Cigarette smoke induces oxidative stress and apoptosis in normal term fetal membranes.

Cigarette smoking and bacterial infections are two major risk factors associated with preterm prelabor rupture of membranes (pPROM). We hypothesized that exposure of fetal membranes to cigarette smoke extracts might induce oxidative stress (OS) and fetal membrane apoptosis, culminating in an alternate pathway to that commonly activated by infection. To test this, we characterized the production of prostanoids and biomarkers of apoptosis in normal term human fetal membrane explant cultures. Fetal membrane explants collected at term (from cesarean deliveries, not in labor) were stimulated with cigarette smoke extract (CSE) for 24 h. Two classes of prostanoids, F2-Isoprostane (F2-IsoP), a marker of OS and PGF2α, a classical uterotonin, were measured by gas chromatography/mass spectrometry. Western blot analyses of tissue lysates were performed to quantify the anti-apoptotic protein Bcl2 and actin (as a control). Fetal membrane apoptosis was detected by immunohistochemistry for active caspase 3 and confirmed by TUNEL staining for nuclear fragmentation. CSE exposure resulted in significantly more F2-IsoP production from fetal membranes (242.8 ± 79.3 pg/ml/mg of total membrane protein) compared to unstimulated controls (131.5 ± 53.1 pg/ml/mg; p < 0.0001). By contrast, PGF2α was not different in CSE vs. controls (1083 ± 527 vs. 1136 ± 835 pg/ml/mg of protein; p = 0.80). CSE-exposed tissues demonstrated a dose-dependent decrease in Bcl2 expression and increases in active caspase 3 and nuclear fragmentation in both amnion and chorion cells compared to controls. In summary, fetal membranes exposed to CSE manifest evidence of OS and apoptosis. The differential pattern of prostanoid production observed in this study supports the hypothesis that an alternate non-inflammatory pathway mediated by OS and apoptosis in pPROM may promote proteolysis resulting in membrane weakening and rupture.

Chorioamnionitis--a complex pathophysiologic syndrome.

Chorioamnionitis, inflammation of the amniochorionic membrane (fetal membranes) is a very common disease but a complex syndrome associated with pregnancy. It presents a clinical impasse due to lack of knowledge of specific etiologies associated with this condition making confident clinical interventions difficult. Recent reports provide insight into genetic, epigenetic, behavioral, psychosocial, molecular and pathophysiological factors that are associated with chorioamnionitis. However, a coordinated approach in understanding causality and lack of early indicators (clinical and biomarkers) has hampered gaining knowledge about the disease status preventing proper intervention. Several reviews have provided in-depth analysis of the histologic and clinical evidence associated with chorioamnionitis. In this review, we provide a novel perspective on chorioamnionitis based on recent evidences from scientific literature on inflammation, apoptosis and genetics.

Increased bioavailability of TNF-alpha in African Americans during in vitro infection: predisposing evidence for immune imbalance.

OBJECTIVE: The objective of this study is to examine TNF-alpha and its soluble and membrane bound receptors in fetal membranes derived from blacks and whites in response to in vitro infectious stimulus, and the balance between TNF-alpha and the receptors. Fetal membranes collected from black and white women at term were maintained in an organ explant system and stimulated with lipopolysaccharide (LPS). TNF-alpha, soluble TNF receptors (sTNFR1 and sTNFR2) in culture media and membrane bound TNF receptors (TNFR1 and TNFR2) in tissue homogenates were measured. Molar ratio (TNF/sTNFR) was calculated between LPS stimulated and unstimulated (controls) cultures in both races. TNF-alpha was increased in both races after LPS stimulation and showed no difference between races (p=0.7). LPS decreased sTNFR1 in blacks, but increased in whites, showing a significant difference between races (p=0.001). In blacks sTNFR2 also decreased and increased in whites, but the results were not significant between races (p=0.4). Both TNFR1 and TNFR2 were increased in blacks after LPS stimulation whereas no such changes were seen in whites compared to controls that were also significant between races. After LPS stimulation TNF-alpha bioavailability was increased in blacks with a drop in soluble receptors and with an increase in membrane receptors. This was not evident in whites because in whites soluble receptors were increased with no change in membrane receptors. Our data demonstrated that LPS stimulation results in a molar ratio switch favoring TNF-alpha biofunction in blacks, but not in whites.

