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.     

Decidual relaxins: gene and protein up-regulation in preterm premature rupture of the membranes by complementary DNA arrays and quantitative immunocytochemistry

OBJECTIVE: This study was undertaken to show both decidual relaxin gene and protein up-regulation in preterm premature rupture of the fetal membranes. STUDY DESIGN: Membranes after preterm premature rupture (n = 4) have been matched in pairs with preterm intact membranes (n = 4). These tissues were from patients without infection, labor, preeclampsia or intrauterine growth restriction, and none of the patients had a latency period of more than 8 hours. The messenger RNAs from these tissues were used on complementary DNA expression arrays; 488 genes were analyzed. Relaxin gene expression was quantitated from the arrays and in additional tissues by Northern analysis. The two relaxin proteins, H1 and H2, in the decidual cells were immunolocalized and quantitated by microdensitometry with the use of specific antisera that were raised to decapeptides over the region of least homologic features. The expression of five other genes that were selected from the arrays were quantitated by Northern analysis. RESULTS: Relaxin gene expression was up-regulated 3.4-fold on the complementary DNA arrays but was not confirmed on Northern analysis. On the other hand, protein analysis for relaxin H1 and H2 in the decidual cells showed them to be significantly up-regulated (P <.0001, for both proteins) in patients with preterm premature rupture of the membranes compared with control subjects. The 20 most highly expressed genes at preterm in tissues without rupture were determined. In addition, analysis of the genes that were up-regulated with preterm rupture of the membranes showed 30 differentially expressed genes. CONCLUSION: Relaxin gene expression in the decidua is up-regulated, and its protein expression is significantly increased with preterm rupture of the fetal membranes.     

基质金属蛋白酶-8和环氧合酶-2在早产、胎膜早破时胎膜中的表达及其意义

目的 :探讨基质金属蛋白酶 8(matrixmetalloproteinase -8,MMP -8)和环氧合酶 2 (cyclooxygenase -2 ,COX- 2 )在早产、胎膜早破 (PROM)发病机制中的作用。方法 :用免疫组化法 (SP法 )检测 8例早产临产、11例早产PROM、2 0例足月PROM、10例足月临产及 12例足月未临产正常产妇 (对照组 )宫颈胎膜和宫体胎膜组织中MMP 8和COX- 2的分布和表达。结果 :(1)MMP- 8和COX -2在羊膜及绒毛膜上皮细胞和间质细胞中均可见表达 ;(2 )前 4组产妇宫颈胎膜组织中MMP 8表达的免疫组化积分分别是 5 .2 5± 0 .89、5 .73±0 .4 7、4 .0 0± 1.95、3.70± 2 .2 6 ,均高于对照组的 2 .0 8± 1.5 6 (P <0 .0 5 ) ;同时亦均高于同组产妇宫体胎膜组织MMP 8的表达 ,分别是 3.38± 1.6 9、2 .2 7± 2 .2 8、2 .2 5± 2 .0 5与 1.70± 1.6 4(P <0 .0 5 ) ;而对照组宫颈和宫体胎膜组织中MMP 8的表达差异无显著性 (P >0 .0 5 ) ;(3) 5组产妇之间宫体胎膜组织中MMP- 8的表达差异无显著性 (P >0 .0 5 ) ;(4)宫颈和宫体胎膜组织中COX -2表达的免疫组化积分 ,早产临产组为 5 .75± 0 .4 4、5 .6 3±0 .5 2 ,足月临产组为 4 .4 0± 1.4 3、3.70± 1.77,均高于对照组的 2 .92± 1.31、2 .2 5± 1.6 6 (P<0 .0 5 ) ;其中早产临产组明显增?     

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.     

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.     

Genes upregulated in human fetal membranes by infection or labor.

