1. Proteomic analysis of AF to identify women with preterm labor and intra-amniotic inflammation/infection (IAI). ? A cross-sectional study was designed to combine a novel computational method of pattern discovery with mass spectrometric proteomic profiling of AF to discover biomarkers of IAI in patients with spontaneous PTL and intact membranes who delivered at term, and those who delivered preterm with IAI. This proteomic profiling was conducted through multiple simultaneous SELDI mass spectrometry conditions. Thirty nine unique mass spectrometric peaks discriminated patients with PTL/delivery with IAI from those with PTL and term delivery, with an overall accuracy of 90.3%. We demonstrated that proteomic analysis of AF allowed the identification of mass spectrometry features, which can distinguish patients with PTL and IAI from those with PTL without IAI who subsequently delivered at term.? ? 2. Detection of microbial biofilms in the amniotic cavity of women with PTL.? Microbial biofilms are communities of sessile microorganisms formed by cells that are attached irreversibly to a substratum or interface or to each other, and embedded in a hydrated matrix of extracellular polymeric substances, and have been recognized to be implicated in more than 80% of human infections. We have previously identified AF sludge using ultrasound, and undertook a study to determine the nature of AF sludge by examining the material. Macroscopically, AF sludge is similar to pus and is associated with a markedly elevated white blood cell count in AF, as well as positive Gram stain and cultures. In a case of spontaneous PTL and clinical chorioamnionitis at 28 weeks of gestation, AF sludge was retrieved and bacteria were identified with scanning electron microscopy and FISH for conserved regions of the microbial genome, and the exopolymeric matrix was identified by histochemistry using the wheat germ agglutinin lectin method. AF sludge was imaged with confocal laser scanning microscopy and scanning electron microscopy, which allowed the identification of bacteria embedded in an amorphous material, and inflammatory cells. This is the first report of microbial biofilms in the AF. ? ? 3. The anti-inflammatory limb of the immune response in PTL, IAI, and spontaneous parturition at term: a role for IL-10. ? The anti-inflammatory limb of the immune response is crucial for dampening inflammation. IL-10 is an anti-inflammatory cytokine that participates in a negative feedback to reduce inflammation. We designed a cross-sectional study to determine whether there is a relationship between AF concentrations of IL-10 and gestational age, parturition (at term and preterm), and IAI. IL-10 was detectable in AF and its concentration did not change with gestational age. The presence of labor at term was associated with a significantly elevated AF IL-10 concentration. Moreover, the presence of IAI was associated with a significantly higher AF concentration of IL-10 in patients at term as well as in those with PTL with intact membranes when compared to those without IAI. These results suggest that IL-10 plays an important role in the control of labor and takes part in the regulation of the immune response in vivo by initiating actions that dampen inflammation.? ? 4. Chorioamnionitis and increased galectin-1 expression in PPROM.? Galectin-1 is an anti-inflammatory protein expressed by chorioamniotic membranes and the decidua that has pleiotropic intra- and extra-cellular functions, is over-expressed in activated immune and endothelial cells, as well as in sites of inflammation, and is considered to regulate immune responses upon infection and inflammation. We set out to determine galectin-1 expression in the chorioamniotic membranes and its changes in women with a normal pregnancy and patients with PPROM with and without histologic chorioamnionitis. Galectin-1 mRNA and protein were localized by in situ hybridization and immunohistochemistry, and determined by qRT-PCR. In patients with PPROM, galectin-1 mRNA expression in fetal membranes was 2-fold higher in those with histologic chorioamnionitis than in those without. Moreover, histologic chorioamnionitis was associated with strong galectin-1 immunostaining in amniotic epithelium, chorioamniotic mesodermal cells, and apoptotic bodies. These findings suggest that galectin-1 may be involved in the regulation of the inflammatory responses to chorioamniotic infection. ? ? 5. Soluble receptor for advanced glycation end products (sRAGE) and endogenous secretory RAGE (esRAGE) in AF.? The receptor for advanced glycation end products (RAGE) participates in the innate and adaptive immune responses. RAGE induces production of pro-inflammatory cytokines and chemokines, as well as neutrophil chemotaxis in a manner that may be suppressed or stimulated by soluble, truncated forms of RAGE including the sRAGE and esRAGE. RAGE is expressed in the amnion epithelium, extravillous trophoblast and decidual cells in patients without chorioamnionitis and in the neutrophils in the choriodecidua in cases of histologic chorioamnionitis. Our group conducted a study to determine if IAI is associated with changes in the AF concentration of sRAGE and esRAGE in the midtrimester of pregnancy as well as in term and PTL, and patients with PPROM. The AF concentrations of sRAGE and esRAGE increased as a function of gestational age, and decreased in the presence of labor at term. The AF concentrations of sRAGE and esRAGE were significantly elevated in the presence of IAI in both patients with PTL and intact membranes and patients with PPROM, suggesting that changes in the AF concentration of sRAGE and esRAGE may represent part of the immune response to IAI.? ? ? 6. Visfatin in AF in normal pregnancy, spontaneous labor at term, PTL and PPROM. ? Visfatin is a novel adipokine produced by visceral adipose tissue. Its gene expression is increased in inflammatory conditions such as sepsis and metabolic syndrome. It is expressed by amniotic epithelium, cytotrophoblast and decidua, and over-expressed in fetal membranes when exposed to mechanical stress and/or pro-inflammatory stimuli. The Branch conducted a study to determine if visfatin is detectable in AF and whether its concentration changes with gestational age, spontaneous labor, PPROM and in the presence of MIAC. We found that visfatin is a physiologic constituent of AF, its concentrations in AF increase with advancing gestational age and in patients with MIAC, regardless of the membrane status. This is the first in vivo study describing the presence of visfatin in AF and its association with acute infection in humans, suggesting that visfatin plays a role in normal gestation, as well as in the inflammatory response associated with infection. ? ? 7. Microbial prevalence, diversity and abundance in AF during PTL.? Unrecognized intra-amniotic infections caused by cultivation-resistant microbes may play a role in PTL. Our group, in collaboration with investigators at Stanford University, used broad-range end-point and real-time PCR assays to amplify, identify and quantify ribosomal DNA (rDNA) of bacteria, fungi and archaea from AF in women in PTL with intact membranes. The combined use of molecular and culture methods revealed a greater prevalence and diversity of microbes in AF than did culture alone. A positive PCR was associated with histologic chorioamnionitis and funisitis. The positive predictive value of PCR for preterm delivery was 100%. A temporal association between a positive PCR and delivery was supported by a shortened amniocentesis-to-delivery interval. A dose-response association was demonstrated between bacterial rDNA abundance and gestational age at delivery. The strength, temporality and gradient with which these microbial sequence types are associated with preterm delivery support a causal relationship.
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