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BACKGROUND: Preterm birth (PTB) is hypothesized to be an inflammatory response disease. However, no single factor alone is likely to explain PTB risk. It is more probable that coordinated networks of cytokines affect risk. METHODS: Therefore, we examined the relationships between amniotic fluid (AF) cytokines/chemokines and related biomarkers in PTB and normal term deliveries in African Americans and Caucasians. Data were obtained from African American (41 preterm labor and 91 term labor) and Caucasian (105 preterm labor and 100 term labor) pregnant mothers. Pro-inflammatory cytokines and related molecules interleukin (IL)-1, IL-6, IL-8, and tumor necrosis factor- (TNF)-α, TNF soluble receptors (sTNFR1 and sTNFR2), and anti-inflammatory cytokine IL-10 that were all previously associated with PTB were studied. Correlations between biomarkers were calculated; differences of correlation coefficients between AF from African American and Caucasian samples in preterm labor and term labor were measured. RESULTS: Multiple differences were observed between African American and Caucasian preterm and term birth groups. In term birth the strongest differences were between pro- and anti-inflammatory correlations, whereas in PTB differences were equally distributed between pro-inflammatory/anti-inflammatory and pro-inflammatory/pro-inflammatory correlations. Three correlation patterns differed significantly between AF from PTB African Americans with and without microbial invasion of the intra-amniotic cavity (MIAC); no differences were observed in Caucasians with MIAC. CONCLUSION: Correlation analyses of cytokine measurements suggest coordinated interplay during pregnancy; significant differences exist between African Americans and Caucasians. Such analyses can serve as a means of understanding risk factors in these populations.

Problem The prematurity rate is higher in African-Americans (AA) compared with Caucasians (C). As spontaneous preterm labor has been hypothesized to be a host inflammatory response disease racial differences in human placental membrane inflammatory cytokine and prostaglandin pathway gene expression patterns between AA and C were examined in this report. Method of study Placental membranes (amniochorion) collected from AA and C women from cesareans at term were maintained in an organ explant system and stimulated with endotoxin (lipopolysaccharide, LPS). Microarray analysis and enzyme-linked immunosorbent assay was performed on mRNAs and culture media from AA- and C-derived membranes to document any differences in mRNA expression and protein production of IL-1, IL-6, IL-8, IL-10 and expression of cyclooxygenase 1 (COX-1), COX-2 and 15-hydroxyprostaglandin dehydrogenase (PGDH). Results Increased mRNA expression of IL-1, IL-8 and COX-2 in AA and IL-6, IL-10, COX-1 and PGDH in C were documented after LPS stimulation. Concentration of IL-1 was significantly higher in media derived from AA whereas IL-6 and IL-10 concentrations were higher in C with no differences observed in IL-8 after LPS stimulation compared with respective unstimulated controls. Conclusion These data document ethnic diversity in placental membrane immune response.

Objective. The objectives of this study were to document the expression of IL-19 and IL-20, localize their expression in human fetal membranes and to examine their influence on the production of other inflammatory cytokines (IL-1, IL-6, IL-8, and TNF-alpha) from placental membranes.Methods. Human fetal membranes collected at term from normal pregnancies were stimulated with either recombinant human IL-19, IL-20, bacterial endotoxin (LPS) alone or the cytokine + LPS. The expression of IL-19 and IL-20 was studied by reverse transcriptase polymerase chain reaction (RT-PCR) and localized using immunohistochemistry. Concentrations of IL-1, IL-6, IL-8, and TNF-alpha were measured with multiplex sandwich immunoassay using microsphere technology.Results. RT-PCR documented IL-19 and IL-20 gene expression in fetal membranes. Immunohistochemistry localized both peptides to amnion and chorion layers. LPS stimulated the production of all four cytokines (IL-1, IL-6, IL-8, and TNF-alpha) from fetal membranes compared to unstimulated controls. No change in IL-1 and IL-8 concentration was seen after IL-19 or IL-20 stimulation, whereas IL-6 concentration was three- and two-fold higher after IL-19 and IL-20 treatment, respectively. TNF levels were unchanged after IL-19 and IL-20 treatment; however, TNF levels were significantly decreased in membranes treated with IL-19 or IL-20 + LPS compared to LPS alone.Conclusion. Fetal membranes are a source of IL-19 and IL-20. These cytokines act as an inhibitory agent to LPS-induced TNF production whereas they stimulate IL-6 production and have no effect on IL-1 and IL-8 production from human fetal membranes. The effect of IL-19 and IL-20 in pregnancy will be dependent on their concentrations and other environmental factors such as infection.