Ethnic differences in interleukin 6 (IL-6) and IL6 receptor genes in spontaneous preterm birth and effects on amniotic fluid protein levels.

Preterm birth (PTB) is a significant neonatal health problem that is more common in African-Americans (AA) than in European-Americans (EA). Part of this disparity is likely to result from the differing genetic architectures of EA and AA. To begin assessing the role of these differences, patterns of genetic variation in two previously proposed candidate genes, encoding interleukin 6 (IL6) and its receptor (IL6R), were analyzed in mothers and fetuses from 496 EA birth-events (149 cases and 347 controls) and 397 birth-events in AA (76 cases and 321 controls). IL-6 levels in amniotic fluid (AF) samples were determined in a subset of these pregnancies. Case-control comparisons revealed a single SNP in IL6R associated with PTB (p=0.04 for allelic and p=0.05 for genotype association). In addition, all of the SNPs studied showed significant frequency differences between AA and EA in at least one comparison, significantly in excess of that expected from general population databases. Higher IL-6 concentrations were associated with the IL6 SNP -661 in EA preterm samples (p=0.0056), and this result seems to be driven by microbial invasion of the amniotic cavity, indicating a gene by infection interaction. These findings indicate that, as a function of IL6 genotype, EA and AA women respond differently to infection with respect to their expression of IL-6. Our data support differential genetic control of levels of IL-6 in amniotic fluid between EA and AA.

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.

Interleukin-10 inhibition of gelatinases in fetal membranes: therapeutic implications in preterm premature rupture of membranes.

OBJECTIVE: To examine the effect of interleukin-10 on production and regulation of gelatinases by amniochorion in an in vitro model of infection. METHODS: We placed amniochorionic membranes collected from eight women who had elective repeat cesareans at term in an organ explant culture system. After 48 hours in culture, the membranes were stimulated with lipopolysaccharide (50 ng/mL), and some were costimulated with interleukin-10 (500 ng/mL). Tissue and media samples were collected after 24-hour stimulation. Quantitative polymerase chain reactions and enzyme-linked immunosorbent assays were used to evaluate matrix metalloproteinase 2 and matrix metalloproteinase 9 messenger RNA and proteins, respectively. RESULTS: Lipopolysaccharide stimulation induced 55.14 transcripts of matrix metalloproteinase 9, compared with 0.83 in control tissues (P <.001). Costimulation with interleukin-10 and lipopolysaccharide significantly reduced matrix metalloproteinase 9 messenger RNA levels to 10 transcripts (P <.001). Lipopolysaccharide stimulation produced 29.25 ng/mL of immunoreactive matrix metalloproteinase 9, which was reduced to 6.3 ng/mL (P(adj) =.016) after costimulation with interleukin-10. Although not significant, matrix metalloproteinase 2 messenger RNA levels were higher in lipopolysaccharide-stimulated tissues (4.37 x 10(6) transcripts) compared with control (2.8 x 10(5) transcripts; P(adj) =.08), with a significant decrease in matrix metalloproteinase 2 messenger RNA levels in interleukin-10- costimulated tissues (2.9 x 10(6); P(adj) =.007). Interleukin-10 costimulation resulted in a significant decrease in matrix metalloproteinase 2 protein production (203.1 [lipopolysaccharide] and 149.75 [with interleukin-10]; P(adj) <.001). CONCLUSION: Interleukin-10 eliminated lipopolysaccharide induction of matrix metalloproteinase 2 and 9 in amniochorion.

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.

