Premature birth is the leading cause of perinatal mortality and morbidity worldwide. The Perinatology Research Branch has defined preterm labor as a syndrome and determined that at least 25% of preterm births are born to women with sub-clinical intrauterine infection. Moreover, we have provided evidence that many premature neonates are critically ill before birth and proposed that the onset of premature labor has survival value in the context of intrauterine infection. The goal of this project is to understand the pathophysiology of premature labor and delivery and the focus of our research this year was to study the role of matrix degrading enzymes in preterm premature rupture of membranes. The focus of the Branch this year was understanding the role of the innate immune system in the mechanisms of preterm parturition. The findings that were noteworthy include: 1. Maternal serum of women with preeclampsia reduces trophoblast cell viability: evidence for an increased sensitivity to fas-mediated apoptosis: Preeclampsia, or """"""""toxemia of pregnancy,"""""""" has been attributed to the presence of a circulating """"""""toxin"""""""" that disappears from peripheral blood after delivery of the placenta. However, the presence, nature and effects of this toxin have eluded characterization. Increased trophoblast apoptosis has been observed in the placenta of women with preeclampsia, and it is possible that this biological phenomenon is important for the genesis of the disease and mediated through a soluble factor(s) present in maternal blood. The Branch conducted a study to determine whether serum from women with preeclampsia changes trophoblast viability. H8 trophoblast cells were cultured with serum obtained from normal pregnant women and patients with preeclampsia. Cell viability was determined by Cell Titer 96 Assay. Fas sensitivity was determined by treating the cells with an anti-Fas antibody or a blocking anti-Fas Ligand antibody. Serum from normal pregnant women did not affect trophoblast cell viability. In contrast, serum from preeclamptic women reduced trophoblast viability, and this was enhanced by treatment with an anti-Fas antibody. This effect was reversed by the treatment with a blocking anti-Fas Ligand antibody. We conclude that serum from women with preeclampsia induces cytotoxicity of first trimester trophoblast cell line (H8). This effect appears to be related to changes in trophoblast sensitivity to Fas-mediated apoptosis. These findings suggest that a factor present in maternal blood of patients with preeclampsia may have a role in the genesis of the syndrome. Future studies will be aimed to determine whether this factor is present prior to the development of the disease. 2. Antimicrobial factors in the amniotic fluid of normal women and those with preterm labor and premature rupture of membranes: The amniotic cavity is considered a sterile compartment, presumably accomplished by the participation of the innate immune system, which includes the cervical epithelium, cervical mucus plug, chorioamniotic membranes, cellular components of decidua, amnion and chorion, including neutrophils, macrophages, NK cells and trophoblast. Although intact chorioamniotic membranes are thought to protect against microbial invasion of the cavity (MIAC), this physical barrier alone cannot be responsible for the sterile nature of amniotic fluid (AF) in normal pregnancy because bacteria can cross the intact membranes. Indeed, patients with intact membranes can have MIAC, as this has been demonstrated in patients in the midtrimester of pregnancy, with preterm labor and intact membranes or even spontaneous labor at term. This suggests that in addition to the physical barrier, other mechanisms must be operative to prevent bacterial proliferation within the amniotic cavity. Antimicrobial peptides, part of the innate limb of the immune response, have been identified in plants, insects and all vertebrates examined thus far. Neutrophils contain several antimicrobial peptides including neutrophil defensins [Human Neutrophil Peptides (HNP) 1, 2, and 3], Bactericidal/Permeability-Increasing Protein (BPI) and calprotectin (MRP8/14). The purpose of this study was to determine if AF contains immunoreactive antimicrobial peptides (HNP 1-3, BPI and MRP8/14) and whether their concentration changes with MIAC, parturition and premature rupture of membranes (PROM). The Branch conducted a series of studies that yielded the following: 1) Intraamniotic infection is associated with a significant increase in AF concentrations of HNP1-3, BPI and calprotectin in both women with preterm labor and intact membranes, and women with preterm PROM. 2) Preterm PROM is associated with a significant increase in AF concentrations of HNP 1-3, BPI and calprotectin. 3) Preterm parturition is associated with a significant increase in AF concentrations of HNP 1-3, BPI and calprotectin, while term parturition is associated with a significant increase in AF concentrations of immunoreactive HNP 1-3. 4) Among patients with preterm labor and intact membranes, elevation of AF HNP 1-3, BPI and calprotectin concentrations was associated with intraamniotic inflammation, histologic chorioamnionitis and short interval to delivery. 3. The effect of antibiotic therapy on intrauterine infection-induced preterm parturition in rabbits: Microbial invasion of amniotic fluid cavity and sub-clinical intrauterine infection have been implicated as one of the mechanisms responsible for preterm labor and delivery. Consequently, investigators have examined whether the administration of antibiotics to women in preterm labor with intact membranes and preterm premature rupture of membranes (PROM) can prolong pregnancy and improve neonatal outcome. There are conflicting results from different randomized clinical trials and meta-analysis. The Cochrane Review concluded that routine use of antibiotics is not effective in the prevention of preterm parturition with intact membranes. In contrast, animal experiments suggest that, in some instances, antibiotic administration can reduce intra-amniotic bacterial load and preterm delivery, as well as improve neonatal survival. One possible explanation for the ineffectiveness in humans is that antibiotics must be administered early to be effective and that when patients present with clinical preterm labor and intraamniotic infection, it is too late to down-regulate the inflammatory response and thus antimicrobial therapy cannot prevent preterm parturition and/or fetal damage. A rabbit model of ascending uterine infection was used to assess whether there is a time-dependent relationship between antibiotic administration and the prevention of preterm parturition. We conducted a study in which rabbits were inoculated with E. Coli without antibiotic treatment delivered prematurely. The median inoculation-to-delivery interval was significantly shorter in the infected group than in the control group [36.3 hours (15-76.5) vs. 219 hours (173-240), p<0.001]. Antibiotic administration within 12 hours, but not after 18 hours, of inoculation increased the duration of pregnancy (by reducing the rate of preterm delivery) and neonatal survival (0% vs. 71%; p<0.005). We found that antibiotic administration can prolong pregnancy and reduce perinatal mortality after an ascending intrauterine infection only if administered early (less than 12 hours) after microbial inoculation. Whether this approach could also reduce neonatal morbidity remains to be determined. The importance of this observation is the need for the rapid and early identification of intraamniotic infection in humans.
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