IL-18, a product of choriodecidual cells, increases during premature rupture of membranes but fails to turn on the Fas-FasL-mediated apoptosis pathway

Problem: IL-18 is a novel cytokine, which promotes inflammation and apoptosis. This study examines its expression pattern, site of production, and levels in the amniotic fluid (AF) during pregnancy complications such as preterm labor and preterm premature rupture of membranes (pPROM). The ability of IL-18 to induce the Fas–Fas ligand (FasL)/caspase-mediated apoptotic pathway is also studied.Methods: Amniochorion collected at term was placed in an organ explant system. IL-18 mRNA expression was studied by RT-PCR. IL-18 mRNA and peptide were localized by in situ hybridization and immunohistochemistry. IL-18 in the AF of women with pPROM, with preterm labor with no rupture of membranes, and at term was measured using ELISA. Multiplex PCR was used to study the expression pattern of proapoptotic genes such as Fas, FasL, caspase 8, and Fas-associated death domain (FADD). ELISA was also used to measure the release of soluble Fas from IL-18-stimulated amniochorion in culture media.Results: IL-18 is a constitutively expressed gene in human chorion and decidua but not in human amnion and increases in the AF of women with pPROM [2.9 ± 3.3 ng/ml (SD)] compared to women with preterm labor (1.1 ± 0.67 ng/ml; P < 0.05) and term (0.9 ± 0.73 ng/ml; P < 0.05). IL-18 induces Fas expression, whereas FADD is a constitutively expressed gene in human fetal membranes. IL-18 failed to induce FasL or caspase 8 expressions. Soluble Fas release from amniochorion was increased after IL-18 stimulation.Conclusion: Chorion and decidua are a source of IL-18, whose concentrations are increased in the AF during pPROM. IL-18 induced Fas expression in amniochorion; however, it failed to turn on other genes in the Fas–FasL apoptosis pathway including FasL and caspase 8.     

Distinct molecular events suggest different pathways for preterm labor and premature rupture of membranes

OBJECTIVE: On a clinical level, the etiologies associated with premature rupture of the membranes and preterm labor are virtually identical, though these conditions end in distinctly different events. This study was designed to determine differences between preterm labor and preterm premature rupture of membranes by using molecular markers of extracellular matrix degradation and apoptosis. STUDY DESIGN: Amniochorion and amniotic fluid samples were collected from gestational age-matched groups of women undergoing cesarean delivery before term. Samples were collected from 2 groups of women, women with premature rupture of membranes and women with preterm labor with no rupture of membranes. Changes in the expression pattern of messenger ribonucleic acid for matrix metalloproteinases (MMP), tissue inhibitor of metalloproteinases (TIMP), and pro-apoptotic (p53 and Bax) and anti-apoptotic (Bcl-2) proteins were identified by quantitative polymerase chain reaction. Enzyme-linked immunosorbent assay was used to determine the levels of these proteins in the amniotic fluid. Multiplex polymerase chain reaction was performed to study the expression of Fas-Fas ligand-associated pro-apoptotic genes. Unpaired nonparametric, 2-tailed Mann-Whitney U test was used to determine statistical significance of quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (P <.05 was considered significant). RESULTS: Quantitative polymerase chain reaction results demonstrated an increased mRNA expression for MMP2, MMP9, and MT1-MMP and a decreased expression for TIMP2 in prematurely ruptured membranes compared with preterm labor membranes. Enzyme-linked immunosorbent assay documented increases in the amniotic fluid concentrations of immunoreactive and bioactive MMP2 and MMP9 and immunoreactive MMP3 and a decreased TIMP2 concentration in fluids obtained from the premature rupture of membranes group compared with the preterm labor group. The pro-apoptotic genes p53 and bax were up-regulated in premature rupture of membranes when compared with preterm labor. Anti-apoptotic gene (Bcl-2 ) expression was increased in preterm labor membranes compared with prematurely ruptured membranes. Interleukin-18 (a pro-apoptotic cytokine) was increased in the amniotic fluid during premature rupture of membranes compared with preterm labor. Prematurely ruptured membranes also demonstrated fragmented deoxyribonucleic acid and expression of Fas and caspase 8 (apoptosis initiator), which were all absent in preterm labor membranes. CONCLUSIONS: We have begun to delineate 2 divergent molecular pathways for premature rupture of membranes and preterm labor. Most likely, this is the beginning of the identification of differences that will become evident with the use of molecular biology.     

