The risks of systemic inflammation measured by distinct profiles of inflammatory proteins in newborn blood associated with placental colonization by specific groups of bacteria are presented as odds ratios in and . The number of samples that tested positive for each organism or group of organisms is stated in the bottom row of each column. The odd ratios indicate the relationship of culture-positive cases with those whose placentas did not harbor any culturable microorganism rather than with all cases studied.
Risk of neonate systemic inflammatory response associated with placental colonization by specific types of microorganisms
Risk of neonate systemic inflammatory response associated with placental colonization by Lactobacillus species alone or mixed with others in contrast to that by BV-associated species
Of the 527 placentas studied, 313 were culture negative, and 214 were culture positive for one or more microorganisms of the groups shown in . Lactobacilli (detected alone or mixed with other microorganisms in 21 cases or in 9.8% of all culture-positive placentas) were associated with a prominently low risk of systemic inflammatory reaction in the preterm newborn in contrast to the groups of pathogenic microorganisms and organisms characteristic of bacterial vaginosis (BV), which were all associated with increased risks of specific sets of elevated proteins compared to placentas that did not harbor any microorganisms (). The group of BV organisms was most frequently detected, with Gardnerella vaginalis, Prevotella bivia, anaerobic Streptococcus, and Peptostreptococcus present alone or in mixed culture in 47% or 22% of all culture-positive placentas, respectively. The BV organisms were associated with a heightened proinflammatory pattern similar to those of the infectious facultative anaerobes Escherichia coli and alpha Streptococcus (each detectable in 28% or 13% of the culture-positive placentas, respectively) and distinct from those of the various genital mycoplasma species (collectively detected in 38% or 18% of the culture-positive placentas, respectively). The mixed BV microorganisms and the infectious facultative anaerobes shared an identical pattern of highest odds of elevated levels of interleukin-1β (IL-1β), E-selectin, and serum amyloid A (SAA). In addition, the organisms within these two groups variably shared odds for elevated levels of IL-6, tumor necrosis factor alpha (TNF-α), TNF receptors, IL-8, intercellular adhesion molecule 1 (ICAM-1), ICAM-3, C-reactive protein (CRP), myeloperoxidase (MPO), vascular endothelial growth factor (VEGF), and VEGF receptor 2 (VEGF-R2). In total, the proinflammatory protein profile of the cases of mixed BV culture included 9 of the 25 proteins studied and was not duplicated by any single BV microorganism, suggesting that the proinflammatory effect should be attributable to the cumulative effects of the BV community rather than that of an individual microbial species.
When BV-associated microorganisms were evaluated separately, each had its own profile of elevated concentrations of proteins in the newborn blood samples (). This analysis included all cases with a specific microorganism recovered (including monocultures and mixed cultures) versus those that did not have any microorganism recovered. While the presence of Prevotella was associated with a relatively low risk of inflammatory response, Gardnerella was marked by an increased risk of TNF-α, IL-8, ICAM-1, and VEGF-R2, anaerobic Streptococcus was associated with a pattern of elevated IL-1β, IL-6, TNF receptor type 1 (TNF-R1), TNF-R2, and E-selectin, and Peptostreptococcus was characterized by a high risk of elevated macrophage inflammatory protein 1β (MIP-1β), ICAM-3, matrix metalloproteinase 9 (MMP-9), MPO, and VEGF and undetectable insulin growth factor binding protein 1 (IGF-BP-1).
Cases with cultured genital mycoplasma species (including Ureaplasma urealyticum and other Mycoplasma spp.) showed a reproducible inflammatory protein pattern that closely resembled only that of Peptostreptococcus and was dominated by increased odds of elevated MIP-1β, I-TAC (interferon-inducible T cell alpha-chemoattractant), ICAM-3, MMP-9, MPO, and VEGF and decreased odds of elevated VEGF-R1 and IGF-BP-1. Interestingly, in contrast to the BV-mixed group and the facultative anaerobes E. coli and alpha Streptococcus, they were not associated with elevated proinflammatory cytokines or the hepatic markers of systemic inflammation CRP and SAA.
Skin-associated organisms (Staphylococcus and Propionibacterium species), detectable in 13 to 17% of the culture-positive placentas, were not associated with elevated or reduced concentrations of inflammatory proteins in the newborn blood samples.
The levels of IL-6 receptor (IL-6R), monocyte chemotactic protein 1 (MCP-1), MCP-4, RANTES (regulated upon activation, normal T cell expressed and [presumably] secreted), and MMP-1 in the newborns’ blood samples did not appear to be significantly affected by the presence of any type of cultural bacteria in their placentas ().
Because of the distinct low inflammatory profile of the Lactobacillus-positive cases () and because most of them (62%) represented mixed cultures, we broke down the group into monoculture and mixed-culture Lactobacillus-positive subgroups, and we further contrasted those two subgroups with cases where BV organisms were found alone or in combination with non-BV organisms (). Again, odds ratios of having top-quartile protein concentrations were calculated by comparison to those of cases with no microorganisms recovered.
Although the number of placentas colonized by Lactobacillus alone was relatively small (n = 8), resulting in lack of statistical power, it was striking that most inflammatory proteins, including those associated with individual or mixed BV microorganisms (IL-1β, IL-6, TNF-R1, TNF-R2, IL-8, MIP-1β, ICAM-1, ICAM-3, E-selectin, MMP-9, CRP, SAA, and VEGF), were completely undetectable in placentas that harbored Lactobacillus alone (). In addition, even when mixed with others (n = 13), lactobacilli continued to be associated with undetectable ICAM-1 and VEGF, and despite the wide confidence intervals due to the overall low prevalence of Lactobacillus in the ELGAN placentas, the mixed Lactobacillus cultures remained at low odds for all studied proteins.
The opposite was observed in the group positive for BV-associated species. The cases that were exclusively BV organism positive (n = 18) represented 38% of the group. When these cases were separately analyzed, they showed an even higher risk of elevated protein concentrations, including IL-1β, IL-6, TNF-α, IL-8, ICAM-1, ICAM-3, vascular cell adhesion molecule 1 (VCAM-1), VEGF-R2, and both hepatic markers of systemic inflammation, CRP and SAA (). In contrast, the group harboring BV mixed with other microorganisms (n = 29), of which a significant part (24%) was mixed with Lactobacillus, showed lower odds for top-quartile concentrations of all cytokines, chemokines, and hepatic markers of inflammation (, last column), possibly due to the modulatory effect of Lactobacillus in the mixed cultures.
The difference between the groups of Lactobacillus-positive and -negative cases is illustrated in by concentration distributions for selected proteins representing the cytokines (IL-6), chemokines (IL-8), and hepatic inflammatory markers (CRP and SAA). Although the analysis of raw concentration values could not have the nominal statistical value of the percentile odds ratio analysis () due to the overall low prevalence of Lactobacillus species cultured from the ELGAN placentas, the data demonstrate the contrast between the observed narrow distribution and lower levels of proteins in the Lactobacillus-positive cases.
FIG 1 Comparison of risks of systemic inflammatory response measured by selected proteins in blood specimens obtained from newborns with Lactobacillus species versus other organisms detected in their placentas. Box plots represent logarithmically transformed (more ...)