Expression of heparin-binding EGF-like growth factor in term chorionic villous explants and its role in trophoblast survival.

Heparin-binding EGF-like growth factor (HBEGF) induces trophoblast extravillous differentiation and prevents apoptosis. These functions are compromised in preeclampsia. Because HBEGF is downregulated in placentas delivered by women with preeclampsia, we have examined its expression and cytoprotective activity in term villous explants. Chorionic villous explants prepared from non-pathological placentas collected by cesarean section at term were cultured at either 20% or 2% O2 and treated with the HBEGF antagonist CRM197 or recombinant HBEGF. Paraffin sections were assayed for trophoblast death, proliferation and HBEGF expression using the TUNEL method, immunohistochemistry for nuclear Ki67 expression and semi-quantitative immunohistochemistry with image analysis, respectively. Trophoblast cell death was increased significantly after 8h of culture with CRM197 or by culture for 2h at 2% O2. Exogenous HBEGF prevented cell death due to hypoxia. Proliferative capacity was not affected by culture at either 20% or 2% O2. Contrary to first trimester placenta, term trophoblasts do not elevate HBEGF expression in response to hypoxia. However, low endogenous levels of HBEGF are required to maintain survival. Therefore, HBEGF-mediated signaling significantly reduces trophoblast cell death at term and its deficiency in preeclampsia could negatively impact trophoblast survival.

Protein kinase C stimulates release of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 by human decidual cells.

OBJECTIVE: Increased concentrations of amniotic fluid matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1 have been observed in the context of premature rupture of membranes (PROM) and microbial invasion of the amniotic cavity. However, the source of the stimuli that contribute to the accumulation of these proteins in amniotic fluid remains to be identified. The present study was conducted to investigate MMP-2, MMP-9 and TIMP-1 secretion by decidual cells in response to activated protein kinase C (PKC). METHODS: Decidual cells were isolated from term placentae, grown to confluence and incubated with control media or 10(-11) to 10(-8) mol/l concentrations of phorbol 12-myristate 13-acetate (PMA). Concentrations of MMP-2, MMP-9 and TIMP-1 in the culture supernatant were determined using sensitive and specific immunoassays. Substrate zymography was conducted to confirm MMP-9 assays. RESULTS: PMA induced a concentration-dependent stimulation of release of MMP-9 (control vs. PMA l0(-9) and 10(-8) mol/l; p < 0.01) and TIMP-1 (control vs. PMA 10(-9) and 10(-8) mol/l; p < 0.001), but not MMP-2. A direct positive correlation was observed between MMP-9 and TIMP-1 release (r = 0.645; p < 0.001). Substrate zymography confirmed increased release of MMP-9 in response to PMA (control vs. PMA 10(-8) and PMA 10(-7) mol/l; p < 0.01). CONCLUSIONS: Activation of PKC within the decidua will result in enhanced MMP-9 release, which upon activation could contribute to degradation of matrices within fetal membranes leading to PROM.

Participation of the novel cytokine interleukin 18 in the host response to intra-amniotic infection.