Support for an infection-induced apoptotic pathway in human fetal membranes.

OBJECTIVE: Lipopolysaccharide and tumor necrosis factor alpha levels are both elevated in the amniotic fluid of women during infection-associated preterm labor and premature rupture of fetal membranes. Our laboratory has shown that apoptosis is associated with premature rupture of fetal membranes but is not associated with preterm labor. The exact pathway that leads to apoptosis-mediated premature rupture of fetal membranes is still unclear. Because infection and increased inflammatory cytokine response are associated with the majority of cases of premature rupture of fetal membranes, we examined the roles of bacterial lipopolysaccharide and tumor necrosis factor alpha in inducing the proapoptotic caspase pathway in fetal membranes. STUDY DESIGN: Amniochorionic membranes collected from women undergoing elective repeat cesarean delivery at term were placed in an organ explant system. At the end of a 48-hour incubation period, membranes were stimulated with lipopolysaccharide (50 ng/mL) and recombinant tumor necrosis factor (50 ng/mL). Total ribonucleic acid extracted from these samples was subjected to reverse transcription and two separate sets of multiple polymerase chain reaction. One set studied the expression of Fas, Fas ligand, caspase 8, Fas-associated death domain, and tumor necrosis factor receptor-associated death domain genes and the second set studied the expression of caspase 2, 4, 6, 7, and 10. Caspase 2, 3, and 9 expression was also studied by reverse transcriptase-polymerase chain reaction. RESULTS: Multiple polymerase chain reactions and reverse transcriptase-polymerase chain reactions documented the induction of Fas and caspase 2, 3, 7, 8, and 9 genes in amniochorion after lipopolysaccharide and tumor necrosis factor stimulation compared with the nonstimulated controls. Neither lipopolysaccharide nor tumor necrosis factor induced Fas ligand expression in human fetal membranes. Caspase 3, 4, and 6, Fas-associated death domain, and tumor necrosis factor receptor-associated death domain expressions were constitutive in all the tissues tested; however, tumor necrosis factor receptor-associated death domain expression appeared stronger in tumor necrosis factor-stimulated tissues. CONCLUSION: The presence of the signal docking proteins tumor necrosis factor receptor-associated death domain and Fas-associated death domain and the induction of caspase cascade initiators (caspase 2, 8, and 10) and effector caspases (caspase 3, 6, 7, and 9) by lipopolysaccharide and tumor necrosis factor suggest that tumor necrosis factor-tumor necrosis factor receptor-mediated apoptosis may occur in the human fetal membrane.

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.

Lipopolysaccharide stimulation of 70 kilo Dalton heat shock protein messenger ribonucleic acid production in cultured human fetal membranes.

OBJECTIVE: The 70 kilo Dalton heat shock protein is up-regulated when cells are under physiological stress. It prevents protein denaturation and incorrect polypeptide assembly, and inhibits apoptosis as well as the transcription of genes coding for pro-inflammatory cytokines. To evaluate if up-regulation of heat shock protein 70 can occur during pregnancy, we examined whether addition of bacterial lipopolysaccharide to human amniochorion membranes in vitro stimulated heat shock protein 70 gene transcription. MATERIALS AND METHODS: Amniochorionic membranes (n = 5), collected at the time of elective repeat cesarean section prior to labor from normal term gestations, were placed in an organ explant system. After 48 hour in culture, the membranes were stimulated with lipopolysaccharide for 24 hours. Total RNA was extracted and subjected to an oligo dT primed reverse transcriptase reaction followed by polymerase chain reaction (PCR) using heat shock protein 70 specific primers. PCR products were hybridized with biotinylated internal probes and identified by enzyme-linked immunosorbent assay (ELISA). Results were analyzed by Mann-Whitney U test. A p < 0.05 was significant. RESULTS: Heat shock protein 70 messenger RNA was expressed by all fetal membrane preparations both prior to and following in vitro culture. Addition of lipopolysaccharide increased the concentrations of heat shock protein 70 messenger RNA in each sample tested from a mean of 35.5 +/- 29.6 ng/milliliter (12.1-80.1 ng/milliliter) to 169.6 +/- 69.9 ng/ml (51.7-218.2 ng/milliliter) (p = 0.03). CONCLUSION: Human fetal membranes constitutively express heat shock protein 70 messenger ribonucleic acid. Bacterial lipopolysaccharide markedly stimulated heat shock protein 70 messenger RNA gene transcription in human fetal membranes. Thus, heat shock protein 70 is inducible in fetal membranes and may facilitate fetal survival under adverse conditions.

