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.     

Role of apoptosis,bcl-2 and bax protein expression in premature rupture of fetal membranes

OBJECTIVE: To examine the degree of apoptosis in human fetal membranes associated with premature rupture of membranes (PROM) as compared with normal pregnancies and to evaluate the expression of proapoptotic bax and antiapoptotic bcl-2 gene products. STUDY DESIGN: Fetal membranes from 50 pregnancies were included in the study. Thirty of 50 pregnancies had PROM. Twenty pregnancies with intact membranes served as controls. Chorioamniotic membrane biopsies were taken from the rupture site of the membrane and periphery of the rupture side. In the control group, membrane biopsies were taken from the artificial rupture site, cervical pole of the membranes and membranes close to the edge of the placenta. In recognizing apoptotic figures, routinely processed samples were stained with hematoxylin and eosin for light microscopic evaluation. Quantification of the apoptotic cells was performed with high-power fields and expressed as the number per 100 cells. The membranes of both groups were then stained with bcl-2 and bax antibodies by using the standard steptavidin-biotin-immunoperoxidase method. Staining with both antibodies were compared between two groups. RESULTS: Apoptotic cells were detected in the amniotic epithelium, in chorionic cells and fibroblastic layer of the fetal membranes. Apoptotic cells were found mostly in the chorionic cells. There was a statistically significant difference between the apoptotic index in PROM and the control group in both rupture and peripheral sites (P < .05), although within each group peripheral and rupture sites showed no difference in terms of apoptotic cell counts. Both bax and bcl-2 expression was observed in 40% of control cases and in 57% and 50% of cases with PROM, respectively, mostly in the chorionic trophoblastic cells. The PROM and control groups showed no statistically significant difference in terms of bcl-2 and bax protein expression. CONCLUSION: Apoptosis may play a role in the pathogenesis of PROM, but the changes in apoptosis do not seem to be mediated by bcl-2 and bax genes in the amniotic membrane. Other regulatory mechanisms must be investigated.     

Increased concentration of pro-matrix metalloproteinase 9 in term fetal membranes overlying the cervix before labor: implications for membrane remodeling and rupture

OBJECTIVES: Regional structural alterations that develop before labor are important in the mechanisms of both physiologic and pathologic membrane rupture, because they are also detected in preterm prelabor rupture of the fetal membranes, the most common cause of preterm birth (as great as 60%). Matrix metalloproteinases are located in the fetal membranes and are the main mediators of extracellular matrix degradation. The objective of this study was to examine whether gelatinases (matrix metalloproteinases 2 and 9) could be involved in the development of these regional structural changes seen at term before labor. STUDY DESIGN: Fetal membranes from patients undergoing elective cesarean delivery were regionally sampled from over the cervix (cervical membranes) and midway between this area and the placental edge (midzone). Fetal membranes obtained after spontaneous labor and delivery at term were also regionally sampled. Matrix metalloproteinase 2 and 9 activities were assessed by gelatin zymography, whereas total matrix metalloproteinase 9 protein was determined by enzyme-linked immunosorbent assay. RESULTS: Zymography only detected activity corresponding to the pro-matrix metalloproteinase 2 (72 kd) and 9 (92 kd) forms in prelabor fetal membranes. Although the levels of pro-matrix metalloproteinase 2 showed no regional differences, the pro-matrix metalloproteinase 9 level was higher in the cervical area than in the midzone (2.5 +/- 0.98 vs 0.76 +/- 0.28 optical density units/20 microg protein; P <.01). The concentration of pro-matrix metalloproteinase 9 protein in the cervical area was also significantly higher than that in the midzone (6.69 +/- 4.8 vs 1.58 +/- 1.14 ng/mg protein; P <.01). After delivery both pro-matrix metalloproteinase 2 and 9 activities were elevated, whereas pro-matrix metalloproteinase 9 protein activity showed no regional difference between the rupture site and midzone (23.47 +/- 4.5 vs 25. 3 +/- 6.2 ng/mg protein). Active bands of matrix metalloproteinases 2 (66 kd) and 9 (83 kd) were also detected after delivery. CONCLUSION: This study suggests that a specific regional induction of pro-matrix metalloproteinase 9 occurs in the cervical area before labor and may play a role in "programming" this area for subsequent rupture after activation during labor.     

