BACKGROUND Preterm birth (PTB) is a leading cause of neonatal morbidity and mortality and is not uncommonly associated with chorioamnionitis. We recently have demonstrated that the placenta harbors a unique microbiome with similar flora to the oral community. We also have shown an association of these placental microbiota with PTB, history of antenatal infection, and excess maternal weight gain. On the basis of these previous observations, we hypothesized that the placental membranes would retain a microbiome community that would vary in association with preterm birth and chorioamnionitis. OBJECTIVE In the current study, we aimed to examine the differences in the placental membrane microbiome in association with PTB in both the presence and absence of chorioamnionitis and/ or funisitis using state-of-the-science whole-genome shotgun metagenomics. STUDY DESIGN This was a cross-sectional analysis with 6 nested spontaneous birth cohorts (n = 9–15 subjects/cohort): Term gestations without chorioamnionitis, term with chorioamnionitis, preterm without chorioamnionitis, preterm with mild chorioamnionitis, preterm with severe chorioamnionitis, and preterm with chorioamnionitis and funisitis. Histologic analysis was performed with Redline's criteria, and inflammatory cytokines were analyzed in the cord blood. DNA from placental membranes was extracted from sterile swabs collected at delivery, and whole-genome shotgun sequencing was performed on the Illumina HiSeq platform. Filtered microbial DNA sequences were annotated and analyzed with MG-RAST (ie, Metagenomic Rapid Annotations using Subsystems Technology) and R. RESULTS Subjects were assigned to cohorts on the basis of gestational age at delivery and independent scoring of histologic chorioamnionitis. We found that preterm subjects with severe chorioamnionitis and funisitis had increases in cord blood inflammatory cytokines. Of interest, although the placental membrane microbiome was altered in association with severity of histologic chorioamnionitis (permutational multivariate analysis of variance P = .005), there was no observable impact with either beta-methasone or antibiotic treatment. In preterm subjects with chorioamnionitis, we found a high abundance of both urogenital and oral commensal bacteria. These alterations in the microbiome were accompanied by significant variation (P < .05) in microbial metabolic pathways important in the glucose-fed pentose phosphate pathway (term subjects), or glycerophopholipid metabolism, and the biosynthesis of the siderophore group nonribosomal peptides (preterm subjects). CONCLUSION Consistent with ours and others previous findings, women who experienced spontaneous PTB harbor placental microbiota that further differed by severity of chorioamnionitis. Integrative meta-genomic analysis revealed significant variation in distinct bacterial metabolic pathways, which we speculate may contribute to risk of preterm birth with and without severe chorioamnionitis.

SUMMARY The human Ureaplasma species are the most frequently isolated microorganisms from the amniotic fluid and placentae of women who deliver preterm and are also associated with spontaneous abortions or miscarriages, neonatal respiratory diseases, and chorioamnionitis. Despite the fact that these microorganisms have been habitually found within placentae of pregnancies with chorioamnionitis, the role of Ureaplasma species as a causative agent has not been satisfactorily explained. There is also controversy surrounding their role in disease, particularly as not all women infected with Ureaplasma spp. develop chorioamnionitis. In this review, we provide evidence that Ureaplasma spp. are associated with diseases of pregnancy and discuss recent findings which demonstrate that Ureaplasma spp. are associated with chorioamnionitis, regardless of gestational age at the time of delivery. Here, we also discuss the proposed major virulence factors of Ureaplasma spp., with a focus on the multiple-banded antigen (MBA), which may facilitate modulation/alteration of the host immune response and potentially explain why only subpopulations of infected women experience adverse pregnancy outcomes. The information presented within this review confirms that Ureaplasma spp. are not simply “innocent bystanders” in disease and highlights that these microorganisms are an often underestimated pathogen of pregnancy.