OBJECTIVE: To determine whether suppression subtractive hybridization can detect genes in fetal membranes that are upregulated by infection, preterm premature rupture of membranes (PROM), or labor. METHODS: Using suppression subtractive hybridization, messenger RNAs from a preterm fetal membrane obtained at cesarean delivery without labor (control) were subtracted from a pool of messenger RNAs of three patients with preterm PROM and vaginal delivery. Eight candidate genes identified as upregulated were quantitated by Northern analysis in each of the tissues and in additional patient subgroups. RESULTS: Eight differentially upregulated genes were identified in preterm labor with PROM. Four of the genes are known to be involved in the response to inflammation or infection, and subsequent histologic examination showed one of the preterm PROM tissues to be infected. F-actin capping protein and chitinase precursor, not previously known to be involved in infection, were also upregulated in the infected tissue from preterm PROM. Northern blots using additional subgroups of patients showed that a regulatory G-protein signaling protein gene was significantly upregulated at term by labor in addition to significant upregulation of interleukin-8. There was a strong correlation between the gene expression for complement factor-B and duration of membrane rupture in the patients with preterm PROM. CONCLUSION: Two novel genes potentially involved in the response to inflammation or infection have been identified. A regulatory G-protein signaling protein and interleukin-8 gene expression were upregulated by labor. Complement factor-B gene expression was directly related to the duration of membrane rupture.     

Evidence for the participation of interstitial collagenase (matrix metalloproteinase 1) in preterm premature rupture of membranes

OBJECTIVE: Rupture of membranes is thought to result from the effects of physical forces in localized areas of the membranes weakened by the degradation of structural collagens. Matrix metalloproteinases are enzymes that degrade extracellular matrix components and have been implicated in membrane rupture. The objective of this study was to determine whether spontaneous rupture of membranes is associated with a change in the amniotic fluid concentration of interstitial collagenase (matrix metalloproteinase 1 [MMP-1]), a major collagenase. STUDY DESIGN: A cross-sectional study was conducted to determine MMP-1 concentrations in amniotic fluid from 353 women in the following categories: (1) term with intact membranes not in labor and in labor, (2) preterm labor who delivered at term, (3) preterm labor who delivered preterm without microbial invasion of the amniotic cavity, (4) preterm labor who delivered preterm with microbial invasion of the amniotic cavity, (5) preterm premature rupture of membranes with and without microbial invasion of the amniotic cavity, (6) term premature rupture of membranes not in labor and in labor, and (7) mid trimester of pregnancy. Microbial invasion of the amniotic cavity was determined by an amniotic fluid culture positive for microorganisms. MMP-1 concentrations in amniotic fluid were determined by means of sensitive and specific immunoassays. RESULTS: (1) MMP-1 was detectable in 81.3% of amniotic fluid samples (287/353), and its concentrations increased with advancing gestational age (r = 0.4; P <.001). (2) Preterm premature rupture of membranes was associated with a significant increase in the median amniotic fluid concentration of MMP-1 (P =.02). (3) Women with term premature rupture of membranes had a significantly lower amniotic fluid MMP-1 concentration than those with intact membranes at term not in labor (P <.001). (4) Microbial invasion of the amniotic cavity in patients in preterm labor with intact membranes and in patients with preterm premature rupture of membranes was also associated with significant increases in the median amniotic fluid MMP-1 concentrations (P <.05 and P <.01, respectively). (5) Patients with preterm premature rupture of membranes and microbial invasion of the amniotic cavity had a significantly higher median amniotic fluid MMP-1 concentration than those with intact membranes and microbial invasion of the amniotic cavity (P =.01). (6) Neither term nor preterm parturition was associated with changes in amniotic fluid MMP-1 concentrations (P =.6 and P =.3, respectively). CONCLUSION: (1) Collagenase 1 (MMP-1) is a physiologic constituent of amniotic fluid. (2) Preterm premature rupture of membranes (in both the presence and absence of infection) was associated with an increase in the amniotic fluid MMP-1 concentrations. (3) Neither term nor preterm parturition was associated with a significant increase in the amniotic fluid concentration of MMP-1.     

