Stephen J. Fortunato

Amniotic fluid infection, inflammation, and colonization in preterm labor with intact membranes

OBJECTIVE The purpose of this study was to compare intraamniotic inflammation vs microbial invasion of the amniotic cavity (MIAC) as predictors of adverse outcome in preterm labor with intact membranes. STUDY DESIGN Interleukin-6 (IL-6) was measured in prospectively collected amniotic fluid from 305 women with preterm labor. MIAC was defined by amniotic fluid culture and/or detection of microbial 16S ribosomal DNA. Cases were categorized into 5 groups: infection (MIAC; IL-6, ≥11.3 ng/mL); severe inflammation (no MIAC; IL-6, ≥11.3 ng/mL); mild inflammation (no MIAC; IL-6, 2.6-11.2 ng/mL); colonization (MIAC; IL-6, <2.6 ng/mL); negative (no MIAC; IL-6, <2.6 ng/mL). RESULTS The infection (n = 27) and severe inflammation (n = 36) groups had similar latency (median, <1 day and 2 days, respectively) and similar rates of composite perinatal morbidity and mortality (81% and 72%, respectively). The colonization (n = 4) and negative (n = 195) groups had similar outcomes (median latency, 23.5 and 25 days; composite morbidity and mortality rates, 21% and 25%, respectively). The mild inflammation (n = 47) groups had outcomes that were intermediate to the severe inflammation and negative groups (median latency, 7 days; composite morbidity and mortality rates, 53%). In logistic regression adjusting for gestational age at enrollment, IL-6 ≥11.3 and 2.6-11.2 ng/mL, but not MIAC, were associated significantly with composite morbidity and mortality rates (odds ratio [OR], 4.9; 95% confidence interval [CI], 2.2-11.2, OR, 3.1; 95% CI, 1.5-6.4, and OR, 1.8; 95% CI, 0.6-5.5, respectively). CONCLUSION We confirmed previous reports that intraamniotic inflammation is associated with adverse perinatal outcomes whether or not intraamniotic microbes are detected. Colonization without inflammation appears relatively benign. Intraamniotic inflammation is not simply present or absent but also has degrees of severity that correlate with adverse outcomes. We propose the designation amniotic inflammatory response syndrome to denote the adverse outcomes that are associated with intraamniotic inflammation.

Inflammatory cytokine (interleukins 1, 6, and 8 and tumor necrosis factor-α) release from cultured human fetal membranes in response to endotoxic lipopolysaccharide mirrors amniotic fluid concentrations ☆ ☆☆ ★

OBJECTIVE This study was conducted to quantitate and compare the amount of cytokines released from human fetal membranes in response to treatment with bacterial lipopolysaccharide and to compare this with amniotic fluid levels. STUDY DESIGN Amniochorionic membranes were collected from women undergoing elective repeat cesarean section and showing no signs of infection- or pregnancy-related complications. Membranes were maintained in an organ explant system and stimulated with bacterial lipopolysaccharide for 24 hours. Media samples were collected and stored at -20 degrees C until cytokine levels were assayed by enzyme-linked immunosorbent assay. RESULTS Enzyme-linked immunosorbent assay results demonstrated that lipopolysaccharide stimulated production of interleukins 1, 6 and 8 and tumor necrosis factor-alpha by the fetal membranes in comparison with the control cultures. A greater release of interleukin-6 and interleukin-8 compared with interleukin-1 and tumor necrosis factor-alpha was noticed. The relationships between cytokine concentrations observed in culture mirror those seen in amniotic fluid. CONCLUSION Amniochorionic membranes can respond to an infectious process with increased secretion of interleukins 1, 6 and 8 and tumor necrosis factor-alpha. Cytokines produced from both amnion and chorion (interleukin-6 and interleukin-8) are released in greater quantities than those cytokines produced from chorion or amnion alone (interleukin-1 and tumor necrosis factor-alpha). These studies support a major role for amnion in infection-induced preterm labor.

The Role of Matrix Degrading Enzymes and Apoptosis in Repture of Membranes

Prematurity is the third leading cause of perinatal death, and pretem premature rupture of the membranes (pPROM) is associated with approximately 20-50% of all preterm births. The etiologic factors described for pPROM and pretem labor (PTL) are the same, although the clinical presentation (pPROM vs PTL) differs among patients. The reason for this disparity is unknown and poses a therapeutic dilemma. Several etiologic factors have been described for PTL and pPROM. PTL and pPROM are associated with overwhelming host inflammatory response. Many of these pro-inflammatory factors (inflammatory cytokine release) are common in both conditions; however, the clinical presentation differs. The objective of this review is to explain the differential expression pattern of matrix metalloproteinases (MMPs) and pro-apoptotic elements in human fetal membranes in pPROM and PTL and how they interact to present different clinical outcomes during pregnancy.