OBJECTIVE: Interleukin 18 is a proinflammatory pleiotropic cytokine that has been implicated in the host defense against infection. This study was undertaken to determine whether interleukin 18 concentrations change in the maternal, fetal, and amniotic fluid compartments with labor (term and preterm) and microbial invasion of the amniotic cavity. STUDY DESIGN: Amniotic fluid was assayed for interleukin 18 in samples obtained from 285 patients in the following groups: (1) term not in labor (n = 22), in labor (n = 19), and with microbial invasion of the amniotic cavity (n = 16); (2) preterm labor who delivered at term (n = 38), who delivered preterm but without microbial invasion of the amniotic cavity (n = 41), and preterm labor with microbial invasion of the amniotic cavity (n = 24); (3) preterm premature rupture of membranes without microbial invasion of the amniotic cavity (n = 30) and with microbial invasion of the amniotic cavity (n = 34); (4) term premature rupture of membranes not in labor (n = 20) and term premature rupture of membranes in labor (n = 19); and (5) midtrimester (n = 22). In addition, cord and maternal plasma samples from women at term not in labor (n = 20) and in labor (n = 20) were assayed for interleukin 18. RESULTS: (1) Interleukin 18 was detectable in all amniotic fluid samples and maternal and umbilical cord blood samples. (2) Interleukin 18 concentrations increased with advancing gestational age (r = 0.47; P <.0001). (3) Microbial invasion of the amniotic cavity in either preterm or term parturition was associated with a significant increase in the amniotic fluid concentration of interleukin 18 (preterm labor without microbial invasion of the amniotic cavity: median, 14.95 pg/mL; range, 3.9-277.0 pg/mL; vs preterm labor with microbial invasion of the amniotic cavity: median, 20.75 pg/mL; range, 5.53-160.21 pg/mL; P <.02; term labor without microbial invasion of the amniotic cavity: median, 18.73 pg/mL; range, 5.09-95.44 pg/mL; vs term labor with microbial invasion of the amniotic cavity: median, 24.35 pg/mL; range, 10.07-144.42 pg/mL; P<.004). (4) Both term and preterm parturition were associated with a modest increase in amniotic fluid interleukin 18 concentrations, although this trend did not reach statistical significance. (5) Rupture of membranes at term was associated with a significant decrease in amniotic fluid interleukin 18 concentrations (intact membranes: median, 14.96 pg/mL; range, <3.89-26.07 pg/mL; vs rupture of membranes: median, 10.1 pg/mL; range, 4.29-21.44 pg/mL; P <.001). CONCLUSION: (1) Interleukin 18 is increased in cases of microbial invasion of the amniotic cavity. (2) Interleukin 18 is detectable in the amniotic, maternal, and fetal compartments. (3) We propose that this novel cytokine plays a role in the host defense against infection.

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

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

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

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

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

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

Interleukin 16 in pregnancy, parturition, rupture of fetal membranes, and microbial invasion of the amniotic cavity.

OBJECTIVE: Interleukin 16 is a proinflammatory cytokine that promotes the recruitment of nonclonotypic T cells and eosinophils to sites of inflammation and induces resistance to activation-induced apoptosis. This peptide has no homology with members of the chemokine family and is produced by epithelial cells. No information is available about the expression of this cytokine during human pregnancy. This study was conducted to determine whether interleukin 16 is present in amniotic fluid and to examine the effects of labor and intrauterine infection on the concentrations of this cytokine. STUDY DESIGN: A cross-sectional study was constructed with 230 women in the following groups: (1) mid- trimester (n = 25); (2) term not in labor (n = 25), term in labor (n = 25), and term premature rupture of membranes not in labor (n = 40); (3) preterm labor and intact membranes with (n = 21) and without (n = 42) intra-ammiotic infection; and (4) preterm premature rupture of membranes in the presence (n = 29) and absence (n = 23) of microbial invasion of the amniotic cavity. Interleukin 16 concentration was measured with sensitive and specific immunoassays validated for amniotic fluid. Data were analyzed with nonparametric statistics. RESULTS: (1) Interleukin 16 was detected in 87.8% (202/230) of amniotic fluid samples. (2) Amniotic fluid interleukin 16 concentrations were significantly higher in women in the midtrimester than in those at term not in labor (median, 321.5 pg/mL; range, 146.9-1185.8 pg/mL; vs median, 85.9 pg/mL; range, <25-409.8 pg/mL; P <.001). (3) Labor at term was not associated with a significant increase in the median amniotic fluid interleukin 16 concentration. (4) Patients with preterm labor who delivered preterm had a significantly higher median amniotic fluid interleukin 16 concentration than those with preterm labor who delivered at term (median, 328.1 pg/mL; range, 38. 9-4660 pg/mL; vs median, 119.8 pg/mL; range, <25-558.5 pg/mL;P <.05). (5) Microbial invasion of the amniotic cavity was associated with a significant increase in median amniotic fluid interleukin 16 concentration in patients with preterm labor and intact membranes (microbial invasion of the amniotic cavity: median, 839 pg/mL; range, <25-8620 pg/mL; vs no microbial invasion of the amniotic cavity: median, 119.8 pg/mL; range, <25-558.5 pg/mL; P <.001) and in patients with preterm premature rupture of the membranes (microbial invasion of the amniotic cavity: median, 1005.8 pg/mL; range, <25-4590 pg/mL; vs no microbial invasion of the amniotic cavity: median, 204.9 pg/mL; range, 42.6-2347 pg/mL; P <.05). (6) Spontaneous rupture of the membranes at term but not preterm was associated with a significant decrease in amniotic fluid concentrations of interleukin 16 (term premature rupture of the membranes: median, <25 pg/mL; range, <25-231.2 pg/mL; vs term intact membranes: median, 85.9 pg/mL; range, <25-409.8 pg/mL; P <.05). CONCLUSIONS: Amniotic fluid interleukin-16 concentrations decreased with advancing gestational age. Women with preterm labor that led to preterm delivery and women with microbial invasion of the amniotic cavity had higher interleukin 16 amniotic fluid concentrations than those with preterm labor who delivered at term or those with sterile amniotic fluid. Microbial invasion of the amniotic cavity but not spontaneous labor at term or rupture of membranes was associated with increased concentrations of interleukin 16 in amniotic fluid.