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.

Amniochorion gelatinase-gelatinase inhibitor imbalance in vitro: a possible infectious pathway to rupture.

OBJECTIVE: To estimate the effect of lipopolysaccharide on gelatinases and tissue inhibitors of matrix metalloproteinase 2 (gelatinase inhibitor) balance in human fetal membranes. METHODS: Amniochorionic membranes in organ explant were stimulated with 1000 ng/mL lipopolysaccharide for 24 hours after a 48-hour preincubation period. Quantitative competitive polymerase chain reaction (PCR) was done to quantitate messenger RNAs for gelatinase A and B (matrix metalloproteinase 2 and 9) and tissue inhibitor of metalloproteinase 2. Protein levels were assayed by enzyme-linked immunosorbant assay. The molar ratio between gelatinases and tissue inhibitor of metalloproteinase 2 was calculated. Statistical evaluation was done by Mann-Whitney U test. RESULTS: Lipopolysaccharide stimulation produced 3.6 x 10(6) and 366 transcripts of gelatinase A and B, respectively, compared with only 5.9 x 10(4) (P = .009) and three transcripts (P = .006), respectively, in the controls. Lipopolysaccharide stimulation released 210 ng/mL compared with 7 ng/mL of gelatinase A and B proteins compared with 120 (P = .01) and 4.6 ng/mL (P = .3) in controls, respectively. Control amniochorion produced 5.7 x 10(5) transcripts of tissue inhibitor of metalloproteinase 2, whereas lipopolysaccharide stimulation produced 4.1 x 10(5) transcripts (P = .69). Lipopolysaccharide reduced the release of this inhibitor from 114 ng/mL to 68 ng/mL (P = .007). The molar ratio between gelatinases and tissue inhibitor of metalloproteinase 2 increased from a balanced ratio of 1:1 to 3.1:1 after 1000 ng/mL of lipopolysaccharide. CONCLUSION: Lipopolysaccharide increased the expression and release of gelatinases and decreased its inhibitor, which shifted the balance in favor of gelatinase activity leading to membrane degradation that predisposes to premature rupture of membranes.

Stromelysins in placental membranes and amniotic fluid with premature rupture of membranes.

OBJECTIVE: To determine the expression and site of production of stromelysins in fetal membranes and to measure stromelysin 1 levels in amniotic fluid and amniochorion culture media. METHODS: Amniochorionic membranes were cultured from organ explant. Membranes were stimulated with lipopolysaccharide for 24 hours after a 48-hour preincubation period. Membranes were also collected from women after vaginal deliveries. RNA samples from those tissues were subjected to reverse transcriptase-polymerase chain reaction using primers specific for stromelysin 1, stromelysin 2, stromelysin 3, and matrilysin. In situ hybridization and immunohistochemistry were used to localize stromelysin mRNA and peptide. Levels of stromelysin 1 in culture media and amniotic fluid collected from women with preterm premature rupture of membranes (PROM) and at term with intact membranes were compared using enzyme-linked immunosorbant assay. RESULTS: Amniochorion in culture and from laboring and nonlaboring women expressed all three stromelysins. In situ hybridization showed stromelysin mRNA in amnion, chorion, and extracellular matrix. Immunohistochemical analysis localized stromelysin 1 protein to those same regions. Amniotic fluid levels of stromelysin 1 were higher in preterm PROM amniotic fluids (median 3.2 ng/mL) compared with term deliveries with intact membranes (median 1.3 ng/mL) (P = .02). Lipopolysaccharide stimulation in culture increased the release of stromelysin 1 from fetal membranes compared with control (median 70.35 versus 15.8 ng/mL, respectively, P = .05). CONCLUSION: Human fetal membranes are a source of stromelysins 1, 2, and 3. Increased stromelysin 1 during preterm PROM and in vitro after lipopolysaccharide stimulation suggests a possible effect of that matrix metalloproteinase in PROM.