Association of type II apoptosis and 92-kDa type IV collagenase expression in human amniochorion in prematurely ruptured membranes with tumor necrosis factor receptor-1 expression

OBJECTIVE: We assessed the presence of tumor necrosis factor receptor-1 (TNF-R1), apoptosis, and simultaneous expression of 92-kDa collagenase type IV (MMP-9) in samples of human chorioamnion from women with premature rupture of membranes (PROM). METHODS: Amniotic membranes from women who underwent normal labor, cesarean delivery, or had PROM at term were studied by immunohistochemistry for localization of TNF-R1 and R2. Transmission electron microscopy and DNA fragmentation analyses by agarose gel electrophoresis were performed to identify apoptosis characteristics. Zymography and in situ zymography were used to assess gelatinolytic activity. RESULTS: We found that TNF-R1 was abundant in membranes from subjects who had normal labor and very abundant in those who had PROM. By contrast TNF-R2 was abundant only in membranes from subjects who had cesarean delivery. Gelatinolytic activity was associated with extracellular matrix rather than cells and was higher in extracts from fetal membranes from PROM and normal labor than in extracts obtained from cesarean deliveries. Transmission electron microscopy of fetal membranes from PROM revealed ultrastructural characteristics in amnion epithelium consistent with type II apoptosis. DNA laddering in agarose gel electrophoresis corroborated results from DNA fragmentation. CONCLUSION: During PROM the fetal membranes undergo type II apoptosis and extracellular matrix degradation in association with TNF-R1 expression.     

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.     

官腔脱落细胞中p53、ki-67、bcl-2、bax基因的表达及意义

目的 采用实时荧光定量PCR法检测p53、ki-67、bcl-2、bax基因在子宫内膜癌及正常人官腔脱落细胞中的表达变化水平,探讨官腔脱落细胞中p53、ki-67、bcl-2、bax基因的表达变化在子宫内膜癌早期诊断中的意义.方法 收集健康志愿者及子宫内膜癌患者官腔脱落细胞样本共90例;提取细胞总RNA,采用实时荧光定量PCR法检测目的基因的表达变化水平;所有实验数据采用SPSS 17.0软件进行统计学分析.结果 荧光定量PCR结果显示,p53基因在正常人官腔脱落细胞中未检测到表达,随着恶性程度升高,表达量显著升高(P＜0.01),ki-67的表达在正常人中表达量最低,随着恶性程度升高,表达量逐渐升高(P＜0.05),bcl-2基因的表达逐渐降低(P＜0.05),bax/bcl-2值随着恶性程度增高而降低.结论 p53、ki-67、bcl-2、bax基因的表达量在子宫内膜癌发生发展过程中呈一定的变化趋势,提示p53、ki-67、bcl-2、bax基因可作为子宫内膜癌早期诊断的分子标志物.     