The tumor necrosis factor alpha and its soluble receptor profile in term and preterm parturition.

OBJECTIVE: The common terminal pathway of parturition describes the anatomic, biochemical, endocrine, and clinical events present in the fetus and mother in both term and preterm labor. Labor at term is thought to result from physiologic activation of this pathway, whereas preterm labor is the result of pathologic activating events. The purpose of this study was to determine whether physiologic and pathologic activation could be discerned by the analysis of a cytokine-receptor signaling system. Tumor necrosis factor alpha and its soluble receptors were used as probes because of their pivotal role in the regulation of several processes activated during parturition. Soluble receptors are thought to buffer the biologic and potentially deleterious effects of tumor necrosis factor alpha in pathologic conditions. STUDY DESIGN: The in vivo concentrations of tumor necrosis factor alpha and its soluble receptors were studied in patients in term labor and preterm labor. Amniotic fluid was retrieved from 175 women and tumor necrosis factor alpha, tumor necrosis factor receptor 1, and tumor necrosis factor receptor 2 concentrations were measured by highly sensitive immunoassays. Patients were classified in the following groups: (1) term labor (n = 29), (2) term not in labor (n = 29), (3) preterm labor leading to term delivery (n = 34), (4) preterm labor without infection resulting in preterm delivery (n = 34), (5) preterm labor with intra-amniotic infection (n = 23), and (6) second trimester (n = 26). RESULTS: Tumor necrosis factor alpha and tumor necrosis factor receptor 1 concentrations decreased with advanced gestational age (r = -0.51 and r = -0.7; P <.01 for each). (1) Patients in spontaneous term labor had a higher median concentration of tumor necrosis factor alpha than those at term not in labor (median, 6.4 pg/mL; range, 2.4->500 pg/mL vs median, 4.1 pg/mL; range, 1.1-22.7 pg/mL; P <.01) but had lower concentrations of tumor necrosis factor receptor 1 (median, 3.2 ng/mL; range, 1.3-9.1 ng/mL vs median, 4.2 ng/mL; range, 1.6-8.3; P <.001) and tumor necrosis factor receptor 2 (median, 5.5 ng/mL; range, 0.73-12.8 ng/mL vs median, 6.8 ng/mL; range, 2.9-12.9 ng/mL; P <.01). (2) In contrast, patients with preterm labor leading to preterm delivery had higher concentrations of tumor necrosis factor alpha (median, 12.3 pg /mL; range, 1.5->500 pg/mL vs median, 4.8 pg/mL; range, 1-60.9 pg/mL; P <.01), tumor necrosis factor receptor 1 (median, 8.8 ng/mL; range, 2.5-38 ng/mL vs median, 6.2 ng/mL; range, 1.4-28 ng/mL; P <.05), and tumor necrosis factor receptor 2 (median, 8.5 ng/mL; range, 3.5-45.4 ng/mL vs median, 6.1 ng/mL; range, 1.99-14.1 ng/mL; P <.01) than patients with preterm labor who delivered at term. (3) Microbial invasion of the amniotic cavity was associated with dramatic increases in the concentrations of tumor necrosis factor alpha (median, 93.5 pg/mL; range, 1.2->500 pg/mL) and its soluble receptors tumor necrosis factor receptor 1 (median, 8.8 ng/mL; range, 2.1-36.7 ng/mL) and tumor necrosis factor receptor 2 (median, 11.8 ng/mL; range, 3.4-46. 3 ng/mL), concentrations that were significantly higher than in those with preterm labor who delivered at term and those who delivered preterm but were not infected. CONCLUSION: The tumor necrosis factor alpha and tumor necrosis factor alpha soluble receptor profiles are different in term and preterm parturition. Our observations provide support for the thesis that preterm parturition is a pathologic condition. Increased tumor necrosis factor alpha soluble receptor concentrations may attenuate the deleterious effects of the excess of tumor necrosis factor alpha found in pathologic labor.