Lactoferrin in intrauterine infection, human parturition, and rupture of fetal membranes

OBJECTIVE: Lactoferrin is an iron-binding protein with antimicrobial properties. This study was undertaken to determine whether amniotic fluid concentrations of this protein change with gestational age, infection, labor, and rupture of membranes. STUDY DESIGN: This cross-sectional study included women who underwent transabdominal amniocentesis (n = 268) in the following groups: (1) mid trimester of pregnancy; (2) preterm labor who delivered at term, preterm labor who delivered preterm with intra-amniotic infection, and preterm labor who delivered preterm without intra-amniotic infection; (3) preterm premature rupture of membranes in the presence or absence of intra-amniotic infection; (4) term with intact membranes not in labor, in labor, and in labor with intra-amniotic infection; and (5) premature rupture of membranes at term not in labor. In addition, lactoferrin concentrations were determined in maternal plasma and cord blood of patients at term not in labor. Lactoferrin concentration was measured with an immunoassay. RESULTS: (1) Lactoferrin was detectable in 85.4% (229/268) of amniotic fluid samples, not detectable in all fluid obtained in the mid trimester, and detectable in all maternal and cord plasma samples. (2) The concentration of lactoferrin increased with advancing gestational age (r = 0.68; P <.0001). (3) Intra-amniotic infection was associated with significant increases in amniotic fluid lactoferrin concentrations in patients with preterm labor (no intra-amniotic infection median, 1641.2 ng/mL; range, <1.24-35,090.0 ng/mL; vs intra-amniotic infection median, 3833.6 ng/mL; range, 746.0-47,020.0 ng/mL; P <.001), term labor (no intra-amniotic infection median, 2085.8 ng/mL; range, 425.0-23,230.0 ng/mL; vs intra-amniotic infection median, 5627.0 ng/mL; range, <1.24-19,220.0 ng/mL; P <. 001), and preterm premature rupture of membranes (no intra-amniotic infection median, 2190 ng/mL; range, <1.24-7456.1 ng/mL; vs intra-amniotic infection median, 3449.3 ng/mL; range, <1.24-83,600. 0; P <.01). (4) Spontaneous labor at term but not preterm was associated with a significant decrease in amniotic fluid lactoferrin concentration (P <.05). (5) Spontaneous term parturition was associated with a significant increase in umbilical cord plasma lactoferrin concentration (P <.005). CONCLUSION: (1) Intra-amniotic infection was consistently associated with dramatically increased concentrations of lactoferrin in amniotic fluid. (2) Term parturition was associated with a significant increase in lactoferrin concentration in the fetal compartment (umbilical cord blood) and a decrease in the amniotic compartment. We propose that lactoferrin is part of the repertoire of host defense mechanisms against intra-amniotic infection.     

The human relaxin receptor (LGR7): expression in the fetal membranes and placenta

The relaxin receptor has been recently described as a leucine-rich repeat G-protein coupled receptor and designated as LGR7. A closely related receptor, LGR8, is co-expressed by some cells. This study explored the expression of the genes for these receptors in the human fetal membranes and placenta by RT-PCR and the LGR7 protein by immunolocalization. The results showed that LGR7 was well expressed in the fetal membranes, with significantly more in the decidua (p<0.05) than in the amnion. On the other hand, relatively low levels were expressed in the placenta. The major splice variant of LGR7 was undetectable in either the placenta or fetal membranes. Expression of LGR8 was also below the level of detectability in either tissue. Immunostaining for LGR7 was conducted with antisera to both its endodomain and ectodomain, in order to seek evidence for a solubilized ectodomain. However, similar staining patterns were obtained with both antisera, with predominant staining in the cells of the amniotic epithelium, chorionic cytotrophoblast and decidua. Full-thickness fetal membranes from preterm deliveries, before and after labor or after preterm premature rupture of the membrane (PPROM) and labor were collected. In addition, membranes at term, both before and after spontaneous labor were used for analysis of LGR7 gene expression. There was significantly greater LGR7 expressed (p=0.01) in the preterm period compared to term, indicating a potentially important role for relaxin at this time. There was a marginal decline in LGR7 gene expression after labor and delivery both at preterm and term, which did not reach significance. Immunostaining patterns showed less inter-patient variability than did gene expression, with more intense staining for LGR7 after labor and delivery.     

