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 neonates 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, in the context of intrauterine infection, the onset of premature labor has survival value. 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 several components of the innate immune system, including neutrophils and Toll-like receptors as well as the role of allergy in causing preterm labor and delivery. The Branch also used a genome-wide approach to identify differentially regulated genes in patients with premature rupture of membranes. The most noteworthy findings are as follows: 1. Pregnancy is associated with changes in the adaptive and innate immune response. Although some investigators have proposed that pregnancy is an """"""""anti-inflammatory state"""""""" thought to be beneficial for survival of the fetal semi-allograft, others have suggested that pregnancy is characterized by ?activation of the innate immune system? to compensate for impaired adaptive immunity and to protect the mother from infection. The following observations were made when studying neutrophils and monocytes of normal pregnant women and those with complications as well as the cell biology that underlies metabolic changes in these cells. A. Phenotypic and metabolic profile of monocytes and granulocytes in normal pregnancy: Granulocytes from normal pregnant women overexpress CD14 and CD64 when compared to granulocytes of nonpregnant women. The baseline intracellular reactive oxygen species, oxidative burst, and stimulation index values were higher in the granulocytes of normal pregnant women compared to that of nonpregnant women. However, phenotypic and metabolic changes were more marked in acute systemic maternal infection indicating that the innate limb of the immune response is not maximally stimulated during normal pregnancy. B. Phenotypic and metabolic profile of monocytes and granulocytes in preeclampsia, preterm labor, and preterm PROM: Flow cytometry studies indicated that these three complications of pregnancy are associated with phenotypic and metabolic changes consistent with activation. C. The controversy about whether or not neutrophil and macrophage function is impaired during pregnancy: In vitro studies suggest that many neutrophil functions are depressed in normal pregnancy, including microbial killing, chemotaxis, phagocytosis and adhesion. Yet, these results appear to conflict with those reported by recent investigations. The Branch conducted a number of studies to explore the reason for the conflicting results. D. Studies of neutrophil NADPH oxidase: Oxidant production by neutrophils is dependent on NADPH oxidase, which can be regulated by mechanisms related to enzyme assembly or substrate level regulation. The effects of different factors including cytokines are collectively expressed as NADPH metabolic oscillations, which can be characterized by defining their amplitude and frequency. These studies are performed using non-perturbative quantitative microfluorometry, which allows real-time single cell assay of reactive oxygen radical production. Neutrophils from normal pregnant women were found to have an increased number of high amplitude oscillations (20 seconds). Maternal infection and preeclampsia had an even higher percentage of cells that displayed this type of oscillations. However, in other complications of pregnancy, the neutrophil metabolic profile showed a reduced number of high amplitude oscillations. E. Unique metabolic characteristics of neutrophils in normal pregnancy: During pregnancy, nneutrophils were found to display high amplitude NADPH oscillations, but they lose the ability to change (increase) their frequency. This state is unique to pregnant women. The Branch has identified a novel and fundamental mechanism for oxidant regulation, which accounts for the higher basal activity but the inability to ?activate? pregnancy neutrophils. Translocation of enzymes in the hexose monophosphate shunt account for reduced production of reactive oxygen metabolites. Studies are in progress to define the precise mechanism responsible for both amplitude and frequency modulation of NADPH oscillations. This model resolves discrepancies in the literature. 2. Induction of premature labor and delivery by allergic reaction and prevention by histamine H1 receptor antagonist: The Branch proposed that a sub-group of patients with premature labor may have an allergic-like mechanism of disease responsible for preterm birth. The studies conducted this year provided evidence that sensitization of animals and subsequent challenge during pregnancy is able to cause premature labor and delivery. At days 40 through 50 of gestation, ovalbumin-sensitized guinea pigs that had not been treated or had been pretreated with an H1 receptor antagonist (ketotifen) or a mast-cell stabilizer (cromolyn sodium) were challenged with either ovalbumin or saline solution. Nonsensitized guinea pigs were challenged with ovalbumin. The duration of gestation was significantly shorter in sensitized animals challenged with ovalbumin than in the other groups. Pre-treatment with ketotifen significantly increased the duration of pregnancy in sensitized animals, compared with untreated animals. Cromolyn sodium had no effect. These observations support the concept that a Type-1 hypersensitivity reaction may be a mechanism of disease in premature labor. This is important because it has been estimated that 40 to 60 million Americans have allergic disorders. 3. Genome-wide expression profiling of fetal membranes reveals a deficient expression of proteinase inhibitor 3 in premature rupture of membranes: Preterm premature rupture of membranes (PPROM) is a leading cause of preterm birth. A growing body of evidence supports the concept that preterm parturition is a syndrome that is defined by the presence of increased uterine contractility, cervical dilatation and decidual/membrane activation with or without PROM that has multiple causes. One approach to dissect the mechanisms of disease in PPROM is genomics. We conducted a study to determine messenger RNA (mRNA) expression profiles with microarrays to classify preterm birth with intact membranes and PPROM in the presence and absence of acute chorioamnionitis. Microarray experiments identified the decreased expression of proteinase inhibitor 3 in the PPROM cases. Quantitative reverse transcriptase-polymerase chain reaction confirmed these results. Immunohistochemistry demonstrated decreased proteinase inhibitor 3 protein expression in PPROM. The significance of this finding is that this protein had not been previously implicated to play a role in parturition and, specifically, in membrane rupture. Our observations provide a novel pathway for investigation in the pathobiologic condition of preterm birth. P13 is a low-molecular weight serine proteinase inhibitor. We propose that the production of P13 in the fetal membranes protects the tissue from damage that could be caused by increased activity and availability of neutrophil elastase. Patients who are not capable of producing adequate amounts of P13 may be predisposed to PPROM.
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