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.

A role for matrix metalloproteinase-9 in spontaneous rupture of the fetal membranes.

OBJECTIVES: Preterm premature rupture of fetal membranes is responsible for 30% to 40% of preterm deliveries. Fetal membranes are composed primarily of collagen. Matrix metalloproteinases are enzymes capable of degrading extracellular matrix macromolecules, including collagens. Expression of matrix metalloproteinase-9 (gelatinase B, 92 kd) and its tissue inhibitor (tissue inhibitor of metalloproteinase-1) has been localized in amnion and chorion. The objective of this study was to determine whether rupture of fetal membranes and intrauterine infection are associated with changes in the expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1. STUDY DESIGN: Two hundred one women in the following categories had amniotic fluid retrieved: (1) preterm labor and intact membranes in the presence (n = 42) or absence (n = 21) of microbial invasion of the amniotic cavity, (2) preterm premature rupture of the membranes with (n = 29) or without (n = 23) microbial invasion of the amniotic cavity, and (3) term gestation with intact membranes (n = 50) or with premature rupture of the membranes (n = 40). Women in groups 1 and 2 were matched for gestational age at amniocentesis. Microbial invasion of the amniotic cavity was defined by a positive amniotic fluid culture for micro-organisms. Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 were measured with use of sensitive and specific immunoassays that were validated for amniotic fluid. RESULTS: Spontaneous rupture of membranes at term is associated with a significant increase in the amniotic fluid concentrations of matrix metalloproteinase-9 (premature rupture of membranes, no labor: median 3.9 ng/mL, range 2. 7 to 11.1 ng/mL vs no premature rupture of membranes, no labor: median <0.4 ng/mL, range <0.4 to 22.4 ng/mL; P <.001). Patients with preterm premature rupture of the membranes had higher median matrix metalloproteinase-9 concentrations than those with preterm labor and intact membranes who were delivered at term (7.6 ng/mL, range <0.4 to 230.81 ng/mL vs <0.4 ng/mL, range <0.4 to 1650 ng/mL; P =.06). Women with microbial invasion of the amniotic cavity had higher median matrix metalloproteinase-9 concentrations than did those without microbial invasion regardless of membrane status (preterm labor: 54.5 ng/mL, range <0.4 to 3910 ng/mL vs <0.4 ng/mL, range <0. 4 to 1650 ng/mL; P <.01; preterm premature rupture of membranes: 179. 8 ng/mL, range <0.4 to 611 ng/mL vs 7.6 ng/mL, range <0.4 to 230.81; P <.001). CONCLUSION: Our data support a role for matrix metalloproteinase-9 in the mechanisms responsible for membrane rupture in term and preterm gestations.

Saralasin-induced inhibition of ovulation in the in vitro perfused rat ovary is not replicated by the angiotensin II type-2 receptor antagonist PD123319.