Expression of inflammatory cytokines (interleukin-1β and interleukin-6) in amniochorionic membranes

OBJECTIVE This study was designed to investigate the expression of inflammatory cytokines (interleukin-1 beta and interleukin-6) by fetal membranes in response to infection in vivo and to endotoxin in organ culture. STUDY DESIGN Amniochorionic membranes were collected from infected and uninfected women and analyzed for cytokine messenger ribonucleic acid and protein. Normal membranes were cultured and exposed to endotoxin. Messenger ribonucleic acid expression was analyzed by reverse transcriptase-polymerase chain reaction. Cellular localization of messenger ribonucleic acid and protein was determined by in situ hybridization and immunocytochemical evaluation, respectively. RESULTS Messenger ribonucleic acid for interleukin-1 beta appeared to be increased in infected or endotoxin-stimulated amniochorionic membranes, whereas interleukin-6 messenger ribonucleic acid was only observed in infected membranes or after endotoxin stimulation. Interleukin-1 beta messenger ribonucleic acid was localized exclusively to chorionic cells, whereas protein was observed in both chorion and amnion. Interleukin-6 messenger ribonucleic acid and protein were produced in both amniotic and chorionic cells. CONCLUSION Amniochorionic membranes are a site of inflammatory cytokine production. These findings may have significance in preterm labor or premature rupture of membranes.

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

Abstract 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 5 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.

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

Abstract 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.

Matrix metalloproteinases-9 in preterm and term human parturition.

OBJECTIVE Spontaneous rupture of the fetal membranes occurs after the commencement of labor in 90% of cases. Recent evidence indicates that the process of parturition requires not only an increase in myometrial contractility and cervical ripening, but also degradation of extracellular matrix in fetal membranes (i.e., leakage of fibronectin into cervico-vaginal secretions). This study was undertaken to determine if parturition is associated with in vivo evidence of increased bioavailability of matrix metalloproteinases-9 (MMP-9) and its inhibitor, tissue inhibitor of metalloproteinases-1(TIMP-1). METHODS A cross-sectional study was conducted with women in the following categories: 1) midtrimester (n = 25); 2) preterm labor and intact membranes in the absence of intraamniotic infection (n = 78); 3) term not in labor (n = 25); and 4) term with intact membranes in labor (n = 25). MMP-9, and TIMP-1 were measured using sensitive and specific immunoassays. RESULTS 1) Spontaneous labor at term was associated with a significant increase in MMP-9 but not in TIMP-1.2) Women with preterm labor who delivered prematurely had significantly higher concentrations of MMP-9 but not TIMP-1 in amniotic fluid than those with preterm labor who delivered at term. 3) The concentrations of TIMP-1 decreased with advancing gestational age. In contrast, MMP-9 concentrations did not change with advancing gestational age. CONCLUSIONS Spontaneous human parturition is associated with specific changes in the enzymatic machinery responsible for extracellular matrix degradation.

Short Fetal Leukocyte Telomere Length and Preterm Prelabor Rupture of the Membranes

Background Rupture of the fetal membranes is a common harbinger of imminent labor and delivery. Telomere shortening is a surrogate for oxidative stress (OS) and senescence. Fetal leukocyte and placental membrane DNA telomere lengths were evaluated to determine their association with preterm prelabor rupture of the membranes (pPROM) or spontaneous preterm births with intact membranes (PTB), compared to term birth. Methods Telomere lengths were quantified in cord blood leukocytes (n = 133) from three major groups: 1) pPROM (n = 28), 2) PTB (n = 69) and 3) uncomplicated full term births (controls, n = 35), using real-time quantitative PCR. Placental membrane specimens (n = 18) were used to correlate fetal leukocyte and placental telomere lengths. Telomere length differences among the groups were analyzed by ANOVA. Pearson correlation coefficients determined relationships between leukocyte and placental membrane telomere lengths. Results In pregnancies with intact membranes, fetal leukocyte telomere length was inversely proportional to gestational age. The mean telomere length decreased as gestation progressed, with the shortest at term. pPROM had telomere lengths (9962±3124 bp) that were significantly shorter than gestational age-matched PTB (11546±4348 bp, p = 0.04), but comparable to term births (9011±2497 bp, p = 0.31). Secondary analyses revealed no effects of race (African American vs. Caucasian) or intraamniotic infection on telomere length. A strong Pearsons correlation was noted between fetal leukocyte and placental membrane telomere lengths (ρ = 0.77; p<0.01). Conclusions Fetal leukocyte telomere length is reduced in pPROM compared to PTB but is similar to term births. pPROM represents a placental membrane disease likely mediated by OS-induced senescence.

Diversity in cytokine response to bacteria associated with preterm birth by fetal membranes

OBJECTIVE This study compared cytokine and prostaglandin (PG) responses by fetal membranes stimulated with 4 different bacterial species associated with preterm birth (PTB). STUDY DESIGN Fetal membranes (n = 13 from normal term cesarean sections [not in labor]) in an organ explant system were stimulated with heat-killed Ureaplasma parvum, Gardanerella vaginalis, Escherichia coli, group B Streptococcus (GBS), or lipopolysaccharide (LPS). Cytokines (interleukin [IL]-1beta, IL-6, IL-8, IL-10, tumor necrosis factor [TNF]-alpha, and interferon-gamma) and PG (PGF(2alpha) and PGE(2)) concentrations were quantitated and compared. RESULTS LPS and E coli increased all cytokine and PG productions compared with controls. Cytokine profiles were similar after G vaginalis and GBS stimulation. G vaginalis increased PGE(2), whereas GBS increased PGF(2alpha). U parvum demonstrated the mildest response with only IL-10 and TNF-alpha concentrations being higher with no detectible effect on PGs. CONCLUSION Fetal membrane cytokine signatures of 4 different bacteria associated with PTB are distinct, suggesting that infection as a potential cause of PTB is not homogeneous in its presentation.

Interleukin-10 inhibition of interleukin-6 in human amniochorionic membrane : Transcriptional regulation

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.