IL-1 beta is a better inducer of apoptosis in human fetal membranes than IL-6

The objective of this study was to compare two of the inflammatory cytokines (IL-1 and IL-6) elevated in both preterm labour and preterm premature rupture of the membranes (pPROM), with respect to their ability to induce fetal membrane apoptosis. Fetal membranes collected from women at term were placed in an organ explant system and stimulated with recombinant human IL-1 beta and IL-6. The expression patterns of pro-apoptotic genes (Fas, FasL, TRADD, FADD) and caspases 2, 3, 8, 9 were studied using PCR. Caspase activity and DNA fragmentation were studied using substrate assays and TUNEL respectively. Caspase 8 and 9 expressions were induced in IL-1 beta and IL-6 treated amniochorion. Caspase 2 expression was seen only in IL-1 beta stimulated tissues. When compared to control, IL-1 beta increased caspase 2, 3, 8 and 9 activities, whereas IL-6 treated membranes did not exhibit a significant change. DNA fragmentation was seen in greater numbers after IL-1 beta treatment than after IL-6 treatment. This study demonstrates that IL-1 beta is a better inducer of apoptosis in normal human fetal membranes than IL-6.     

Programmed cell death (apoptosis) as a possible pathway to metalloproteinase activation and fetal membrane degradation in premature rupture of membranes

OBJECTIVE: Increased matrix metalloproteinase 2 expression and activity are associated with premature rupture of fetal membranes. A proapoptotic protein produced in response to deoxyribonucleic acid fragmentation, p53, can bind to the matrix metalloproteinase 2 gene promoter and cause increased gene expression. It promotes apoptosis by inducing the expression of the proapoptotic bax gene and inhibiting the antiapoptotic bcl-2 gene. This study was undertaken to investigate the expression pattern of apoptotic elements in pregnancy complications that may cause increased expression of the gene for matrix metalloproteinase 2. STUDY DESIGN: Amniochorial membranes were collected from the following groups of women: (1) women with premature rupture of fetal membranes, (2) women with preterm labor and intact membranes, and (3) women with term labor after vaginal delivery. Deoxyribonucleic acid fragmentation was tested with ligation-mediated polymerase chain reaction and the terminal deoxynucleotidyl transferase-mediated biotinylated deoxyribonucleoside triphosphate end-labeling assay. Matrix metalloproteinase 2, p53, bcl-2, and bax gene expression patterns were studied with quantitative competitive polymerase chain reaction. Statistical analysis was performed with the Tukey-Kramer multiple comparison test. RESULTS: Quantitative competitive polymerase chain reaction documented a 10-fold increase in the expression of the gene for matrix metalloproteinase 2 in premature rupture of fetal membranes with respect to term and preterm labor. This induction coincided with an increase in the expressions of the proapoptotic genes p53 and bax and a drop in the expression of the antiapoptotic gene bcl-2. Ligation-mediated polymerase chain reaction revealed deoxyribonucleic acid fragmentation in specimens from premature rupture of fetal membranes and not in those from preterm labor or labor at term. Histochemical analysis documented fragmented deoxyribonucleic acid in chorionic and amniotic cells. CONCLUSION: This study suggests that apoptosis is associated with premature rupture of fetal membranes. Deoxyribonucleic acid fragmentation, associated with elevations in the levels of the two proapoptotic gene products evaluated (p53 and bax ) and a drop in the level of the antiapoptotic bcl-2, was seen in premature rupture of the fetal membranes. Induction of matrix metalloproteinase 2 may be a function of p53 gene expression increase in premature rupture of fetal membranes.     