OBJECTIVE: Our aim was to explain the effect of the nonspecific angiotensin II antagonist saralasin and the specific angiotensin II type-2 receptor antagonist PD123319 on ovulation. STUDY DESIGN: Saralasin, 1 micromol/L (n = 5), and PD123319 10 micromol/L (n = 6), were administered to in vitro perfused rat ovary. Prostaglandin (prostaglandin E2, prostaglandin F2alpha, 6-keto-prostaglandin F1alpha), hydroxy-eicosatetraenoic acid (12-hydroxy-eicosatetraenoic acid, 15-hydroxy-eicosatetraenoic acid), estradiol, and progesterone levels in the perfusate and the ovulation rate were compared (Mann-Whitney U test) with controls. RESULTS: Saralasin significantly (P < .01) inhibited the ovulation rate (3.0 +/- 1.4) versus control (13.1 +/- 1.0) and reduced prostaglandin E2 (at 3 hours P < .01 and 20 hours P < .05) and 6-keto-prostaglandin F1alpha (at 20 hours P < .05) levels. Saralasin did not alter prostaglandin F2alpha, hydroxy-eicosatetraenoic acids, or steroid levels. PD123319 decreased 15-hydroxy-eicosatetraenoic acid levels at 3 hours (P < .05) but had no effects on other eicosanoids, steroid levels, or the ovulation rate. CONCLUSION: Angiotensin II plays an important role in ovulation in the rat and is associated with ovarian prostaglandin synthesis. This effect is not selectively regulated via the angiotensin II type-2 receptor.

A fetal systemic inflammatory response is followed by the spontaneous onset of preterm parturition.

OBJECTIVE: There is no evidence for the participation of the human fetus in the mechanisms responsible for the onset of preterm labor. We propose that preterm labor in the setting of infection results from the actions of proinflammatory cytokines secreted as part of the fetal and/or maternal host response to microbial invasion. The objective of this study was to determine whether a systemic fetal inflammatory response, defined as an elevation of fetal plasma interleukin-6 concentrations, has a temporal relationship with the commencement of labor. STUDY DESIGN: After informed consent was obtained, amniocentesis and cordocentesis were performed in 41 patients with preterm premature rupture of membranes who were not in labor on admission. Amniotic fluid was cultured for both aerobic and anaerobic bacteria, as well as for mycoplasmas. Fetal plasma interleukin-6 was assayed by a sensitive and specific immunoassay. Statistical analyses included contingency tables and survival analysis with time-dependent Cox regression hazard modeling. RESULTS: Microbial invasion of the amniotic cavity was present in 58.5% (24/41) of patients. Fetuses with fetal plasma interleukin-6 concentrations > 11 pg/mL had a higher rate of spontaneous preterm delivery within 48 and 72 hours of the procedure than those with fetal plasma interleukin-6 levels < or = 11 pg/mL (88% vs 29% and 88% vs 35%, respectively; P < .05 for all comparisons). Moreover, patients with initiation of labor and delivery within 48 hours of the procedure had a higher proportion of fetuses with plasma interleukin-6 values > 11 pg/mL than patients delivered > 48 hours (58% [7/12] vs 8% [1/13], respectively; P < .05). Survival analysis indicated that fetuses with elevated fetal plasma interleukin-6 levels had a shorter cordocentesis-to-delivery interval than those without elevated fetal plasma interleukin-6 concentrations (median 0.8 days [range 0.1 to 5] vs median 6 days [range 0.2 to 33.6], respectively; P < .05). Time-dependent Cox regression hazard modeling indicated that fetal plasma interleukin-6 level was the only covariate significantly associated with the duration of pregnancy after we adjusted for gestational age, amniotic fluid interleukin-6 level, and the microbiologic state of the amniotic cavity (P < .01). CONCLUSION: A systemic fetal proinflammatory cytokine response is followed by the onset of spontaneous preterm parturition in patients with preterm premature rupture of membranes.

Elevated interleukin-8 concentrations in amniotic fluid of mothers whose neonates subsequently develop bronchopulmonary dysplasia.