Matrilysin (matrix metalloproteinase 7) in parturition, premature rupture of membranes, and intrauterine infection

OBJECTIVE: Matrix metalloproteinases are enzymes capable of degrading extracellular matrix components. Matrilysin (matrix metalloproteinase 7), a novel member of this family, degrades fibronectin and proteoglycans. The objective of this study was to determine whether parturition (either term or preterm), premature rupture of the membranes, and microbial invasion of the amniotic cavity are associated with changes in the amniotic fluid concentration of matrilysin. STUDY DESIGN: A cross-sectional study was conducted with 275 women in the following categories: (1) second trimester, (2) term not in labor, (3) term in labor, (4) term with microbial invasion of the amniotic cavity, (5) preterm labor with intact membranes without microbial invasion of the amniotic cavity who delivered at term, (6) preterm labor without microbial invasion of the amniotic cavity who delivered preterm, (7) preterm labor with microbial invasion of the amniotic cavity, (8) preterm premature rupture of membranes with and without microbial invasion of the amniotic cavity, and (9) term premature rupture of membranes not in labor and without microbial invasion of the amniotic cavity. Matrilysin concentrations were measured with a sensitive specific immunoassay that was validated for amniotic fluid. RESULTS: Matrilysin was detectable in 97.4% (268/275) of the samples. The concentration of matrilysin increased with advancing gestational age (r = 0.8; P <.001). Parturition at term was not associated with a significant increase in amniotic fluid concentration of matrilysin. Preterm parturition in the absence of microbial invasion of the amniotic cavity was associated with a significant increase in amniotic fluid concentration of matrilysin (preterm labor with preterm delivery: median, 1.7 ng/mL; range, 0.45-21.6 mg/mL; vs preterm labor with term delivery: median, 1.2 ng/mL; range, 0.17-42. 1 ng/mL; P <.05). Premature rupture of membranes without microbial invasion of the amniotic cavity (either term or preterm) was not associated with a significant change in the amniotic fluid matrilysin concentration. Intra-amniotic infection was associated with a significant increase in amniotic fluid matrilysin among both patients with preterm labor and patients with preterm premature rupture of membranes (preterm labor with microbial invasion of the amniotic cavity: median, 3.2 ng/mL; range, 0.16-21.9 ng/mL; vs preterm labor and delivery without microbial invasion of the amniotic cavity: median, 1.7 ng/mL; range, 0.45-21.6 ng/mL; vs preterm labor with term delivery: median, 1.2 ng/mL; range, 0.17-42. 1 ng/mL; P <.01 for each comparison; and preterm premature rupture of membranes without microbial invasion of the amniotic cavity: median, 1.7 ng/mL; range, 0.29-13.9 ng/mL; vs preterm premature rupture of membranes with microbial invasion of the amniotic cavity: median, 3.6 ng/mL; range, 0.59-20.3 ng/mL; P <.01). CONCLUSION: Matrilysin is a physiologic constituent of amniotic fluid, and its concentration increases with advancing gestational age. Microbial invasion of the amniotic cavity in preterm gestations was associated with a significant increase in amniotic fluid concentration of matrilysin. Matrilysin therefore may play a role in the host defense mechanism.     

胎膜组织BMP-2及MMP-9的表达及与胎膜早破的关系

目的:探讨胎膜组织骨形成蛋白-2(BNP-2)及基质金属蛋白酶-9(MMP-9)与胎膜早破(PROM)发生的关系.方法:选择足月临产产妇、足月PROM产妇、早产临产产妇、早产PROM产妇及足月未临产产妇(对照组)各18例,分娩后取破口处胎膜组织,采用免疫组织化学法染色,并使用病理影像多媒体图文操作系统对BMP-2、MMP-9阳性表达物的面积积分光密度(AIOD)进行半定量分析.结果:①胎膜组织中BMP-2和MMP-9阳性表达的AIOD在足月临产组、足月PRON组、早产临产组、早产PROM组均高于对照组,差异均有统计学意义(P<0.0071);②足月PROM组BMP-2和MMP-9高于足月临产组(P=0.0065,P=0.0069);早产PROM组BNP-2和NMP-9高于早产临产组(P=0.0069,P=0.0052);早产PROM组BMP-2和MMP-9高于足月PROM组(P=0.0037,P=0.0065);③5组胎膜组织中BMP-2和MMP-9的表达均呈明显正相关(r_s=0.76159,P<0.0001).结论:BMP-2和MMP-9与胎膜破裂的发生密切相关,胎膜组织中BMP-2与MMP-9的表达水平相关.     