细胞凋亡与胎膜早破的相关性研究

目的:探讨细胞凋亡增加是否为胎膜早破的一个发病机理或危险因素。方法:随机采集临产前剖宫产分娩的初产妇肘静脉血、羊水及胎膜,其中胎膜早破者34例,正常完好胎膜17例。①用ELISA测定各组血清、羊水中TNF-α的浓度:②用TUNEL原位标记术精确测定各组胎膜细胞凋亡指数;③用免疫组化法检测各组胎膜组织中Bax、Bcl-2、Fas、Caspase-3表达情况。结果:①胎膜早破组血清、羊水中TNF-α浓度明显高于正常对照组水平(P<0.001);且血清、羊水中TNF-α浓度有相关性,r=0.386。胎膜感染组血清、羊水中TNF-α浓度较无感染组高(P<0.001)。②胎膜早破组胎膜感染发生率高于对照组(P<0.05)。③胎膜早破组细胞凋亡指数明显高于正常组(P<0.001);感染组细胞凋亡指数较无感染组高(P<0.001)。④胎膜早破组促凋亡蛋白Bax、Fas、Caspase-3阳性表达率高于对照组(P<0.05),但抑凋亡蛋白Bcl-2阳性表达率两组间无统计学差异(P>0.05);胎膜感染组Bax、Bcl-2、Caspase-3阳性表达率与无感染组比较无统计学差异(P>0.05),但感染组Fas阳性表达率高于无感染组(P<0.05)。结论:基因、环境因素相互作用下的细胞凋亡增加可能是胎膜早破的一个重要发病机制,可独立致病,亦或与其他相关危险因素(如感染)协同致病。     

母血羊水中IL—6和IL—8水平与绒毛膜羊膜炎关系的研究

目的 探讨白细胞介素－６（ＩＬ－６）和白细胞介素－８（ＩＬ－８）在监测胎早破中的作用。方法 采用酶联免疫吸附实验对４６例胎盘早破孕妇母血清,羊水中ＩＬ－６和ＩＬ－８水平进行监测,并以正常足月妊娠孕妇２０例做对照组。结果 胎膜早破孕妇母血清ＩＬ－６,ＩＬ－８和羊水中ＩＬ－６和ＩＬ－８水平均较正常足月妊娠组高。差异差异（Ｐ〈０．０１,Ｐ〈０．０５）;随着破膜时间延长母血ＩＬ－６、ＩＬ－８和羊水中ＩＬ－     

人胎膜组织中E26转录因子1的表达及其与胎膜早破的关系

目的 检测人胎膜组织中E26转录因子1(Ets-1)的表达及定位,探讨其与胎膜早破发生的关系.方法 选择2007年2-11月在重庆医科大学附属第一医院住院分娩的产妇100例为研究对象,按足月与否、有无胎膜早破、分娩方式将其分为早产临产、未足月胎膜早破(PPROM)、足月临产、足月胎膜早破(TPROM)组,以足月择期刮宫产产妇为对照组,每组各20例,分娩后采集其胎膜组织,用免疫组化链霉菌抗生物素蛋白-过氧化物酶连接(SP)法检测Ets-1蛋白的表达水平及定位,每组选取6例以逆转录(RT)-PCR技术检测Ets-1 mRNA的表达水平,并进行半定量分析.结果 (1)各组胎膜组织中Ets-1 mRNA的表达:早产临产、PPROM、足月临产、TPROM、对照组胎膜组织中Ets-1mRNA表达水平分别为0.342±0.016、0.603±0.027、0.325±0.013、0.582±0.075、0.139±0.012,PPROM组和TPROM组均高于对照组.分别比较,差异均有统计学意义(P<0.05);早产临产组与足月临产组,PPROM组与TPROM组比较,差异均无统计学意义(P>0.05).(2)Ets-1蛋白在胎膜组织的定位:Ets-1蛋白集中在羊膜和绒毛膜的间质层、滋养层细胞的胞质和胞核中表达;细胞内可见清晰的棕黄色颗粒.在羊膜上皮细胞中未见Ets-1蛋白表达.(3)各组胎膜组织中Ets-1蛋白的表达:早产临产、PPROM、足月临产、TPROM、对照组胎膜组织中Ets-1蛋白表达水平分别为0.552±0.018、2.853±0.174、0.538±0.042、2.731±0.090、0.214±0.013,PPROM组与TPROM组均高于对照组,分别比较,差异均有统计学意义(P<0.05);但早产临产组与足月临产组,PPROM组与TPROM组胎膜组织分别比较,差异均无统计学意义(P>0.05).结论 Ets-1在人类胎膜组织中有表达,且在胎膜早破时表达水平升高.     