Objective This study compares the immune responsiveness of amniochorionic membranes (AC) derived from African American (AA) and white (C) women to an infectious stimulus ex vivo. Study design AC derived from AA and C women were placed in an organ explant culture for 48 hours and then stimulated with endotoxin. Enzyme-linked immunosorbent assay measured the concentration of matrix metalloproteinase 9 (MMP9), tumor necrosis factor-α (TNF-α), and soluble TNF receptors (sTNFR1and sTNFR2) in culture media from stimulated and unstimulated AC. Results The C group produced 8-fold more TNF-α after stimulation than did the AA group. Both soluble receptor (R1 and R2) production increased in the C group and decreased in the AA group after stimulation. Although the C group–derived membranes produced more MMP9 at rest, a 6-fold increase in MMP9 concentration was seen in the AA group–derived membranes after stimulation. No change in MMP9 concentration was seen after stimulation of the C group–derived membranes. Conclusion Although the C group produced more TNF, they also produce higher sTNFRs, which may serve a protective role. The increased MMP9 release by the AA group may be suggestive of the greater risk of premature rupture of membranes in the AA group.

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-α (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.

To examine the effect of interleukin-10 on production and regulation of gelatinases by amniochorion in an in vitro model of infection.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.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).Interleukin-10 eliminated lipopolysaccharide induction of matrix metalloproteinase 2 and 9 in amniochorion.

Objective: Lipopolysaccharide and tumor necrosis factor α 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 α 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. (Am J Obstet Gynecol 2001;184:1392-8.)

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.

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 × 106 and 366 transcripts of gelatinase A and B, respectively, compared with only 5.9 × 104 (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 × 105 transcripts of tissue inhibitor of metalloproteinase 2, whereas lipopolysaccharide stimulation produced 4.1 × 105 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.

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 immunohisto-chemistry 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.

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.

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-β (50/50, 50/100), transforming growth factor-β (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 × 10 6 molecules per microliter of interleukin-8 messenger ribonucleic acid compared with 6 × 10 3 molecules per microliter in controls. Transforming growth factor-β alone and lipopolysaccharide plus transforming growth factor-β stimulation produced 6 × 10 5 and 6 × 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 × 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-β–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-β 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. (Am J Obstet Gynecol 1998;179:794-9.)

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.

OBJECTIVE: This study was designed to detect the regulatory effect of the immunoinhibitory cytokines interleukin-10 and transforming growth factor-β on the amniochorion production of tumor necrosis factor-α. STUDY DESIGN: Amniochorionic membranes were collected from women undergoing elective repeat cesarean section with no history of infection. Membranes were placed in organ explant culture for 48 hours and then stimulated with lipopolysaccharide (50 ng/ml), lipopolysaccharide plus interleukin-10 (50/50, 50/100 ng/ml), interleukin-10 (50 and 100 ng/ml), lipopolysaccharide plus transforming growth factor-β (50/50 and 50/100 ng/ml), and transforming growth factor-β (50 and 100 ng/ml). At the end of a 24-hour stimulation tissue samples were frozen for ribonucleic acid analysis and media samples were frozen for enzyme-linked immunosorbent assay. Quantitation of the messenger ribonucleic acid was accomplished by quantitative competitive polymerase chain reaction, and tumor necrosis factor-α protein was assayed by use of enzyme-linked immunosorbent assay. RESULTS: Lipopolysaccharide stimulation of fetal membranes produced approximately 60,000 molecules of tumor necrosis factor-α messenger ribonucleic acid, whereas control tissue produced none. Lipopolysaccharide plus interleukin-10 stimulation resulted in a dose-dependent decrease in tumor necrosis factor-α messenger ribonucleic production (transcriptional regulation) to 6000 (50/50) and 600 (50/100) molecules. Enzyme-linked immunosorbent assay performed on media samples from these experiments demonstrated a dose-dependent reduction in tumor necrosis factor-α peptide release. Stimulation of membranes with lipopolysaccharide plus transforming growth factor-β had minimal effects on tumor necrosis factor-α messenger ribonucleic acid and protein production compared with lipopolysaccharide-treated samples. Membranes stimulated with interleukin-10 alone showed no effect on messenger ribonucleic acid or protein levels and remained similar to the levels seen in control tissues. In the absence of lipopolysaccharide, transforming growth factor-β treatment produced a dramatic decrease in tumor necrosis factor-α peptide levels without affecting messenger ribonucleic acid levels. CONCLUSION: In the presence of a stimulatory agent, interleukin-10 down-regulates tumor necrosis factor-α release from cultured human amniochorionic membranes. Transforming growth factor-β seems to have some stimulatory effect on transcription, and no effect on translation was seen with concurrent lipopolysaccharide stimulation. However, down-regulation of tumor necrosis factor-α peptide by transforming growth factor was seen in fetal membranes when not overridden by an inflammatory stimulant. This study suggests that interleukin-10 and transforming growth factor-β can regulate tumor necrosis factor-α release from amniochorion under different conditions and by a different mechanism. (Am J Obstet Gynecol 1997;177:803-9.)