OBJECTIVE: To determine if an intrauterine sub-clinical inflammatory process is a risk factor for the development of bronchopulmonary dysplasia. METHODS: A cohort study was conducted in patients who met the following criteria: (1) Singleton gestation; (2) preterm labor or preterm premature rupture of the membranes; (3) amniocentesis for microbiologic studies of the amniotic fluid and (4) delivery between 24 and 28 weeks of gestation. Bronchopulmonary dysplasia was defined as the need for supplemental oxygen for 28 days or longer after birth, associated with compatible chest radiographic findings. Amniotic fluid interleukin-8, was measured using a specific immunoassay. Logistic regression analysis and bootstrap procedure were used for statistical purposes. RESULTS: Forty-seven patients met the inclusion criteria for this study. Among these patients, the prevalence of bronchopulmonary dysplasia was 23.4% (11/47). Amniotic fluid culture was positive in 21 out of 47 (44.7%) patients. Median (range) amniotic fluid interleukin-8 concentration was higher in patients whose neonates subsequently developed bronchopulmonary dysplasia than in those who did not (17 [9.8-583.7] ng ml(-1) versus 9.6 [0.91-744] ng ml(-1), P=0.057). An amniotic fluid IL-8 level greater than 11.5 ng ml(-1) was far more common in mothers whose fetuses went on to develop bronchopulmonary dysplasia than in those who did not (10/11 [90.9%] versus 17/36 [47%]; P=0.01). This relationship remained significant even after correcting for the effect of gestational age and birthweight (Odds ratio: 11.9; P<0.05). CONCLUSION: Sub-clinical intrauterine inflammation is a risk factor for the subsequent development of bronchopulmonary dysplasia. We propose that in utero aspiration of fluid with high concentration of pro-inflammatory mediators may contribute to the lung injury responsible for the development of bronchopulmonary dysplasia.

The lipoxygenase inhibitor, nordihydroguaiaretic acid, inhibits ovulation and reduces leukotriene and prostaglandin levels in the rat ovary.

Eicosanoids, the active metabolites of arachidonic acid, are grouped into cyclooxygenase products (prostaglandins [PGs] and thromboxanes) and lipoxygenase products (leukotrienes [LTs] and lipoxins). Numerous studies suggest a role for the lipoxygenase system in ovulation. The aim of this study was to further characterize the effects of lipoxygenase inhibition and the interactions of the lipoxygenase and cyclooxygenase systems in the rat ovary during ovulation. The lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), was administered in vivo and in the isolated perfused rat ovary to determine its effect on ovulation rate. The in vivo study confirmed the inhibitory effect of NDGA, and in the perfusion experiments, NDGA caused a dose-dependent reduction in the ovulation rate. To further define the interaction between the lipoxygenase and cyclooxygenase systems, a second set of perfusions was performed with NDGA (10 microM) and the combination of NDGA (10 microM) plus a nonselective cyclooxygenase inhibitor, indomethacin (10 microM). NDGA significantly reduced the number of ovulations compared to that in controls. The ovulation rate for the combination of NDGA+indomethacin was also significantly lower than in controls but not different from that in the NDGA-treated group. Steroidogenesis was decreased only in the NDGA+indomethacin perfusions. Ovarian tissue PGE2 and PGF2alpha levels in the NDGA-treated ovaries were significantly suppressed compared to those in controls. Almost a complete block of PGE2 and PGF2alpha was seen in the NDGA+indomethacin group. LTB4 levels in the 10-h-perfused ovarian tissues were significantly decreased by NDGA compared to those in control tissues. Furthermore, LTB4 (3 microg added twice) completely reversed the inhibitory effect of 0.1 microM NDGA on ovulation rate and partially reversed the effect of 10 microM NDGA in the perfusion model. These results demonstrate that the products of the lipoxygenase pathway, especially LTB4, are important in the process of ovulation in this cyclically ovulating species. The interconnected lipoxygenase and cyclooxygenase pathways may optimize ovulation and facilitate steroidogenesis.

Ceramide stimulates prostaglandin production by human amnion and decidual cells.

OBJECTIVE: To determine whether ceramide regulates prostaglandin (PG) production by cultured human amnion cells and decidual cells independently of interleukin-1 beta (IL-1 beta). METHODS: Cells were grown in monolayer culture and then incubated with varying concentrations of ceramide, IL-1 beta, ceramide in the presence and absence of IL-1, and control media. Production of PGE2 was determined using a specific radioimmunoassay. RESULTS: Ceramide induced a significant concentration-dependent increase in PGE2 production by amnion cells and decidual cells. However, PGE2 production induced by IL-1 beta was significantly more than with ceramide alone, and there was no potentiation of PGE2 production with coincubation of ceramide and IL-1 beta. CONCLUSION: We suggest that term human amnion cells and decidual cells are responsive to ceramide independent of IL-1 beta and that generation of these substances in response to an infection in the uterus may lead to increased PG production by human gestational tissues, indicating that there are several mechanisms leading to PG production by these cells.