Polymorphisms in the tumor necrosis factor-alpha gene at position -308 and the inducible 70 kd heat shock protein gene at position +1267 in multifetal pregnancies and preterm premature rupture of fetal membranes

OBJECTIVE: The purpose of this study was to determine the relationship between preterm premature rupture of membranes, tumor necrosis factor-alpha, and heat shock protein-70 gene polymorphisms in multifetal gestations. STUDY DESIGN: Buccal swabs from 101 mother-neonate pairs of multifetal pregnancies were tested for single nucleotide polymorphisms at position -308 of the tumor necrosis factor-alpha gene and +1267 of the heat shock protein-70 gene. Pregnancy outcome data were obtained subsequently. RESULTS: Tumor necrosis factor-alpha allele 2 carriage by the first-born occurred in 10 of 27 pregnancies (37.0%) that resulted in preterm premature rupture of membranes compared with 6 of 67 pregnancies (9.0%) without preterm premature rupture of membranes ( P = .002). The allele frequency of tumor necrosis factor-alpha allele 2 and heat shock protein-70 allele 2 in the first born was higher in pregnancies that were complicated by preterm premature rupture of membranes (18.5% vs 4.5%; P = .003; and 57.7% vs 41.3%; P = .04, respectively). There was no relationship between tumor necrosis factor-alpha allele 2 or heat shock protein-70 allele 2 carriage by the second fetus or mother and preterm premature rupture of membranes. CONCLUSION: Tumor necrosis factor-alpha allele 2 and/or heat shock protein-70 allele 2 carriage by the first-born fetus is associated with preterm premature rupture of membranes in multifetal pregnancies.     

Ⅲ型胶原及CTGF在胎膜早破发病机制中的作用

目的探讨Ⅲ型胶原、结缔组织生长因子（CTGF）在人胎膜组织中的表达及在胎膜早破发病机制中的作用。方法38例胎膜早破孕妇为胎膜早破组，其中未足月胎膜早破组（pPROM组）18例，足月胎膜早破组（tPROM组）20例；与胎膜早破组孕周相对应的非胎膜早破孕妇作为对照组，早产对照组18例，足月对照组20例。应用免疫组化及图像分析法检测胎膜Ⅲ型胶原、CTGF表达水平，以阳性区平均灰度值为检测依据。将各组孕妇胎膜Ⅲ型胶原、CTGF测定结果进行直线相关分析。结果①四组胎膜中均存在不同程度的Ⅲ型胶原、CTGF的表达；②pPROM组Ⅲ型胶原（88．81±5．25）、CTGF水平（85．45±6．91）均低于早产对照组（95．99±8．41，90．30±5．74），差异有统计学意义（P〈0．01，P〈0．05）；tPROM组Ⅲ型胶原（94．53±6．43）、CTGF（88．15±4．93）均低于足月对照组（100．80±9．77，93．20±5．33），（P〈0．05）；pPROM组Ⅲ型胶原（88．81±5．25）表达低于tPROM组（94．53±6．43），两者比较差异有统计学意义（P〈0．01）；pPROM组CT—GF灰度值（85．45±6．91）表达低于tPROM组（88．15±4．93）．但无统计学差异（P〉0．05）；③Ⅲ型胶原、CTGF水平在pPROM组中的表达呈正相关性，r=0．830（P〈0．01），而tPROM组中两者则无相关性。结论pPROM组与tPROM组中存在Ⅲ型胶原与CTGF的低表达。tPROM的发生可能与胎膜的退行性变有关，而pPROM的发生与胎膜本身结构病变密切相关。     

