1. Widespread microbial invasion of the chorioamniotic membranes is a consequence and not a cause of intra-amniotic infection. The pathway of microbial invasion leading to intra-amniotic infection has not been elucidated. The traditional view is that bacteria from the lower genital tract traverse the cervix and gain access to the decidua, generating a diffused infection of the decidua and membranes, and only after this, enter the amniotic cavity. This view was inconsistent with a number of observations made by our Branch in the recent past. Therefore, we conducted a series of studies to determine whether substantial bacterial propagation in the chorioamniotic membranes (CAMs) precedes microbial invasion of the amniotic cavity (MIAC). Bacteria (detected with a live/dead stain and FISH) were more frequently detected in the AF than in the CAMs of patients with positive amniotic fluid (AF) cultures for microorganisms. Bacteria were detected more frequently in the CAMs as the severity of chorioamnionitis worsened. A fraction of patients with chorioamnionitis (or MIAC) did not have bacteria in the CAMs. The median 16S rRNA gene copy number in the amnion was significantly greater than in the chorion. Bacteria were not detected in the CAMs or the AF of women at term without labor (this is in contrast with previous reports). Collectively, these findings indicate that MIAC does not follow widespread infection of CAMs, but precedes it: the initial stage is MIAC through a discrete region of the CAMs, followed by intra-amniotic proliferation, and bacterial invasion of the CAMs primarily extends from the AF. This study emphasizes the importance of assessing the intra-amniotic compartment for diagnosis and treatment of preterm birth. 2. A primate subfamily of galectins expressed at the maternal-fetal interface that promote immune cell death. Galectins are glycoproteins that regulate immune responses through the recognition of cell-surface glycans. Investigators at the PRB found that 16 human galectin genes are expressed at the maternal-fetal interface and demonstrate that a cluster of 5 galectin genes on human chromosome 19 emerged during primate evolution. We show that human placenta-specific galectins are predominantly expressed by the syncytiotrophoblast, a primary site of metabolic exchange where, during early pregnancy, the fetus comes in contact with immune cells circulating in maternal blood. Because ex vivo functional assays demonstrate that placenta-specific galectins induce the apoptosis of T lymphocytes, we propose that these galectins reduce the danger of maternal immune attacks on the fetal semi-allograft, presumably conferring additional immune tolerance mechanisms, and in turn sustaining hemochorial placentation during the long gestation of anthropoid primates. 3. Villitis of unknown etiology (VUE) is associated with a distinct pattern of chemokine up-regulation in the feto-maternal and placental compartments: implications for conjoint maternal allograft rejection and maternal anti-fetal graft-versus-host disease. VUE is a destructive inflammatory lesion of the placenta characterized by participation of Hofbauer cells and maternal T cells. The fundamental immunopathology of VUE is unknown. The PRB conducted this study to characterize the placental transcriptome between term placentas with and without VUE and to determine whether VUE was associated with systemic maternal and/or fetal inflammatory responses. Differential expression of 206 genes was found and associated with pathways related to the immune response. The mRNA expression of a subset of chemokines and their receptors was higher in VUE placentas than in normal placentas (p<0.05). Blood cell mRNA showed a higher expression of CXCL9 and CXCL13 in the mother, and CXCL11 and CXCL13 in the fetus of VUE cases (p<0.05). We propose that VUE be a unique state combining maternal allograft rejection and maternal antifetal graft-vs-host disease mechanisms. 4. Expression patterns of microRNAs in the chorioamniotic membranes: a role for microRNAs in human pregnancy and parturition. MicroRNAs (miRNAs) are involved in the post-transcriptional regulation of gene expression during development. We conducted this study to determine gestational age-dependent changes in miRNA expression in the chorioamniotic membranes and to assess the significance of miRNAs in human pregnancy and parturition. The expression profile of 455 miRNAs was compared between patients at term without labor (TNL), in labor (TL), and preterm labor (PTL) using microarrays. A total of 39 miRNAs were differentially expressed between term and preterm cases (31 were down-regulated at term). The expression of ten miRNAs, including miR-338, was decreased at term. Computational analyses identified PLA2G4B, a phospholipase implicated in parturition, as a putative target of miR-338. Inhibition of endogenous miR-338 with anti-miR-338 increased the mRNA and protein expression of PLA2G4B in decidual cells (confirmed by Luciferase assay with reporter constructs). Interestingly, the expression of Dicer, a key miRNA-processing enzyme, was markedly decreased in TL. These novel findings suggest that post-transcriptional regulation of genes by miRNAs plays a role in pregnancy and parturition. 5. Isobaric labeling and tandem mass spectrometry: A novel approach for profiling and quantifying proteins differentially expressed in amniotic fluid in preterm labor with and without intra-amniotic infection/inflammation. The purpose of this study was to apply, for the first time, the combination of Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) labeling and tandem mass spectrometry to identify proteins differentially regulated in amniotic fluid (AF) samples of women with spontaneous preterm labor (PTL) with and without intra-amniotic infection/inflammation (IAI). We observed differential expression of proteins in AF of patients who delivered preterm in the absence of IAI in comparison with those who delivered at term such as Mimecan precursor and Resistin. Gene ontology enrichment analysis was employed to reveal families of proteins participating in distinct biological processes, including host defense, anti-apoptosis, metabolism/catabolism and cell and protein mobility, localization and targeting. Using iTRAQ, we discovered 82 proteins differentially expressed in 3 clinical subgroups of PTL, 67 which were heretofore unknown. Of particular importance is the identification of proteins differentially expressed in AF from women who delivered preterm in the absence of IAI. This is the first report of the positive identification of biomarkers in this subgroup of patients. 6. A subset of patients destined to develop spontaneous preterm labor has an abnormal angiogenic/anti-angiogenic profile in maternal plasma. An imbalance between angiogenic and anti-angiogenic factors in maternal blood has been observed in several obstetrical syndromes. Vascular lesions have been identified in a subset of patients with spontaneous preterm labor (PTL). We propose that PTL may be one of the manifestations of an anti-angiogenic state. This study was conducted to determine if patients prior to the clinical diagnosis of PTL leading to preterm delivery had plasma concentrations of angiogenic and anti-angiogenic factors different from normal pregnant women before the development of the disease. The plasma sEng concentration in patients destined to develop PTL was higher than that of normal pregnant women from 15-20 weeks onward. The difference became statistically significant at 28 weeks, or approximately 5-10 weeks prior to the diagnosis of PTL. The changes in sEng are demonstrable several weeks prior to the onset of preterm parturition. In contrast, the changes in the other angiogenic proteins are present close to the onset of PTL and delivery.
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