A role for the 72 kDa gelatinase (MMP-2) and its inhibitor (TIMP-2) in human parturition, premature rupture of membranes and intraamniotic infection

OBJECTIVE: Degradation of the extracellular matrix in fetal membranes has been implicated in the process of parturition and rupture of membranes. Matrix metalloproteinases (MMPs) are enzymes capable of degrading extracellular matrix including collagen. Tissue inhibitors of matrix metalloproteinases (TIMPs) inhibit the activity of MMPs by covalently binding to the enzymes. MMP-2 degrades Type IV collagen and TIMP-2 is its specific inhibitor. The objective of this study was to determine if human parturition, rupture of membranes (term and preterm) and microbial invasion of the amniotic cavity (MIAC) are associated with changes in the concentrations of MMP-2 and TIMP-2 in amniotic fluid. STUDY DESIGN: A cross-sectional study was conducted with women in the following categories: 1) term with intact membranes, in labor and not in labor; 2) preterm labor and intact membranes who delivered at term, who delivered preterm and preterm labor with MIAC; 3) preterm premature rupture of membranes (PROM) with and without infection; 4) term and preterm PROM not in labor; and 5) midtrimester. MMP-2 and TIMP-2 concentrations in amniotic fluid were determined using sensitive and specific immunoassays. RESULTS: The concentration of TIMP-2 increased with advancing gestational age (r = 0.6, p < 0.001). No correlation was found between MMP-2 concentrations and gestational age. Human parturition and rupture of membranes (term and preterm) and in patients with intact membranes were not associated with changes in the amniotic fluid MMP-2 concentrations. In contrast, 1) patients with spontaneous labor (term and preterm) had significantly lower median concentrations of TIMP-2 compared to those not in labor (p < 0.05 for both); 2) MIAC in women with preterm labor and preterm PROM was associated with a significant decrease in amniotic fluid TIMP-2 concentrations (p < 0.04 for both comparisons); 3) Rupture of the membranes (term and preterm) was also associated with a significant decrease in the amniotic fluid TIMP-2 concentrations (p < 0.05 and p < 0.03, respectively). CONCLUSIONS: Human parturition (preterm and term), rupture of fetal membranes (term and preterm) and intraamniotic infection are associated with a significant decrease in amniotic fluid TIMP-2 concentrations.     

Pre-B-cell colony-enhancing factor,a novel cytokine of human fetal membranes

OBJECTIVE: Our purpose was to determine whether pre-B-cell colony-enhancing factor (PBEF) is expressed in the human fetal membranes during normal gestation and parturition in the absence of infection and to show its effects on the expression of interleukin (IL)-6 and IL-8. STUDY DESIGN: PBEF was immunolocalized in the fetal membranes from early pregnancy, at preterm, and at term. Its expression was quantitated by Northern analysis in separated uninfected amnion, chorion, decidua, and placenta of patients at term before labor and in full-thickness membranes before and after spontaneous labor at preterm and at term. Amnion-like epithelial (WISH) cells and fetal membrane explants were treated with recombinant PBEF (rhPBEF), and the expression of IL-6 and IL-8 was quantitated. RESULTS: PBEF was immunolocalized throughout gestation in the amniotic epithelium and mesenchymal cells as well as the chorionic cytotrophoblast and parietal decidua. Northern analysis showed significantly more (P <.01) PBEF expressed in the amnion than in either chorion or placenta. Its expression increased after labor at both preterm and term and correlated with that of IL-8 (r = 0.87). rhPBEF treatment of WISH cells significantly increased IL-6 (P <.05) and IL-8 (P <.01) gene expression after 4 hours and of IL-8 protein after 24 hours (P <.01); similar 4-hour treatment of fetal membrane explants significantly increased IL-6 (P <.01) and IL-8 (P <.05) gene expression. CONCLUSION: PBEF is a novel cytokine constitutively expressed by the fetal membranes during pregnancy. It increased the expression of IL-6 and IL-8 and may be important in both normal spontaneous labor and infection-induced preterm labor.     