MMP/TIMP imbalance in amniotic fluid during PROM: an indirect support for endogenous pathway to membrane rupture.

OBJECTIVE: We theorize that excessive degradation of the fetal membrane extracellular matrix (ECM) by specific matrix metalloproteinases (MMPs) results in preterm premature rupture of the membranes (PROM). Active, inhibitor free MMP2 and 9 (gelatinase A and B respectively) can degrade the amniochorion basement membrane Type IV collagen to initiate rupture. This study examines the levels of the gelatinases and their natural inhibitors (tissue inhibitor of matrix metalloproteinases-TIMPs) in the amniotic fluid during PROM, preterm labor (PTL) and at term. METHODS: A total of 51 AF samples were collected from the following groups of patients. Group 1: Women with PTL and no ROM (n = 16) Group 2: Women with PROM (n = 16) irrespective of labor status Group 3: Women at term with intact membranes undergoing cesarean delivery irrespective of labor status (n = 19). ELISA was used to assay MMP2, MMP9, TIMP1 and TIMP2 levels in the amniotic fluid. The active, TIMP free levels of MMP2 were quantitated by zymography followed by computerized densitometry. Active MMP9 was measured using a bioassay that specifically detects MMP9 activity. Statistical analysis was performed by Tukey-Kramer multiple comparison method. RESULTS: PROM is associated with increased MMP2 levels (mean 2125 ng/ml;) when compared with term (mean 1455 ng/ml; p < 0.01) or PTL where a non significant increase was seen (mean 1862 ng/ml; p = ns). MMP9 levels were higher in PROM (mean 15.03 ng/ml) than at term (mean 1.14 ng/ml; p < 0.001) or PTL (mean 3.75 ng/ml; p < 0.01). TIMP1 levels were slightly increased during PROM (mean 3143 ng/ml) compared to term (mean 1892 ng/ml; p < 0.05) pr PTL where a non significant change was seen (mean 2406 ng/ml; p = ns). TIMP2 levels were decreased in PROM (mean 98 ng/ml) compared with term (mean 176 ng/ml; p < 0.05) and PTL (mean 236 ng/ml; p < 0.001). Active, TIMP free MMP2 levels were increased during PROM (mean 233 pg/ml) compared to those at term (mean 132 pg/ml; p < 0.05) or PTL (mean 132 pg/ml; p < 0.05). Active forms of MMP9 were seen only during PROM (mean 632 pg/ml). CONCLUSION: Active, TIMP free forms of MMP2 and 9 are increased in the amniotic fluid of women with PROM. These MMPs can degrade the amniochorion basement membranes and other ECM components resulting in PROM.

The effect of transforming growth factor and interleukin-10 on interleukin-8 release by human amniochorion may regulate histologic chorioamnionitis.