Preterm birth is a major cause of neonatal mortality and morbidity worldwide. Bacterial infection and the subsequent inflammatory response are recognised as an important cause of preterm birth. It is hypothesised that these organisms ascend the cervical canal, colonise placental tissues, cause chorioamnionitis and in severe cases infect amniotic fluid and the foetus. However, the presence of bacteria within the intrauterine cavity does not always precede chorioamnionitis or preterm birth. Whereas previous studies observing the types of bacteria present have been limited in size and the specificity of a few predetermined organisms , in this study we characterised bacteria found in placental tissues from a cohort of 1391 women in rural Malawi using 16S ribosomal RNA gene sequencing. We found that specific bacteria found concurrently on placental tissues associate with chorioamnionitis and delivery of a smaller newborn. Severe chorioamnionitis was associated with a distinct difference in community members, a higher bacterial load and lower species richness. Furthermore, Sneathia sanguinengens and Peptostreptococcus anaerobius found in both matched participant vaginal and placental samples were associated with a lower newborn length-forage Z-score. This is the largest study to date to examine the placental microbiome and its impact of birth outcomes. Our results provide data on the role of the vaginal microbiome as a source of placental infection as well as the possibility of therapeutic interventions against targeted organisms during pregnancy.

Objective-To determine effects in late gestation of U. parvum serovar 3 colonization, and effects, preterm, of U. parvum serovar 6. Study design-Ewes received an intra-amniotic (IA) injection of U. parvum serovar 6 (20x10 6 cfu; n=9), U. parvum serovar 3 (20x10 3 cfu; n=6), vehicle (n=10) or saline (n=4) on day 80 of pregnancy (d). Lambs were delivered at 125d (ureaplasma, n=9; saline or media controls, n=9) or 145d (ureaplasma, n=6; media controls, n=5) for assessment of inflammation and lung maturation.Results-IA ureaplasmas caused histologic chorioamnionitis but not preterm delivery. Fetal lung epithelium was colonized with ureaplasmas at both gestational ages and pulmonary IL-8 levels had doubled in the ureaplasma-colonized animals compared to controls at 145d. Surfactant levels in bronchoalveolar lavage fluid had increased 8 fold and 2.5 fold at 125 and 145d, respectively after ureaplasma injection. Conclusions-Fetal

Bronchopulmonary dysplasia is associated with chorioamnionitis and fetal lung inflammation. Ureaplasma species are the bacteria most frequently isolated from chorioamnionitis. Very chronic ureaplasma colonization of amniotic fluid causes low-grade lung inflammation and functional lung maturation in fetal sheep. Less is known about shorter exposures of the fetal lung. Therefore, we hypothesized that ureaplasmas would cause an acute inflammatory response that would alter lung development. Singleton ovine fetuses received intra-amniotic Ureaplasma parvum serovar 3 or control media at 110, 117, or 121 days and were delivered at 124 days gestational age (term = 150 days). Inflammation was assessed by 1) cell counts in bronchoalveolar lavage fluid (BALF), and 2) cytokine mRNA measurements, immunohistochemistry, and flow cytometry for inflammatory cells and elastin and α-smooth muscle actin (α-SMA) staining in lung tissue. Neutrophils were increased in BALF 3 days after exposure to ureaplasmas (P = 0.01). Myeloperoxidase-positive cells increased after 3 days (P = 0.03), and major histocompatibility complex (MHC) class II-positive cells increased after 14 days of ureaplasma exposure (P = 0.001). PU.1 (macrophage marker)- or CD3 (T lymphocyte marker)-positive cells were not induced by ureaplasmas. CD3-positive cells in the posterior mediastinal lymph node increased in ureaplasma-exposed animals at 3, 7, and 14 days (P = 0.002). Focal elastin depositions decreased in alveolar septa at 14 days (P = 0.002), whereas α-SMA increased in arteries and bronchioli. U. parvum induced a mild acute inflammatory response and changed elastin and α-SMA deposition in the lung, which may affect lung structure and subsequent development.