水通道蛋白9在人胎盘和胎膜中的表达

目的 检测正常人胎盘与胎膜中水通道蛋白9（aquaporin 9，AOP9）的表达。方法 收集5例足月剖宫分娩的胎盘和胎膜样本，运用RT—PCR方法从mRNA水平检测AQP9在胎盘与胎膜的表达；运用免疫组织化学和Western印迹方法检测AQP9蛋白在胎盘与胎膜中的表达。结果 RT—PCR结果显示AQP9mRNA在胎盘和胎膜均有表达；Western印迹显示两条带在相对分子量为30kD及45kD左右；免疫组织化学显示AQP9表达于胎盘的合体滋养细胞、羊膜上皮细胞及平滑绒毛膜滋养细胞。结论 AQP9在母胎液体交换、胎儿代谢物的排出及羊水平衡等的分子机制中可能发挥重要作用。     

Fetal membrane healing after spontaneous and iatrogenic membrane rupture: a review of current evidence

In view of the important protective role of the fetal membranes, wound sealing, tissue regeneration, or wound healing could be life saving in cases of preterm premature rupture of the membranes. Although many investigators are studying the causes of preterm premature rupture of membranes, the emphasis has not been on the wound healing capacity of the fetal membranes. In this review, the relevant literature on the pathophysiologic condition that leads to preterm premature rupture of membranes will be summarized to emphasize a continuum of events between rupture and repair. We will present the current knowledge on fetal membrane wound healing and discuss the clinical implications of these findings. We will critically discuss recent experimental interventions in women to seal or heal the fetal membranes after preterm premature rupture of membranes.     

细胞间黏附分子-1在早产胎膜早破中的表达及临床意义

目的:研究细胞间黏附分子-1(ICAM-1)在早产胎膜早破中的表达及临床意义.方法:选取38例早产胎膜早破(PPROM)孕妇为研究组(PPROM组),36例与PPROM组孕周相对应之胎膜完整的自发早产(PTL)孕妇为对照组(PTL组),应用sP法检测各组胎膜ICAM-1的表达,并进行临床病理的相关性分析.结果:PPROM组中有55.26%(21/38)存在绒毛膜羊膜炎,PIL组中为36.11%(13/36),两组比较差异有统计学意义(P<0.05);两组胎膜中均存在不同程度的ICAM-1阳性表达,两组比较差异有统计学意义(P<0.05),PPROM组和PTL组ICAM-1的阳性表达与绒毛膜羊膜炎的相关系数分别为0.90和0.72,且病理结果中炎症越重的患者其胎膜上ICAM-1的阳性表达越明显.结论:ICAM-1的表达与感染相关,异常表达在早产胎膜早破的发病过程中起一定作用.感染越重的患者,ICAM-1的表达越明显,提示妊娠结局的严重化.     

羊膜腔输液治疗在产时应用的临床分析

目的 :分析羊膜腔输液 (amnioinfusion,AI)治疗产时羊水过少、胎膜早破和胎粪性羊水的临床意义。方法 :选择产程中发生胎心异常合并羊水过少、胎膜早破和羊水胎粪污染的孕产妇 1 0 1例 ,随机分为治疗组 51例 ,对照组 50例。治疗组在胎心监护下行羊膜腔输液或羊水置换 ;对照组给予吸氧 ,改变体位 ,静滴 5% Na HCO3等治疗。结果 :治疗组经羊膜腔输液 50 0～ 1 0 0 0 ml,胎心可变减速 (variable deceleration,VD)和长时减速 (period long deceleration,PL D)消失或明显改善 44例 ,有效率占 86 .3% ,明显高于对照组 (2 2 % ) ,两组比较差异有显著性 (P<0 .0 1 )。治疗组 1 9例胎粪性羊水行羊水置换 ,有 1 7例羊水转为清亮或 度混浊。治疗组产程时间比对照组缩短 ,治疗组和对照组剖宫产率分别为 1 3.7%和 34.0 % ,新生儿窒息率分别为 3.9%和 42 .0 % ;对照组胎粪吸入性肺炎 9例 ,新生儿死亡 3例 ,治疗组仅一例发生胎粪吸入性肺炎 ,无新生儿死亡。产褥病率两组比较 ,差异无显著性 (P>0 .0 5)。结论 :羊膜腔输液是治疗产时羊水过少、胎膜早破、胎粪性羊水的有效方法     

