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We have previously described the placental microbiome and its association with PTB. Others have used histology (Gram staining) to grossly detect bacteria in the membranes and in the basal plate decidua. However, methodology linking DNA-based microbial detection with advanced imaging to (1) identify intact bacteria, (2) accurately and precisely ascribe their location, and (3) quantify their abundance has heretofore been lacking. In this study, we sought to close this gap by probing conserved regions of the bacterial RNA using novel branched DNA technology and confocal high resolution imagery to amplify signal from in situ hybridization (ISH) probes.
Placental tissue was rigorouosly sterile collected from spontaneous preterm (sPTB) (n= 7), PPROM (n=4), indicated preterm (n=6), chorioamnionitis (n=3) and term control (n=4) births. Formalin fixed and paraffin embedded samples were probed using ISH for conserved regions of bacterial RNA. Signals were amplified in a series of hybridizations using branched DNA conjugated to alkaline phosphatase. Bacteria were visualized in both bright field and fluorescence using Fast Red at high resolution.
Using novel advanced imaging methodologies (Figure), bacteria were readily identified within the parenchyma of 5/7 (71.4%) sPTB, 4/4 (100%) PPROM, 3/6 (50%) indicated PTB, 3/3 (100%) chorioamnionitis, and 2/4 (50%) term placentas. Generally, the distribution of bacteria was heterogeneous (mostly clusters). Large clusters were observed in chorioamnionitis and PPROM subjects, scant but present clusters of bacteria observed in term or indicated preterm pregnancy. Choriamnionitis clusters were averaged to an estimated 4.7 x 10ˆ6 bacteria/mm3, which was greater than PPROM (1.1 x 10ˆ6), spontaneous sPTB (9 x 10ˆ5), and both normal term pregnancy (2 x 10ˆ5) and indicated PTB (2 x 10ˆ5; p=0.03).
Intact bacteria were visualized in most pregnancies examined and appear to be in greater concentration in PPROM pregnancies. Generally, the concentration of placental bacteria is estimated to be similar to seawater (1x10ˆ5-1x10ˆ6), but much less than saliva ∼1x10ˆ8 at its most concentrated. The high concentrations observed suggest placental bacteria may well influence placental pregnancy outcome.