The chorioamnionitis associated with preterm delivery is often polymicrobial with ureaplasma being the most common isolate. To evaluate interactions between the different pro-inflammatory mediators, we hypothesized that ureaplasma exposure would increase fetal responsiveness to LPS. Fetal sheep were given intra-amniotic injections of media (control) or Ureaplasma parvum serovar 3 either 7d or 70d before preterm delivery. Another group received an intraamniotic injection of E.coli lipo-polysaccharide (LPS) 2d prior to delivery. To test for interactions, intraamniotic U. parvum exposed animals were challenged with intraamniotic LPS and delivered 2d later. All animals were delivered at 124±1d gestation (Term=150d). Compared to the 2d LPS exposure group, the U. parvum 70d+LPS group had: 1) decreased lung pro and anti-inflammatory cytokine expression 2) fewer CD3+ T-lymphocytes, CCL2+, myeloperoxidase+, and PU.1+ cells in the lung. Interestingly, exposure to U. parvum for 7d did not change responses to a subsequent intraamniotic LPS challenge, and exposure to intraamniotic U. parvum alone induced mild lung inflammation. Exposure to U. parvum increased pulmonary TGFβ1 expression but did not change mRNA expression of either the receptor TLR4 or some of the downstream mediators in the lung. Monocytes from fetal blood and lung isolated from U. parvum 70d+LPS but not U. parvum 7d+LPS animals had decreased in vitro responsiveness to LPS. These results are consistent with the novel finding of down-regulation of LPS responses by chronic but not acute fetal exposures to U. parvum. The findings increase our understanding of how chorioamnionitis exposed preterm infants may respond to lung injury and postnatal nosocomial infections.

IntroductionXanthine oxidase (XO) is distributed in mammals largely in the liver and small intestine, but also is highly active in milk where it generates hydrogen peroxide (H2O2). Adult human saliva is low in hypoxanthine and xanthine, the substrates of XO, and high in the lactoperoxidase substrate thiocyanate, but saliva of neonates has not been examined.ResultsMedian concentrations of hypoxanthine and xanthine in neonatal saliva (27 and 19 μM respectively) were ten-fold higher than in adult saliva (2.1 and 1.7 μM). Fresh breastmilk contained 27.3±12.2 μM H2O2 but mixing baby saliva with breastmilk additionally generated >40 μM H2O2, sufficient to inhibit growth of the opportunistic pathogens Staphylococcus aureus and Salmonella spp. Oral peroxidase activity in neonatal saliva was variable but low (median 7 U/L, range 2–449) compared to adults (620 U/L, 48–1348), while peroxidase substrate thiocyanate in neonatal saliva was surprisingly high. Baby but not adult saliva also contained nucleosides and nucleobases that encouraged growth of the commensal bacteria Lactobacillus, but inhibited opportunistic pathogens; these nucleosides/bases may also promote growth of immature gut cells. Transition from neonatal to adult saliva pattern occurred during the weaning period. A survey of saliva from domesticated mammals revealed wide variation in nucleoside/base patterns.Discussion and ConclusionDuring breast-feeding, baby saliva reacts with breastmilk to produce reactive oxygen species, while simultaneously providing growth-promoting nucleotide precursors. Milk thus plays more than a simply nutritional role in mammals, interacting with infant saliva to produce a potent combination of stimulatory and inhibitory metabolites that regulate early oral–and hence gut–microbiota. Consequently, milk-saliva mixing appears to represent unique biochemical synergism which boosts early innate immunity.

Ureaplasma species are the bacteria most frequently isolated from human amniotic fluid in asymptomatic pregnancies and placental infections. Ureaplasma parvum serovars 3 and 6 are the most prevalent serovars isolated from men and women. We hypothesized that the effects on the fetus and chorioamnion of chronic ureaplasma infection in amniotic fluid are dependent on the serovar, dose, and variation of the ureaplasma multiple-banded antigen (MBA) and mba gene. We injected high- or low-dose U. parvum serovar 3, serovar 6, or vehicle intra-amniotically into pregnant ewes at 55 days of gestation (term = 150 days) and examined the chorioamnion, amniotic fluid, and fetal lung tissue of animals delivered by cesarean section at 125 days of gestation. Variation of the multiple banded antigen/mba generated by serovar 3 and serovar 6 ureaplasmas in vivo were compared by PCR assay and Western blot. Ureaplasma inoculums demonstrated only one (serovar 3) or two (serovar 6) MBA variants in vitro, but numerous antigenic variants were generated in vivo: serovar 6 passage 1 amniotic fluid cultures contained more MBA size variants than serovar 3 (P = 0.005), and ureaplasma titers were inversely related to the number of variants (P = 0.025). The severity of chorioamnionitis varied between animals. Low numbers of mba size variants (five or fewer) within amniotic fluid were associated with severe inflammation, whereas the chorioamnion from animals with nine or more mba variants showed little or no inflammation. These differences in chorioamnion inflammation may explain why not all women with in utero Ureaplasma spp. experience adverse pregnancy outcomes.