OBJECTIVE: Amniochorion is a source of interleukin-8 during infection and inflammation. In this study we investigate the role of 2 immunoinhibitory cytokines, transforming growth factor and interleukin-10, in regulating interleukin-8 production from human fetal membranes and define their mechanism of regulation. STUDY DESIGN: Amniochorion was placed in an organ explant system for 72 hours. Tissues were stimulated with lipopolysaccharide (50 ng/mL), lipopolysaccharide plus transforming growth factor-beta (50/50, 50/100), transforming growth factor-beta (50 and 100 ng/mL), lipopolysaccharide plus interleukin-10 (50/50 and 50/100), and interleukin-10 (50 and 100 ng/mL) in culture. Tissue and media samples were frozen until quantitation of interleukin-8 messenger ribonucleic acid and protein. Quantitation of messenger ribonucleic acid was performed by quantitative competitive polymerase chain reaction and protein by enzyme-linked immunoassay, respectively. RESULTS: Lipopolysaccharide-stimulated tissues produced approximately 6 x 10(6) molecules per microliter of interleukin-8 messenger ribonucleic acid compared with 6 x 10(3) molecules per microliter in controls. Transforming growth factor-beta alone and lipopolysaccharide plus transforming growth factor-beta stimulation produced 6 x 10(5) and 6 x 10(4) molecules of interleukin-8 messenger ribonucleic acid per microliter, respectively. Tissues stimulated with lipopolysaccharide plus 50 ng/mL interleukin-10 produced approximately 600 molecules per microliter of interleukin-8 messenger ribonucleic acid, whereas no amplifiable messenger ribonucleic acid was detected in tissues treated with lipopolysaccharide plus 100 ng/mL interleukin-10. Tissues treated with interleukin-10 alone produced 6 x 10(3) molecules of messenger ribonucleic acid, similar to control levels. Enzyme-linked immunosorbent assay data showed similar levels of interleukin-8 peptide release from lipopolysaccharide and lipopolysaccharide plus transforming growth factor-beta-treated fetal membranes. A dose-dependent decrease in interleukin-8 peptide release was seen in tissues treated with lipopolysaccharide plus interleukin-10, whereas stimulation with transforming growth factor or interleukin-10 alone resulted in interleukin-8 peptide release similar to that of control levels. CONCLUSION: Transforming growth factor-beta seems to have no effect on interleukin-8 protein production in the presence of an infectious agent; however, a drop in messenger ribonucleic acid levels was observed. Interleukin-10 in the presence of lipopolysaccharide showed down-regulation of interleukin-8 messenger ribonucleic acid expression and peptide production. These data suggest that fetal membrane interleukin-8 production can be controlled by interleukin-10 during an infectious process.

Expression of a progelatinase activator (MT1-MMP) in human fetal membranes.

PROBLEM: The finding of MMP-2 (which degrades type IV collagen) and TIMP-2 (the tissue inhibitor of MMP) in fetal membranes suggests the possibility of membrane self-destruction as an etiology of premature rupture of fetal membranes. MMP-2 is activated by a membrane-bound MMP (MT1-MMP). This study was undertaken to detect the presence of MT1-MMP in human fetal membranes. METHOD OF STUDY: Fetal membranes were placed in an organ explant system and stimulated with lipopolysaccaride (LPS). MT1-MMP expression was studied in frozen tissues by reverse transcriptase (RT)-polymerase chain reaction (PCR) using primers designed in our laboratory. DNA sequence analysis was performed to verify the specificity of PCR products. In situ hybridization and immunocytochemistry were used to localize MT1-MMP mRNA and peptide, respectively. RESULTS: RT-PCR data indicated the presence of mRNA for MT1-MMP in fetal membranes. Although PCR is not quantitative, no differences in mRNA band intensities were noticed after LPS stimulation. MT1-MMP expression was constitutive throughout the culture period. In situ hybridization demonstrated amnion, chorionic laeve, cytotrophoblast cells, and the cells in the reticular and spongy layer of the extracellular matrix as the origin of MT1-MMP mRNA and peptide. CONCLUSIONS: This is the first study documenting the amniochorionic membrane as a source of MT1-MMP mRNA and peptide. Activation of progelatinase A requires the presence of this membrane-associated MMP. The finding of MT1-MMP in a tissue already known to produce MMP-2 and TIMP-2 documents the full system for activation and inhibition of this gelatinase. During infection, an imbalance in the expression of MT1-MMP, MMP-2 and TIMP-2 may constitute an endogenous pathway of membrane degradation.