The patient who presents with advanced cervical dilatation and herniating membranes without clear signs of preterm labor prior to the time of fetal viability represents an obstetric dilemma. Nonintervention can be expected to result in inevitable preterm delivery and 20% fetal survival. This study represents the use of a McDonald cerclage in patients with advanced dilatation and herniation of membranes. The study population comprised 14 singleton pregnancies between 17 and 25 weeks gestation at 4 cm dilatation or greater. The operative technique included Foley catheter membrane retraction and McDonald cerclage with 5 mm mersilene tape. Results: The cervix was successfully closed in 13/14 patients (93%). Perioperative complication rate was 21%. Ten of 13 patients in whom the cerclage was successfully placed were delivered of healthy neonates who are alive and well (fetal survival rate 77%). The average duration of pregnancy after successful cerclage placement was 9.3 weeks (range 2 days-17 weeks). Conclusi...

Williamsia serinedens has been isolated from soil but has not yet been implicated in human disease. We report the first case of perinatal sepsis caused by a dual-morphotype form of Williamsia serinedens in a 31-year-old pregnant woman hospitalized with preterm labor.

Racial disparity in spontaneous preterm birth (PTB) between African Americans and Caucasians in the US is unexplained, but is probably related to differences in amniotic fluid (AF) inflammatory cytokine profiles. Therefore, this study analyzed the association of 34 single nucleotide polymorphisms (SNPs) in TNF-alpha and its receptor genes (TNFR1 and TNFR2) with AF TNF-alpha and soluble TNF receptor (R1 and R2) concentrations in PTB. Samples consisted of African American and Caucasian cases (PTB), and controls (term birth) for which both cytokine, and maternal and fetal genotype data were available. Analyses were performed with genotype, case, and maker-status interaction in the model for log transformed cytokine concentrations. In Caucasians, two interactions between genotype and pregnancy outcome associated with cytokine concentrations, whereas 14 gene variants in African Americans showed interactions with pregnancy outcome, and 13 showed association with genetic markers. In conclusion, cytokine concentrations in African American preterm births can be partially explained by interactions between pregnancy outcome, SNPs and infection. This does not appear to be the case in Caucasians. These findings may be important in understanding disparity in rates of PTB between the two populations.

This study compares the immune responsiveness of amniochorionic membranes (AC) derived from African American (AA) and white (C) women to an infectious stimulus ex vivo.AC derived from AA and C women were placed in an organ explant culture for 48 hours and then stimulated with endotoxin. Enzyme-linked immunosorbent assay measured the concentration of matrix metalloproteinase 9 (MMP9), tumor necrosis factor-alpha (TNF-alpha), and soluble TNF receptors (sTNFR1 and sTNFR2) in culture media from stimulated and unstimulated AC.The C group produced 8-fold more TNF-alpha after stimulation than did the AA group. Both soluble receptor (R1 and R2) production increased in the C group and decreased in the AA group after stimulation. Although the C group-derived membranes produced more MMP9 at rest, a 6-fold increase in MMP9 concentration was seen in the AA group-derived membranes after stimulation. No change in MMP9 concentration was seen after stimulation of the C group-derived membranes.Although the C group produced more TNF, they also produce higher sTNFRs, which may serve a protective role. The increased MMP9 release by the AA group may be suggestive of the greater risk of premature rupture of membranes in the AA group.