Regulation of decidual cell and chorion cell production of interleukin-10 by purified bacterial products.

PROBLEM: To determine whether cultured human decidual cells and chorion cells produce interleukin-10 (IL-10) after incubation with purified bacterial products. METHOD OF STUDY: Decidual cell cultures and chorion cell cultures were established by standard techniques. With confluence, monolayers of each culture were incubated with purified bacterial products, including various concentrations of lipopolysaccharide (LPS), lipid A, and lipoteichoic acid (LTA) for 16 hr in quadruplicate. Culture supernatants were collected and assayed for immunodetectable IL-10 by enzyme-linked immunoadsorbent assay (ELISA). RESULTS: Both decidual cell cultures and chorion cell cultures produced significant quantities of IL-10 after stimulation with LPS, lipid A, and LTA. Cultures of decidual cells produced more IL-10 than did chorion cell cultures. CONCLUSIONS: Our data indicate that both maternal decidual cells and fetally derived chorion cells can produce IL-10 after incubation with bacterial virulence factors. This finding contrasts with our previous findings in which chorion cells did not produce IL-10 after stimulation with IL-1 beta, suggesting that chorion cell production after incubation with bacterial products is independent of IL-1 beta. We speculate that the contribution of anti-inflammatory IL-10 production by human gestational tissues to the inflammatory process in these tissues may be overcome or abrogated by the pro-inflammatory process.

Expression of interleukin-10 in human gestational tissues.

OBJECTIVE: To determine production of interleukin-10 (IL-10) by interleukin-1 beta (IL-1 beta) in cultured decidual, chorion, and amnion cells and whether IL-10 is produced in gestational tissues under the setting of infection-associated preterm labor. METHODS: Decidual, chorion, and amnion cells were isolated from term placentas and grown in primary culture. The cells were incubated with various concentrations of IL-1 beta and then culture supernatants were assayed for IL-10 by enzyme-linked immunosorbent assay. In subsequent studies, gestational membranes were isolated from a normal-term pregnancy and a preterm pregnancy complicated by chorioamnionitis. Tissues were evaluated for IL-10 expression by immunohistology and in situ hybridization. Human gestational tissues were collected from 38 women experiencing: 1) term cesarean delivery without labor; 2) normal-term vaginal delivery; 3) preterm cesarean delivery without labor; 4) preterm vaginal delivery without chorioamnionitis; and 5) preterm vaginal delivery with concomitant chorioamnionitis. Amnion, chorion, and decidua were isolated, total RNA from each tissue was extracted, and the presence of IL-10 mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Decidual cells in culture produced IL-10 in response to IL-1 beta, but chorion and amnion cells produced no IL-10 protein. In vivo protein expression by immunohistology showed that most protein was detected within decidua while cells within amnion and chorion rarely had detectable IL-10 protein. In vivo RT-PCR samples demonstrated the strongest IL-10 mRNA signal from decidua samples, although IL-10 mRNA was also noted in chorion and amnion of placentas obtained after preterm labor. CONCLUSION: Maternal decidual cells can potentially produce IL-10, but fetal membranes (amnion and chorion) appear to have limited capabilities to produce IL-10. The relative inability of fetal tissues to produce IL-10 may play an important role in the pathophysiology of infection-associated preterm labor.

Regulation of decidual cell chemokine production by group B streptococci and purified bacterial cell wall components.

