, in which AB is reduced by the cells, was measured by absorbance readings at 540 and 630 nm. For cell migration and invasion, cells were cultured onto uncoated or fibronectin-coated inserts, respectively. AB reduction of migrated cells was normalized to that of control cells to calculate percentages of migration. This model was also tested in the presence of a reported inhibitor, transforming growth factor (TGF) beta. RESULTS: The curve of %AB reduction versus cell number was linear, with intra- and inter-assay Coefficient of Variations of 1.88%and 2.94%, respectively. AB reduction increased with both seeding concentrations and incubation time with AB. TGFbeta treatment caused a modest decrease in AB reduction in both JEG-3 and BeWo cells. TGFbeta treatment also decreased migration in BeWo, but not in JEG-3, cells. CONCLUSIONS: AB assay is a simple and reliable method for quantifying trophoblast viability, migration and invasion.     

Interaction of Decidual CD56+ NK with Trophoblast Cells during Normal Pregnancy and Recurrent Spontaneous Abortion at Early Term of Gestation

The immunological paradox of pregnancy, when maternal immune system recognizes but does not reject the semiallogenic foetus, is not yet fully understood. The aim of this work was to detail the mechanisms of the interaction of decidual CD56+ NK, infiltrating the maternal part of placenta, and trophoblast cells of foetal origin. Samples of the endometrial tissue from 13 healthy non‐pregnant women, 37 placentas, obtained after medical abortion of viable pregnancy at 7–10 weeks of gestation, and 26 samples of placentas from first‐trimester recurrent spontaneous abortion (RSA) were used as the material for investigation. Phenotype of NK was assessed by flow cytometry. The influence of trophoblast cells upon IFNγ and GrB mRNAs expression by dNK was investigated by RT‐PCR. The influence of dNK upon trophoblast cells migration and invasion was studied using collagen and Matrigel systems. In RSA group comparing to the normal pregnancy, the decrease of dNK with inhibitory receptors (NKG2A) and elevation of activated dNK were seen. In normal pregnancy, but not in RSA, trophoblast cells increased the expression of IFNγ and GrB mRNAs by CD56+ dNK. Both in normal and RSA pregnancy, dNK inhibited the migration and invasion of trophoblast cells. Initially, low invasive and migration capacities of trophoblast cells were seen during RSA. Thus, unbalanced activation of dNK can lead to the impairment of dNK and trophoblast cells interaction during RSA.     

Villous explant culture using early gestation tissue from ongoing pregnancies with known normal outcomes: the effect of oxygen on trophoblast outgrowth and migration

BACKGROUND: Early placental and embryo development occur in a physiologically low oxygen environment, with a rise in oxygen tension within the placenta towards the end of the first trimester. Oxygen is implicated in the regulation of trophoblast differentiation and invasion. This study examined the effects of oxygen tension on extravillous trophoblast outgrowth and migration from normal pregnancies free of significant pathology. METHODS: Early gestation villous tissue (11-14 weeks gestation), obtained by chorionic villus sampling, was cultured in 3 or 20% oxygen. Maternal and fetal outcomes were ascertained for all samples. The frequency and amount of trophoblast outgrowth and migration from villi were measured for up to 192 h. RESULTS: Significantly fewer explants produced outgrowths in 3% compared with 20% oxygen. The number of sites of trophoblast outgrowth and the extent of migration were also significantly less in 3% compared with 20% oxygen. In vitro hypoxia/reoxygenation further reduced trophoblast growth compared with 3% oxygen alone. HLA-G expression in extravillous trophoblasts was not affected by oxygen tension, with HLA-G positive extravillous trophoblasts being universally Ki67 negative. CONCLUSION: Human placental villi and extravillous trophoblasts in the late first trimester of pregnancy are sensitive to oxygen tension, with low oxygen inhibiting extravillous trophoblast outgrowth and migration.     

Simvastatin has deleterious effects on human first trimester placental explants

BACKGROUND: Statins inhibit 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMG-CoA reductase), the rate-limiting enzyme of the mevalonate pathway, and have been used successfully in the treatment of hypercholesterolaemia. Animal models have provided evidence for the teratogenic effects of statins on pregnancy outcome. Thus statins are contraindicated during pregnancy. However, conflicting data are available from inadvertent use of statins in human pregnancy. Therefore we decided to explore the effects of simvastatin on the placenta in an in vitro human placental model. METHODS: Human first trimester placental explants that were grown on matrigel were exposed to medium supplemented with simvastatin. Migration of extravillous trophoblast cells was assessed by visual observation. Proliferative and apoptotic events of the trophoblast cells were assesed by immunohistochemical examination using anti-Ki67 and anti-activated caspase-3 antibodies respectively. Hormone levels were measured. RESULTS: Simvastatin sharply inhibited migration of extravillous trophoblast cells from the villi to the matrigel (P < 0.05). Moreover, simvastatin inhibited half of the proliferative events in the villi (P < 0.05) and increased apoptosis of cytotrophoblast cells compared to control. Moreover, simvastatin significantly decreased secretion of progesterone from the placental explants (P < 0.01). CONCLUSION: Simvastatin adversely affects human first trimester trophoblast.     

Pravastatin does not prevent antiphospholipid antibody-mediated changes in human first trimester trophoblast function

STUDY QUESTION: What is the effect of pravastatin on antiphospholipid antibody (aPL) modulation of human first trimester trophoblast function? SUMMARY ANSWER: Pravastatin does not prevent the effects of aPL on human first trimester trophoblast cell function. WHAT IS KNOWN ALREADY: Antiphospholipid syndrome (APS) is associated with recurrent pregnancy loss and late pregnancy complications, such as pre-eclampsia, owing to direct targeting of the placenta by aPL. While treatment with heparin reduces the rate of pregnancy loss, the risk for severe pre-eclampsia remains high. Thus, there is a need to find alternative treatments for the prenatal management of patients with APS. Statins have recently been shown to prevent aPL-mediated fetal loss in mice but their effects on a human pregnancy model of APS have not yet been studied. DESIGN, DATA COLLECTION, METHODS: The human first trimester trophoblast cell line, HTR8, and human first trimester trophoblast primary cultures were incubated with or without a mouse anti-human beta 2 glycoprotein I (β(2)GPI) monoclonal antibody in the presence or absence of pravastatin. Cytokine and angiogenic factor secretion were measured by enzyme-linked immunosorbent assay and multiplex analysis. Cell migration was measured using a colorimetric two-chamber migration assay. MAIN FINDINGS: Using the human first trimester trophoblast cell line, HTR8, pravastatin significantly augmented, compared with no treatment, aPL-dependent secretion of interleukin (IL)-8 (P< 0.05), IL-1β (P< 0.05) and soluble endoglin (P< 0.01) but had no effect on aPL-induced up-regulation of vascular endothelial growth factor, placenta growth factor or growth-related oncogene alpha secretion. Furthermore, pravastatin alone limited basal HTR8 cell migration (P< 0.01), and did not mitigate the adverse effect of aPL on trophoblast migration. Pravastatin also had no impact on the secretion of pro-inflammatory cytokines and angiogenic factors by primary human first trimester trophoblast cells exposed to aPL. LIMITATIONS AND WIDER IMPLICATIONS OF THE FINDINGS: While our in vitro findings suggest that pravastatin may not be effective in preventing pregnancy complications in patients with APS, the in vivo condition may be more complex, and thus, more studies are needed to determine the effectiveness of pravastatin in the prevention of aPL-associated pregnancy complications in humans. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the American Heart Association.