Reverse end diastolic velocity represents the most extreme alteration of the umbilical artery waveform. It has been associated with a high incidence of catastrophic pregnancy outcomes with a stillbirth rate of 33%. We report a case of sudden fetal death within 48 h of a reassuring biophysical profile in a triplet gestation at 31 weeks in which reverse end diastolic velocity was noted in the fetus which died. The postmortem finding of in utero congestive heart failure in this case supports the hypothesis that the alarming rate of stillbirths found in fetuses with absent reverse end diastolic velocity are not necessarily due to asphyxia. This experience illustrates a potential problem with following the fetus who exhibits reverse diastolic flow by traditional markers of fetal well-being such as the biophysical profile.

Objective: To further delineate the differences between the preterm labor and premature rupture of the membrane pathways, we investigated the role of the inflammatory cytokines as activators of matrix metalloproteinases 2 and 9 in human fetal membranes. Study Design: Normal amniochorionic membrane that is maintained in an organ explant system was stimulated with interleukin-1β, tumor necrosis factor-α, or interleukin-6. The expression and activity of matrix metalloproteinases 2 and 9 in amniochorion was documented with reverse transcriptase-polymerase chain reaction and specific substrate activity assays. The matrix metalloproteinase inhibitor, tissue inhibitor of metalloproteinase-1, concentration was measured by enzyme-linked immunosorbent assay. Results: Interleukin-1β, tumor necrosis factor-α, and interleukin-6 induced the expression of matrix metalloproteinase-9 messenger RNA, whereas matrix metalloproteinase-2 expression was constitutive in control and cytokine-stimulated tissues. Matrix metalloproteinase-2 activity did not change after cytokine stimulation. Active matrix metalloproteinase-9 was significantly higher in tumor necrosis factor-stimulated tissues, which conversely were not changed after interleukin-1 or interleukin-6 stimulation. Tissue inhibitor of metalloproteinase-1 levels were decreased after interleukin-1 and tumor necrosis factor stimulation but changed after interleukin-6 stimulation. Conclusion: Only tumor necrosis factor-α increases matrix metalloproteinase-9 activity in amniochorion. (Am J Obstet Gynecol 2002;187:1159-62.)

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. (Am J Obstet Gynecol 2000;182:1468-76.)

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, TIMPI 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). TIMPI 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.

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 MTI-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 MTI-MMP, MMP-2, and TIMP-2 may constitute an endogenous pathway of membrane degradation.

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.

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. (Am J Obstet Gynecol 1997;177:731-41.)

OBJECTIVE: Our purpose was to study the regulatory effects of recombinant interleukin-10 on interleukin-6 messenger ribonucleic acid and protein production by human fetal membranes. STUDY DESIGN: Amniochorionic membranes were collected from women undergoing elective cesarean section. Membranes were maintained in an organ explant system and stimulated with media containing lipopolysaccharide (50 ng/ml) and various amounts of recombinant interleukin-10 (10, 50, 100 ng/ml). Experiments were conducted in a dose- and time-dependent manner. Transcription and translation of interleukin-6 were monitored with quantitative reverse transcriptase - polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: Interleukin-10 stimulation of amniochorionic membranes in culture produced a dose-dependent decrease in the production of interleukin-6 messenger ribonucleic acid and protein. Quantitative polymerase chain reaction was used to document a decrease in interleukin-6 messenger ribonucleic acid, which paralleled the decrease in peptide levels as detected with enzyme-linked immunosorbent assay. The interleukin-10 effect was present only when tissue was concurrently stimulated with lipopolysaccharide. Interleukin-10 inhibition could not be produced in the absence of lipopolysaccharide stimulation. CONCLUSION: Addition of interleukin-10 to culture media leads to transcriptional regulation of interleukin-6, which results in decreased production of both messenger ribonucleic acid and protein by human amniochorionic membranes. The decrease in interleukin-6 is a dose-dependent effect of interleukin-10. This finding may have important implications with respect to a possible role for interleukin-10 or an interleukin-10 stimulatory factor in the management of preterm labor associated with the presence of inflammatory cytokines. (Am J Obstet Gynecol 1996;175:1057-65.)