IL-15, a novel cytokine produced by human fetal membranes, is elevated in preterm labor.

PROBLEM: Interleukin (IL)-15 is a novel cytokine known to have functions similar to those of IL-2 in the cell-mediated immune response. The objectives of this study were to determine whether IL-15 levels change in labor or preterm labor and to identify the regulatory agents and the site of production of IL-15. METHOD OF STUDY: Amniochorionic membranes were cultured in an organ explant system and were stimulated with lipopolysaccharides (LPSs). Samples were subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) using specific primers for IL-15 and IL-2. The localization of mRNA and protein was accomplished by in situ hybridization and immunocytochemistry. IL-15 was measured in culture media and amniotic fluid from term and preterm gestations by enzyme-linked immunoadsorbent assay (ELISA). RESULTS: RT-PCR indicated the expression of IL-15 mRNA in the amniochorion. In situ hybridization and immunocytochemistry documented that mRNA and peptide for IL-15 are found in amnion, chorion, and decidual cells. ELISA results indicated no significant increase of IL-15 peptides in the culture media after LPS stimulation. Maximum levels of this cytokine were seen in the amniotic fluid (AF) of women with preterm labor compared to term labor. AF levels were not higher in preterm-labor patients with proved infection compared with those without infection. RT-PCR-based detection also showed the presence of two isoforms of IL-15 mRNA known to code for two different leader peptide sequences. IL-2 mRNA expression was not observed in the fetal membranes. CONCLUSIONS: The presence of IL-15 mRNA and peptide in the amniochorion and decidua and its increased presence in the AF during preterm labor suggests a possible role for IL-15 in preterm labor. Amniochorion is also shown to possess two IL-15 isoform leader sequences, the differential expression of which may be involved in the regulation of IL-15 secretion.

Presence of four tissue inhibitors of matrix metalloproteinases (TIMP-1, -2, -3 and -4) in human fetal membranes.

PROBLEM: Matrix metalloproteinases play a critical role in fetal membrane extracellular matrix (ECM) homeostasis. Remodeling of the ECM during normal placental development is a balanced activity between various matrix metalloproteinases and their tissue-specific counter-regulatory proteins (tissue inhibitors of matrix metalloproteinases [TIMPs]). We have reported the presence of TIMP-1 and TIMP-2 in placental membranes in culture. In this study we have investigated the membrane expression of TIMP-1 and TIMP-2 during labor and nonlabor conditions and also the presence of two novel TIMP family members (TIMP-3 and TIMP-4). METHOD OF STUDY: Amniochorionic membranes collected from women undergoing Cesarean section and were cultured in an organ explant system. Membranes were also collected from laboring women after vaginal delivery. Samples were subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) using primers specific for TIMP-1, TIMP-2, TIMP-3, and TIMP-4. Localization of TIMP mRNAs was accomplished by in situ hybridization, and peptides were localized by immunocytochemistry. RESULTS: RT-PCR data demonstrated the expression of all the TIMPs in tissues from laboring and nonlaboring women as well as in cultured membranes. TIMP-4 expression was seen in RT-PCR, however, only a faint band was visible in all the tissues tested. In situ hybridization localized the TIMP mRNAs to the amnion, chorion, and to scattered cells in the connective tissue. CONCLUSION: Human fetal membrane cells (amniochorion and decidua) express mRNA for all the TIMPs studied so far.

Immunoreactivity of human fetal membranes to peptidoglycan polysaccharide (PGPS): cytokine response.