15-Hydroxyprostaglandin dehydrogenase protein expression in human fetal membranes with and without subclinical inflammation

Prostaglandins play a central role in the stimulation and maintenance of both term and preterm labor. 15-Hydroxyprostaglandin dehydrogenase (PGDH), localized primarily to chorion trophoblasts, is the key enzyme responsible for the metabolism of prostaglandins. In preterm chorion, levels of PGDH protein and activity were lower when compared to term and were further reduced with the presence of infection, but effects of subclinical inflammation and membrane rupture on PGDH expression are not known. Our objectives were (1) to determine the relative expression of PGDH in amnion and chorion and (2) to determine the effect of preterm premature rupture of membranes (PPROM) and (3) subclinical inflammation on PGDH protein expression in preterm fetal membranes. Fetal membranes were collected from women with idiopathic preterm labor. Patients were divided into preterm birth (1) <32 weeks with PPROM (n = 6), (2) <32 weeks with intact membranes (n = 11), (3) >or=32 and <37 weeks with PPROM (n = 10), and (4) >or=32 and <37 weeks with intact membranes (n = 10). Different antibodies were used to detect protein expression and localization of PGDH in amnion and chorion from these patients using both Western blotting and immunohistochemistry. Antibody T (AbT) localized PGDH to chorion trophoblasts, whereas antibody C (AbC) detected immunoreactive (ir) PGDH predominantly in the amnion mesenchyme. By Western blot, AbT showed a stronger 29-kDa ir-PGDH band whereas with AbC, a stronger 55-kDa ir-PGDH signal was detected. 55-kDa ir-PGDH was significantly higher in PPROM amnion, specifically in the <32 weeks group (P < .05) and with PPROM >24 hours (P < .05). No change was detected in the 29-kDa ir-PGDH in either amnion or chorion with gestational age or the presence and absence of PPROM. In addition, neither form of ir-PGDH was altered significantly with or without subclinical inflammation. ir-PGDH is detectable in both chorion trophoblasts and amnion, especially in the mesenchyme; however, the predominant form of the enzyme differs in the 2 tissues. PPROM and subclinical inflammation do not appear to affect the levels of 29-kDa ir-PGDH protein in the fetal membranes. The differential expression of 55-kDa ir-PGDH in preterm amnion with and without PPROM supports the need for a better understanding of the different forms of PGDH.     

Evidence of in vivo differential bioavailability of the active forms of matrix metalloproteinases 9 and 2 in parturition, spontaneous rupture of membranes, and intra-amniotic infection

OBJECTIVE: Matrix metalloproteinases (MMP-9 and MMP-2) have been implicated in the digestion of fetal membranes. The purpose of this study was to determine the amniotic fluid concentrations of active forms of MMP-2 and MMP-9 and to explore the participation of these enzymes in labor (term and preterm), rupture of membranes (term and preterm), and microbial invasion of the amniotic cavity. STUDY DESIGN: A cross-sectional study was conducted with 291 women in the following categories: (1) term not in labor, (2) term in labor, (3) preterm labor and intact membranes who delivered at term, (4) preterm labor who delivered preterm, (5) preterm labor with microbial invasion of the amniotic cavity, (6) preterm premature rupture of membranes without microbial invasion of the amniotic cavity, (7) preterm premature rupture of membranes with microbial invasion of the amniotic cavity, (8) term premature rupture of membranes not in labor, and (9) mid trimester. Active forms of MMP-2 and MMP-9 were measured by a novel assay that uses a substrate developed by protein engineering. RESULTS: (1) MMP-2 and MMP-9 were detected in 88% and 96% of amniotic fluid samples, respectively (255/291 and 279/291). (2) The concentrations of active forms of MMP-2 and MMP-9 changed with advancing gestational age. (3) Spontaneous term parturition was associated with a significant increase in the median concentration of the active forms of MMP-9 (P <.005) and a significant decrease in the median concentration of the active forms of MMP-2 (P <.003). (4) Preterm labor with intact membranes leading to preterm delivery in the absence of infection was associated with a significant increase in the median concentration of the active forms of MMP-9 (P <.005) but not of the active forms of MMP-2 (P =.2). (5) Rupture of membranes (either term or preterm) was associated with a significant increase in the concentration of the active forms of MMP-9 and with a significant decrease in the concentration of the active forms of MMP-2 (P <.005 for term and P <.03 and P <.003 for preterm, respectively). (6) Microbial invasion of the amniotic cavity in women with preterm premature rupture of membranes was also associated with a significant increase in the concentration of the active forms of MMP-9 (P <.03) and a decrease in the concentration of the active forms of MMP-2 (P <.05). (7) Microbial invasion of the amniotic cavity in patients with preterm labor was associated with a significant increase in the median concentration of the active forms of MMP-9 (P <.005) but not of the active forms of MMP-2 (P =.6). CONCLUSION: Spontaneous rupture of membranes (either term or preterm), parturition (either term or preterm), and microbial invasion of the amniotic cavity were associated with significant increases in the amniotic fluid concentration of the active forms of MMP-9. In contrast, the concentration of the active forms of MMP-2 either decreased or remained the same in these conditions. Our observations provide evidence for a novel regulation of gelatinolytic activity in vivo.     