Objective This study examined the differences in the inflammatory cytokine interleukin (IL)-6 and the immunoinhibitory cytokine IL-10 in the amniotic fluid of black and white women in spontaneous preterm birth. Methods In this study, 321 amniotic fluids from cases (preterm birth 36 or fewer weeks' gestation) and controls (normal term delivery longer than 37 weeks' gestation) were collected (147 cases [49 blacks and 98 whites] and 174 controls [85 blacks and 89 whites]) at the time of active labor. IL-6 and IL-10 concentrations were measured by immunoassays. Using normal-term delivery as controls, logistic regression models were used to estimate odds ratios (OR) and 95% confidence intervals (CIs) for preterm birth. Results A significant difference in IL-6 concentration was observed in white cases (cases: 3773 pg/mL; controls: 1682 pg/mL; P = .0003), compared with controls, but not in blacks (cases: 2042 pg/mL; controls: 2366 pg/mL; P = .6). In a combined multivariable analysis, when the highest and the lowest quartiles of IL-6 were compared in whites, the ORs (95% CI) for preterm birth across quartiles were 1.74 (0.62-4.88), 1.09 (0.39-3.02), and 5.68 (2.15-15.0). No such association was found in blacks. IL-10 concentration was not different between cases and controls in either race. Conclusions Race-specific associations exist between IL-6 but not IL-10 concentration and preterm birth. Elevated IL-6 concentrations are associated with preterm birth in whites but not blacks.

Objective The objective of the study was to study the genetic risk factors of spontaneous preterm birth (PTB) in African Americans. Study Design Case-control analyses were performed using maternal and fetal deoxyribonucleic acid from 279 African American birth events (82 PTB and 197 term) and 1432 single-nucleotide polymorphisms from 130 candidate genes. Single-locus association and haplotype analyses were performed. Results The most significant associations were in the maternal interleukin (IL)-15 (rs10833, allele P = 2.91 × 10 −4 , genotype P = 2.00 × 10 −3 ) gene and the fetal IL-2 receptor B (IL-2RB) (rs84460, allele P = 1.37 × 10 −4 , genotype P = 6.29 × 10 −4 ) gene. The best models for these markers were additive (rs10833, odds ratio [OR], 0.30; 95% confidence interval [CI], 0.14-0.62; P = 1.0 × 10 −3 ; rs84460, OR, 2.32; 95% CI, 1.47-3.67; P −3 ). The largest number of significant associations was found in genes related to infection and inflammation. There were overall a larger number of significant associations in infants than in mothers. Conclusion These results support a strong role for genes involved in infection and inflammation in the pathogenesis of PTB, particularly IL-12 and IL-12RB, and indicate that in African Americans there may be complementarity of maternal and fetal genetic risks for PTB.

RACIAL DIFFERENCES IN THE FETAL MEMBRANE INFLAMMATORY RESPONSE: A POSSIBLE EXPLANATION FOR THE ETHNIC DISPARITY IN PREMATURITY STEPHEN FORTUNATO, SALVATORE LOMBARDI, RAMKUMAR MENON, The Perinatal Research Center, Maternal-Fetal Medicine, Nashville, Tennessee, Maternal-Fetal Medicine, Nashville, Tennessee, Perinatal Research Center, Nashville, Tennessee OBJECTIVE: The prematurity rate is higher in African-Americans (AA) compared to Caucasians (C) (18% vs. 9%). Spontaneous preterm delivery (PTD) has been shown to be a host inflammatory response disease. Racial differences in placental membrane inflammatory gene expression patterns (cytokines, MMPs and caspases) were examined in this report. STUDY DESIGN: Fetal membranes collected from AA and C women from cesareans at term were maintained in an organ explant system and stimulated with of endotoxin. mRNAs were subjected to microarray analysis with C derived samples labeled with Cy5 dye (wavelength 635 nm) and AA samples labeled with Cy3 dye (wavelength 538 nm). The ratio of median intensities for each wavelength was calculated and data was generated using GenePix 4.0 software. ELISA measured the culture media concentrations of many of the interested inflammatory agents. RESULTS: Microarray data is shown in Table. Concentrations of IL-1 and MMP9 were higher in media derived from AA whereas IL-6 and TNF concentrations were higher in C and no changes were seen IL-8 and TIMP1 concentrations between the two groups after stimulation. CONCLUSION: The list of upregulated genes that are involved in proinflammatory response is greater AA derived membranes compared to C. Inflammatory cytokines, MMPs and both initiator and effecter caspases are upregulated in AA but not in C. AA show a pro inflammatory shift with not much change in the inhibitor/antagonists expression. In contrast there is an up regulation of inhibitors in C. This indicates a pro-inflammatory response in AA and a more balanced response in C. This disparity in membrane response to infection may be a factor for the high rate of PTD in AA.