OBJECTIVE: Group-B Streptococcus has been associated with preterm labor and other pregnancy related complications. This study was performed to evaluate the effect of peptidoglycan polysaccharide (PGPS) derived from a beta hemolytic Streptococcal cell wall on amniochorion cytokine production and to compare PGPS effects with lipopolysaccharide (LPS), which is the Gram negative counterpart of PGPS. STUDY DESIGN: Amniochorionic membranes collected from women not in labor, and undergoing elective repeat C-section were placed in an organ explant system. Membranes were stimulated separately with 50 ng/ml of small (100p), large (10s) fractions of PGPS or LPS respectively immediately after collection and after a stabilization period of 48 hrs. Media samples were collected at 3, 6, 9, 12 and 24 hrs for protein analysis after each stimulation. Media samples were analyzed by ELISA for IL-6 and IL-8. RESULTS: Both forms of PGPS and LPS stimulated IL-6 and IL-8 production by human fetal membranes. Of note is that LPS stimulated IL-6 to a greater degree than IL-8, while PGPS stimulated IL-8 to a greater degree than IL-6. No statistical difference was seen in the levels of either one of these cytokines for the larger or smaller fragments of PGPS. Time course studies documented a 3-hour lag phase when tissues are stimulated directly after collection which was absent when tissues are stimulated after a 48-hour stabilization period. CONCLUSION: Both PGPS and LPS stimulate cytokine production differently from fetal membranes. This supports the theory that different bacteria may affect the host in contrasting ways which may lead to a distinct host response, i.e. PROM vs. PTL.

Collagenolytic enzymes (gelatinases) and their inhibitors in human amniochorionic membrane.

OBJECTIVE: This study was designed to investigate the presence of matrix metalloproteinase-2 (gelatinase A), matrix metalloproteinase-9 (gelatinase B), and their natural inhibitors in both cultured amniochorionic membrane and membrane obtained from women with infection-associated preterm labor. STUDY DESIGN: Amniochorionic membranes were collected from women with documented intraamniotic infection and from women not in labor undergoing elective repeat cesarean section with no signs of infection or other complications of pregnancy. Normal membranes were cultured and exposed to endotoxin and peptidoglycan polysaccharide. Messenger ribonucleic acid expression for gelatinase A, gelatinase B, and tissue inhibitors of matrix metalloproteinase types 1 and 2 was studied with use of reverse transcriptase-polymerase chain reaction and localization of messenger ribonucleic acid was accomplished with use of in situ hybridization. Release of gelatinases from the membranes was studied with gelatin zymography. Tissue inhibitors of matrix metalloproteinase peptides were localized with use of immunocytochemistry. RESULTS: The expression of matrix metalloproteinase types 2 and 9 was seen in amniochorionic membranes in culture. Matrix metalloproteinase-2 was seen in membranes from nonlaboring women and in women with intraamniotic infection, whereas matrix metalloproteinase-9 was seen only in membranes from women with intraamniotic infection. The matrix metalloproteinase-9 expression could also be induced by lipopolysaccharide or peptidoglycan polysaccharide stimulation in culture. In situ hybridization localized messenger ribonucleic acid for these matrix metalloproteinases to both amnion and chorion. Zymogram studies showed the activity of matrix metalloproteinase-2 in normal resting membrane and cultured membrane. Matrix metalloproteinase-9 was induced by culture conditions. Tissue inhibitor of matrix metalloproteinase-1 and tissue inhibitor of matrix metalloproteinase-2 messenger ribonucleic acid was seen in normal, infected, and cultured membranes. In situ hybridization data indicated that these messages were mainly produced by chorion, but they were also seen in amnion. Immunohistochemistry demonstrated the presence of tissue inhibitor of matrix metalloproteinase-1 and tissue inhibitor of matrix metalloproteinase-2 peptides in both amnion and chorion and in cells of the reticular layer of the matrix. CONCLUSION: Normal amniochorionic membrane is a source of matrix metalloproteinase-2 and tissue inhibitors of matrix metalloproteinases. Culture conditions and infection induce matrix metalloproteinase-9 expression and release from amniochorion. These findings suggest that these collagenolytic enzymes may play a role in premature rupture of the membranes in infection, which can lead to preterm labor.