Human neutrophil collagenase (matrix metalloproteinase 8) in parturition, premature rupture of the membranes, and intrauterine infection

OBJECTIVES: The mechanisms by which microbial invasion of the amniotic cavity leads to membrane weakening and rupture are poorly understood. Recently, endogenous host enzymes have been implicated in this process. Matrix metalloproteinases are a family of potent enzymes that degrade components of the extracellular matrix. Collagen type I provides the main tensile strength of the fetal membranes. Matrix metalloproteinase 8 (MMP-8), or neutrophil collagenase, degrades interstitial collagens, acting preferentially on collagen type I. This study was undertaken (1) to determine whether MMP-8 is present in amniotic fluid and whether its concentrations are changed in preterm and term labor and membrane rupture with and without intra-amniotic infection and (2) to determine whether the amniotic fluid concentrations of MMP-8 in labor at term are different in the lower and upper uterine compartments. STUDY DESIGN: A cross-sectional study was conducted and transabdominal amniocentesis was performed in women in the following categories: (1) midtrimester (n = 25), (2) preterm labor in the presence and absence of microbial invasion of the amniotic cavity (n = 86), (3) preterm premature rupture of the membranes in the presence and absence of microbial invasion of the amniotic cavity (n = 51), (4) term patients in labor and not in labor (n = 51), and (5) term premature rupture of membranes (n = 20). Additional paired samples of amniotic fluid were retrieved by transabdominal amniocentesis (upper compartment) and transvaginal amniocentesis (lower or forebag compartment) from 14 term patients (28 samples) in spontaneous labor with intact membranes. Amniotic fluid MMP-8 concentrations were determined with a sensitive and specific immunoassay. RESULTS: MMP-8 was detected in 95.4% (249/261) of all samples. (1) Spontaneous human parturition was associated with a significant increase in amniotic fluid concentrations of MMP-8 in both term and preterm gestation. Term (no labor median, 3.3 ng/mL; range, <0.06-38.6 ng/mL; vs labor median, 16.6 ng/mL; range, 0. 33-1650 ng/mL; P <.05). Patients with preterm labor who delivered preterm (in the absence of microbial invasion of the amniotic cavity) had a significantly higher median amniotic fluid MMP-8 concentration than those with preterm labor who delivered at term (preterm labor, term delivery median, 3.1 ng/mL; range, <0.06-415.1 ng/mL; vs preterm labor, preterm delivery median, 32.5 ng/mL; range, <0.06-6006.6 ng/mL;P <.003). (2) Spontaneous rupture of membranes in preterm gestation but not in term gestation was associated with elevated amniotic fluid concentrations of MMP-8. Preterm gestation (preterm labor, intact membranes median, 3.1 ng/mL; range, <0.06-415. 1 ng/mL; vs preterm premature rupture of membranes median, 35.1 ng/mL; range, 0.71-1184.1 ng/mL; P <.05). Term gestation (intact membranes median, 3.3 ng/mL; range, 0.24-38.6 ng/mL; vs rupture of membranes median, 5.6 ng/mL; range, 0.22-19.8 ng/mL; P =.9). (3) Microbial invasion of the amniotic cavity was associated with a significant increase in amniotic fluid MMP-8 concentration in patients with preterm labor and intact membranes, as well as in patients with preterm premature rupture of membranes. Preterm labor (no microbial invasion of the amniotic cavity, preterm delivery median, 32.5 ng/mL; range, <0.06-6006.6 ng/mL; vs microbial invasion of the amniotic cavity median, 208.1 ng/mL; range, 4.2-14,600 ng/mL; P <.001). Preterm premature rupture of membranes (no microbial invasion of the amniotic cavity median, 35.1 ng/mL; range, 0.71-1184. 1 ng/mL; vs microbial invasion of the amniotic cavity median, 317.9 ng/mL; range, 2.16-14,500 ng/mL; P <.01). (4) The median amniotic fluid MMP-8 concentrations were significantly higher in fluid obtained from the forebag compartment than in that obtained from the upper compartment (median, 66.2 ng/mL; range, 7.4-170 ng/mL; vs median, 13.3 ng/mL; range, 2-170 ng/mL; respectively; P <.01). (ABSTRACT TRUNCATED)     