TNF-alpha promotes caspase activation and apoptosis in human fetal membranes

Purpose: Increased amniotic fluid tumor necrosis factor (TNF) is a marker of infection when associated with preterm labor and preterm premature rupture of the amniochorionic membranes (PROM). We have noted increased apoptosis in membranes derived from women with PROM. This study examines the role of TNF in promoting fetal membrane apoptosis. Methods: Amniochorion (n = 8), collected at the time of elective repeat cesarean section prior to labor from normal term gestation, were placed in an organ explant system. After 48 h in culture, the membranes were stimulated with recombinant TNF- (20 ng/mL) for 24 h. Tissue frozen after stimulation was subjected to RT-PCR to study the expression of TNF-induced caspase genes. ELISA assayed the levels of proapoptotic p53 in tissues and cell death related nuclear matrix protein (NMP) in tissue culture supernatants. The activity of caspases in tissue homogenates was measured using substrates specific for caspase 2, 3, 6, 8, and 9. Results were analyzed by using the Wilcoxon nonparametric test for paired samples. A p < 0.05 was considered significant. Results: RT-PCR showed induction of caspases 2, 8, and 9 (caspase cascade initiators) in human fetal membranes after TNF stimulation. Caspases 3 and 6 (effector caspases) expression was constitutive in both TNF stimulated- and control membranes. Caspases, 2, 3, 8, and 9 activity was significantly higher in TNF-stimulated tissues compared with control, whereas, no significant change in caspase 6 activity was noticed. TNF-stimulated tissues released increased levels of NMP (24.03 U/mL) compared with control (13.5U/mL) (p = 0.03). TNF also increased p53 levels in the tissues (0.05 ng/mL) compared with control cultures (0.03 ng/mL; p = 0.02). Conclusions: TNF increases proapoptotic p53 levels and caspase activities in fetal membranes. Increased NMP reflects cell death.     

Evaluation of the expression and enzyme activity of matrix metalloproteinase-7 in fetal membranes during premature rupture of membranes at term in humans

Amnion, chorion, and decidua were separated from fetal membranes at term from women with no labor (cesarean delivery [CS], n = 10), labor (normal delivery, n = 10), and labor during premature rupture of membranes (PROM; n = 8) for evaluation of matrix metalloproteinase (MMP)-7. The expression of pro-MMP-7 was immunohistochemically demonstrated in amnion, chorion, and decidua. Interestingly, however, Western blotting revealed that pro-MMP-7 and MMP-7 expression was the lowest in amnion from PROM, whereas it was the highest in chorion and decidua from PROM. Importantly, the enzymatic activity of MMP-7 determined with an MMP-7-specific substrate was higher in amnion from PROM than that from CS. Moreover, the tissue inhibitor of metalloproteinase (TIMP)-1 level was lower in amnion from PROM than that from CS. Thus, MMP-7 is expressed in fetal membranes (amnion, chorion, and decidua), and its activity is increased in amnion of PROM at term, accompanied with the reduced level of TIMP-1, which may suggest the possible involvement of MMP-7 in PROM.     

Pretreatment assessment of prognostic indicators in endometrial cancer

OBJECTIVE: The object of this study was to assess the association of histologic, cytokinetic, and molecular variables in preoperative endometrial samples with extrauterine disease, recurrence, and survival among patients with endometrial cancer. STUDY DESIGN: In a case-cohort study of 125 women, ploidy, S-phase fraction, proliferative index, deoxyribonucleic acid index, proliferating cell nuclear antigen, MIB-1 proliferation marker, p53 tumor suppressor gene, and cytoplasmic HER-2/neu oncogene and bcl-2 expressions were quantitated. RESULTS: A model with only one independent term predicted progression-free survival; that variable was p53 (P <. 0001; relative risk, 5.60). A model with two independent terms predicted disease-related survival; these variables were p53 (P =. 0002; relative risk, 7.39) and MIB-1 (P =.03; relative risk, 3.27). Among patients with tumors with both p53 and MIB-1 expression exceeding 33%, a total of 32% had died of disease by 2 years. A model for predicting extrauterine disease selected two independent variables: p53 (odds ratio, 3.20; P =.01) and ploidy (odds ratio, 2. 16; P =.04). An advanced surgical stage was encountered in 26% to 35% of cases in which either the p53 expression exceeded 33% or the deoxyribonucleic acid content was nondiploid and in 53% of cases in which both variables were unfavorable. CONCLUSIONS: Preoperative evaluation of quantifiable cytokinetic and molecular variables can assist in identifying tumor types that are predisposed toward a more aggressive clinical course.     

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?     