Male gender promotes an increased inflammatory response to lipopolysaccharide in umbilical vein blood

Objectives: To establish gender-specific differences in maternal and fetal immune response in healthy human fetuses at term. Methods: Forty-five women with elective caesarean sections for uncomplicated singleton pregnancies were recruited for two studies. Using a multiplex biomarker immunoassay system, unstimulated maternal and fetal plasma concentrations of interleukin (IL)-1β, IL-1ra, IL-6, IL-8, macrophage inflammatory protein (MIP)-1α, and tumor necrosis factor (TNF)-α were measured from one study population. Lipopolysaccharide (LPS)-stimulated cytokine response was measured in a second study. Results: There were no significant gender differences in either maternal or fetal unstimulated plasma cytokine concentrations, but concentrations of the proinflammatory cytokines IL-1β and IL-6 were significantly greater in male fetal LPS-stimulated samples than in female fetal samples. Conclusions: Blood of male fetuses mounts a larger pro-inflammatory response to lipopolysaccharide (LPS). This heightened response could be a critical pathway in promoting premature rupture of membranes (PPROM) and may be associated with life long differential gender response to infection.     

Further observations on the fetal inflammatory response syndrome: a potential homeostatic role for the soluble receptors of tumor necrosis factor alpha

OBJECTIVE: The fetal inflammatory response syndrome is a subclinical condition frequently present in preterm labor and preterm premature rupture of the membranes and is associated with increased perinatal morbidity and mortality. Tumor necrosis factor alpha is a mediator of septic shock and death, and it exerts its biologic effects by interacting with 2 receptors, TNF-R1 and TNF-R2. Soluble tumor necrosis factor receptors can buffer the biologic activity and protect against the deleterious effects of tumor necrosis factor alpha. The purpose of this study was to determine the behavior of soluble tumor necrosis factor receptors in fetuses with and without fetal inflammatory response syndrome. STUDY DESIGN: Fetal blood sampling was performed in patients with preterm labor (n = 95) and preterm premature rupture of the membranes (n = 39). Control samples were obtained from fetuses who were undergoing blood sampling for clinical indications and had normal outcomes (n = 21). Fetal inflammatory response syndrome was defined as a fetal plasma interleukin 6 concentration >11 pg/mL. Concentrations of interleukin 6 and TNF-R1 and TNF-R2 were determined by use of sensitive and specific immunoassays. Analysis of covariance was used for statistical analysis. RESULTS: (1) TNF-R1 and TNF-R2 were detectable in all samples, and their concentrations decreased with advancing gestational age (r = -0.8 and r = -0.7; P<.0001 and P<.001, respectively). (2) The mean fetal plasma concentrations of TNF-R1 and TNF-R2 were significantly higher in fetuses with fetal inflammatory response syndrome than in those without the syndrome after adjustment for gestational age and fetal membrane status (TNF-R1: no fetal inflammatory response syndrome, mean +/- SE, 3473.7+/-128.8 pg/mL; vs fetal inflammatory response syndrome, mean +/- SE, 4079.9+/-190.7 pg/mL; P<.005; TNF-R2: no fetal inflammatory response syndrome, mean +/- SE, 6033.2+/-235.4 pg/mL; vs. fetal inflammatory response syndrome, mean +/- SE, 7783.1+/-342.8 pg/mL; P<.0001). (3) Fetuses of patients who delivered within 72 hours of cordocentesis had significantly higher concentrations of TNF-R1 and TNF-R2 receptors than those with longer latency periods (P<.05 for each). CONCLUSION: The fetal inflammatory response syndrome is associated with increased availability of the soluble receptors of tumor necrosis factor alpha in fetal plasma. These factors may attenuate the deleterious effects of tumor necrosis factor alpha.