Preterm premature rupture of membranes: vascular endothelial growth factor and its association with histologic chorioamnionitis

OBJECTIVE: We hypothesize that vascular endothelial growth factor, a known angiogenic and permeability factor that is locally expressed in fetal membranes and decidua, may be the primary regulator in the pathway that eventually leads to preterm premature rupture of membranes. Our objective was to test the hypothesis that, both in the presence and in the absence of histologic chorioamnionitis, there is an increased expression of the vascular endothelial growth factor gene and its receptor Flt-1 in the human fetal membranes. STUDY DESIGN: Membranes were sampled from a region that was distinct as the rupture site from three groups of patients with preterm premature rupture of membranes. Groups 1 and 2 differed only in the length of the latency period from rupture of the membranes to delivery. Group 3 included preterm patients with intact membranes, who acted as control subjects. All patients who were selected for the study lacked clinical signs of chorioamnionitis and were delivered by cesarean delivery. Tissue samples were analyzed for interleukin-6 gene expression by Northern blot analysis and for the presence of interleukin-6 protein by immunocytochemistry. The expression of vascular endothelial growth factor and Flt-1 genes was analyzed by in situ hybridization. RESULTS: All tissue samples from group 1 and five tissue samples from group 2 (designated as group 2A) showed expression of the interleukin-6 gene and the presence of interleukin-6 protein in the fetal membranes (P <.001) and were therefore identified as inflamed. Five tissue samples from the patients in group 2 (designated as group 2B) and all control tissue samples showed neither evidence of interleukin-6 gene expression nor the presence of its protein and therefore were identified as not inflamed. Vascular endothelial growth factor and Flt-1 gene expression were increased significantly in the fetal membrane and decidua samples that were obtained from the noninflamed tissues from group 2B (P <.005) yet showed further enhancement in expression in the inflamed tissues. CONCLUSION: The expression patterns of vascular endothelial growth factor and Flt-1 genes are indicative of a molecular pathologic condition of fetal membranes, regardless of their inflammatory status, which suggests their role as a primary regulator of preterm premature rupture of membranes.     

Uterine activity after preterm premature rupture of the membranes

Preterm premature rupture of the membranes complicates few pregnancies but is a major contributor to overall perinatal morbidity and mortality. Although a reduced incidence of preterm premature rupture of fetal membranes has been reported in women who had antepartum uterine activity monitoring, there are few data regarding uterine activity after preterm premature rupture of fetal membranes. Therefore daily uterine activity monitoring was performed in 101 consecutive women with preterm premature rupture of fetal membranes between 26 and 34 weeks' gestation. The mean gestational ages at rupture and delivery were 31.4 +/- 2.3 and 33.7 +/- 4.5 weeks, respectively. A significant increase in contraction frequency was identified within 24 hours of onset of preterm labor (p less than 0.005). A contraction frequency of four or more per hour predicted the onset of labor within 24 hours with a sensitivity of 72%, a specificity of 90%, a positive predictive value of 54%, and a negative predictive value of 95%. These results indicate that most women with preterm premature rupture of fetal membranes exhibit a baseline contraction frequency that is similar to that of women with intact membranes and premature labor. An abrupt increase in contraction frequency is a warning of impending labor.