妊娠肝内胆汁淤积症患者胎盘细胞凋亡及调控基因的研究

目的　通过观察妊娠肝内胆汁淤积症 (ICP)患者胎盘细胞凋亡调控基因p5 3、bax和bcl 2在胎盘中的表达 ,探讨细胞凋亡基因在胎盘细胞凋亡中的作用。方法　采用原位末端标记法(TUNEL)和免疫组织化学方法对 3 1例ICP患者 (ICP组 )的胎盘组织中p5 3、bax和bcl 2基因表达及调亡指数进行检测 ,并以 3 1例正常妊娠妇女的胎盘组织作对照 (对照组 )。结果　 (1)ICP组胎盘组织中细胞滋养细胞、合体滋养细胞、蜕膜细胞及间质细胞中细胞凋亡指数分别为 (49 1± 9 1) %、(46 6±9 8) %、(3 5 1± 9 5 ) %、(3 8 7± 9 7) % ,明显高于对照组的 (2 2 5± 6 2 ) %、(2 1 6± 5 2 ) %、(17 9±6 2 ) %、(17 0± 4 7) %。两组比较 ,差异有极显著性 (P <0 0 1)。 (2 )而调控基因p5 3的表达 ,ICP组明显高于对照组 ,两组比较 ,差异有极显著性 (P <0 0 1) ;bax的表达在两组滋养细胞中比较 ,差异无显著性 (P >0 0 5 )。但在合体滋养细胞、蜕膜细胞及间质细胞中 ,ICP组明显高于对照组 ,两组比较 ,差异有极显著性 (P <0 0 1) ;bcl 2的表达在ICP组胎盘组织细胞滋养细胞、合体滋养细胞、蜕膜细胞及间质细胞中 ,均明显低于对照组 (P <0 0 1)。 (3 )p5 3、bax和bcl 2在ICP组合体滋养细胞中的表达分别是 (75 9± 8 2 ) %、(65     

The role of extracellular inducer of matrix metalloproteinases in premature rupture of membranes

Objective: To investigate the role of matrix metalloproteinases (MMP-2, MMP-9) and their inducer (CD147) in premature rupture of membranes (PROM) at term labor.

Methods: In a cross-sectional study, 24 women aged 19–39, with 37–40-week pregnancy, and no clinical and histological signs of chorioamnionitis, were divided into two groups with and without PROM. The histological and immunohistochemical study of the fetal membranes was performed with polyclonal rabbit antibodies to MMP-2/MMP-9 and monoclonal rabbit antibodies to CD147.

Results: The analysis of MMP revealed the increase of MMP-9 expression in the amniotic epithelium during premature membrane rupture both in rupture area, and beyond it, and increased MMR-2 expression in the mesodermal cells. We also found high level of CD147 in the amniotic epithelium in PROM group. The above-mentioned changes were found in all areas of fetal membranes, regardless of the rupture localization.

Conclusions: The study results demonstrate the increased expression of MMR-2 and MMR-9, which regulate the catabolism of fetal membrane extracellular matrix proteins, in amniotic membranes of women with PROM at term labor. The increased expression of CD147 may be one of the mechanisms triggering PROM in the absence of infection.     

Reduction-oxidation (redox) state regulation of matrix metalloproteinase activity in human fetal membranes