OBJECTIVE: Our purpose was to determine whether cultured human decidual cells produce chemokines in response to different strains of group B streptococci and purified bacterial cell wall components. STUDY DESIGN: Human decidual cells were cultured from term placentas by standard techniques. Different strains of group B streptococci were isolated from neonates with early-onset group B streptococci sepsis. Confluent cell monolayers were incubated with these different strains of group B streptococci and various concentrations of purified bacterial cell wall components (including lipoteichoic acid, sialic acid, lipopolysaccharide, and lipid A) for 16 hours at 37 degrees C. Culture supernatants were collected and assayed for macrophage inflammatory protein-1 alpha and interleukin-8. Statistical analysis was by analysis of variance. RESULTS: We found that cultured human decidual cells produced significant amounts of the two chemokines macrophage inflammatory protein-1 alpha and interleukin-8 in a strain-specific fashion to the various different strains of group B streptococci tested, from 215% to 421% over baseline production (p < 0.05 by analysis of variance). Also, we found that incubation of decidual cells with various concentrations of lipoteichoic acid, sialic acid, lipopolysaccharide, and lipid A resulted in significant concentration-dependent increases in decidual cell macrophage inflammatory protein-1 alpha and interleukin-8 production (p < 0.05.) CONCLUSIONS: Decidual cells produced significant amounts of the chemokines macrophage inflammatory protein-1 alpha and interleukin-8 in response to intact group B streptococci in a strain-specific fashion and in response to various concentrations of different bacterial cell wall components. Because chemokines are important mediators signaling migration of different immune effector cells into areas of inflammation, we suggest that decidual cell chemokine production in response to bacteria and bacterial cell wall components may be a key early event in the pathogenesis of infection-associated preterm labor.

Variable effects on murine pregnancy of immunoglobulin G fractions from women with antiphospholipid antibodies.

OBJECTIVE: Initial studies showed that passive immunization with human immunoglobulin G fractions containing antiphospholipid antibodies can result in murine fetal loss. We intended to use the murine model to study mechanisms of fetal loss associated with antiphospholipid antibodies. However, we have since found variable effects of antiphospholipid antibodies on murine pregnancy. The objective of this study was to determine the consistency of murine pregnancy loss from antiphospholipid antibody containing immunoglobulin G fraction. STUDY DESIGN: Pregnant C3H/HeN (mated with C57B1/6 males) and BALB/c (mated with BALB/c males) mice were passively immunized with antiphospholipid antibody containing human immunoglobulin G fraction from 20 women with antiphospholipid syndrome. The mice received either a single dose of 10 to 30 mg on day 12 of pregnancy or 10 mg per day on days 12 to 14 of gestation. Some mice receiving each dose of immunoglobulin G fraction were bled to confirm serum levels of anticardiolipin antibodies. Mice were killed on day 15 and the fetal status was determined. RESULTS: Overall, passive immunization with individual antiphospholipid antibody containing immunoglobulin G fractions resulted in 801 live pups (75%), 232 fetal deaths (22%), and 38 resorptions (3%) in 131 mice. The effect of immunoglobulin G fractions from individual patients was highly variable. Immunoglobulin G fraction from eight women resulted in high rates of fetal loss. However, in spite of high levels of anticardiolipin antibodies, fetal outcome was normal in mice immunized with immunoglobulin G fraction from the majority of women. The rate of fetal death did not uniformly increase with increasing doses of immunoglobulin G fraction and was unrelated to the donor's medical history. Fetal outcome was similar for both C3H/HeN and BALB/c mice. CONCLUSIONS: Human antiphospholipid antibodies have variable effects on murine pregnancy outcome. Characterization of antiphospholipid antibodies that do and do not cause murine fetal loss may provide insight into epitopes relevant to fetal loss associated with antiphospholipid syndrome.

Pathogenesis of preterm labor and preterm premature rupture of membranes associated with intraamniotic infection.

We have proposed a model in which the initiation of human parturition in the presence of infection is controlled by the host. Systemic maternal infections such as pyelonephritis or localized infections such as deciduitis can trigger parturition by the activation of the monocyte and macrophage system in peripheral blood and human decidua. Preterm labor and preterm PROM can, according to this, be considered events that occur when the intrauterine or maternal environment is hostile and threaten the survival of the fetal-maternal pair. From this point of view, the initiation of preterm labor may have survival value. Why does intrauterine infection result in preterm labor in some cases and PROM in others? It is possible that regulation of different components of the host response has an important role to play in determining clinical presentation. Thus, if preferential activation of the host response leads to the secretion of uterotonic agents (i.e., prostaglandins), preterm labor will result. On the other hand, if the activation of the host response results predominantly in the production of proteases (i.e., leukocyte elastase and MMPs), patients are more likely to experience PROM. Preterm labor and preterm PROM can be considered expressions of the same basic phenomenon: activation of the host-defense macrophage system. Although we have provided evidence that infection is an important factor in the pathogenesis of these conditions, preterm parturition should be considered as a syndrome with multiple causes.