OBJECTIVE: The mechanisms underlying membrane rupture at term and preterm are obscure. Collagenolytic activity of matrix metalloproteinases in amniochorionic membranes increases during spontaneous term and preterm labor associated with intra-amniotic infection. We sought to test the hypothesis that reduction-oxidation homeostasis, which is altered in inflammatory states, directly regulates amniochorionic matrix metalloproteinases. STUDY DESIGN: Membranes were collected from 7 patients undergoing elective cesarean delivery at term, rinsed thoroughly, and immediately incubated in phosphate-buffered sodium chloride solution at 37 degrees C for 24 hours. Matrix metalloproteinase activity in the culture medium was assayed by substrate-gel electrophoresis and normalized against the dry weight of the tissue incubated. Superoxide anions were generated in the presence of membranes by a xanthine (2 mmol/L) and xanthine oxidase (20 mU/mL) mixture and monitored by reduction of ferri-cytochrome c to ferro-cytochrome c. Incubations were performed in the presence of xanthine alone, a xanthine-xanthine oxidase mixture, superoxide dismutase (500 U/mL), a xanthine-xanthine oxidase-superoxide dismutase mixture, nitro-L-arginine (a nitric oxide synthase inhibitor, 1 mmol/L), xanthine-xanthine oxidase-nitro-L-arginine, S-nitroso-N -acetylpenicillamine (a nitric oxide donor, 10 mmol/L), xanthine-xanthine oxidase-S-nitroso-N -acetylpenicillamine, N -acetylcysteine (a thiol-containing antioxidant, 0.1, 1, or 10 mmol/L), lipopolysaccharide (100 ng/mL), or lipopolysaccharide-N -acetylcysteine. Intracellular generation of superoxide anions was monitored by the reduction of nitroblue tetrazolium to formazan. RESULTS: Basal matrix metalloproteinase 9 and matrix metalloproteinase 2 levels were detected in all samples. Superoxide anions significantly increased matrix metalloproteinase 9 activity but did not increase matrix metalloproteinase 2 activity, which effect was reversed by the addition of superoxide dismutase. N-acetylcysteine reduced basal activity of both matrix metalloproteinase 9 and matrix metalloproteinase 2 to 20%. Importantly, N-acetylcysteine completely inhibited intracellular formazan formation in cultured membranes both in the absence and in the presence of lipopolysaccharide. Neither nitric oxide synthase inhibition nor the nitric oxide donor S-nitroso-N -acetylpenicillamine had any effect on fetal membrane matrix metalloproteinase activity. CONCLUSION: Matrix metalloproteinase activity in human fetal membranes is reduction-oxidation (redox)-regulated. Matrix metalloproteinase 9 activity in human fetal membranes is directly increased by superoxide anion, a byproduct of macrophages and neutrophils. Neither nitric oxide donors nor nitric oxide synthase inhibitors significantly affect matrix metalloproteinase activity in human fetal membranes. The glutathione precursor N-acetylcysteine dramatically inhibits amniochorionic matrix metalloproteinase activity in addition to inhibiting intrinsic superoxide generation within the tissue. Thus thiol-reducing agents, such as N-acetylcysteine, may be beneficial in preventing preterm premature rupture of the 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.     

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.     

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.     

Screening of novel matrix metalloproteinases (MMPs) in human fetal membranes

Objective : Endogenous activation of matrix metalloproteinase (MMP) in human fetal membranes is hypothesized to contribute to membrane weakening leading to early rupture and is also involved in the initiation of labor. Our laboratory and several others have studied the source and action of some of these MMPs. The objective of this study is to document the expression pattern of most of the MMPs cloned and sequenced so far in amniochorion during preterm premature rupture of membranes (pPROM), at term not in labor and during term labor. Materials and Methods : Placentas were collected from women with PROM, term not in labor after C-sections and from women after term vaginal delivery. Membranes were separated from the placenta and a section away from the rupture site was selected. Amniochorion were separated from the placenta. RT-PCR was performed to study the expression pattern of MMP15 (MT2-MMP), MMP16 (MT3-MMP), MMP17 (MT4-MMP), MMP18, MMP20, MMP23, MMP24 (MT5-MMP), MMP25 (MT6-MMP), and MMP 26 using specific primers. Results : A differential pattern of expression was noted for some of the novel MMPs screened in this study in human fetal membranes. mRNA for most of the MMPs were expressed by amniochorion. MMP16 [membrane type metalloproteinase 3], MMP20 [enamelysin], and MMP26 [matrilysin] were not expressed. Conclusion : Amniochorion expresses several of the MMP genes at the time of pPROM, term not in labor and during active labor. We have previously reported the expression pattern of other MMPs and their inhibitors and their potential role in PROM. These findings support our hypothesis that amniochorion has a fully functional MMP system.