Resolution and characterization of subgroups of Gardnerella vaginalis and description of the vaginal microbiota of women with preterm premature rupture of membranes

The vaginal microbial community is critical to a woman’s health and the health of her family. Bacterial vaginosis (BV) is a polymicrobial syndrome characterized by a shift of the vaginal microbiota from a Lactobacillus dominated community to a dense biofilm containing a complex mixture of organisms. Although BV is an important risk factor for poor reproductive health outcomes, the etiology of BV is poorly understood. Gardnerella vaginalis is a hallmark species of BV. Phylogenetic analysis of cpn60 universal target sequences from metagenomic studies of the vaginal microbiome and from G. vaginalis isolates resolved four subgroups within the species. This subdivision, supported by whole genome similarity comparisons, demonstrated that these subgroups might represent different species. Among a group of African women, only G. vaginalis subgroup B was significantly more abundant in women with BV relative to women with Nugent scores not consistent with BV. To characterize the subgroups further, several phenotypic and molecular factors of G. vaginalis subgroups were assessed. Proteomic profiles of isolates within each subgroup formed unambiguous clusters. Sialidase gene sequences were detected in all subgroups, however enzymatic activity was detected only in subgroup B. Two isolates of subgroup B isolates (N153 and N101) were incapable of growth in 7% CO2. Given the well-known relationship between an anaerobic microbiota and BV, anaerobic isolates of G. vaginalis are potentially important players in the vaginal microbial community. To determine genome content differences that could account for the phenotypic difference, whole genome sequences of four G. vaginalis subgroup B isolates representing facultative and anaerobic phenotypes were determined. Comparison of genomes led to the identification of genes predicted to encode proteins involved in cell wall biogenesis and protection from oxidative damage that might account for the observed phenotypes. The cpn60 universal target based methodology that improved resolution of the vaginal microbiota including G. vaginalis was applied in a prospective study of the vaginal microbiome of women with preterm premature rupture of membranes (PPROM). The objectives were to characterize the vaginal microbiota of women following PPROM, and to determine if microbiome composition at the time of rupture predicts latency duration and perinatal outcomes. Only 13/70 samples collected from 36 women were dominated by Lactobacillus spp., the expected profile for healthy women, while Megasphaera type 1 and Prevotella spp. were detected in all samples. Microbiome profiles at the time of membrane rupture did not cluster by gestational age at PPROM, or latency duration. Microbial profiles were unstable over the latency period, with dramatic shifts in composition between weekly samples, and an overall decrease in Lactobacillus abundance. Mollicutes were detected by PCR in 81% (29/36) of women, and these women had significantly lower gestational age at delivery and correspondingly lower birth weight infants than Mollicutes negative women. Taken together, the results presented in this thesis demonstrate the value of high resolution profiling of the vaginal microbiome using cpn60 UT sequences. The resolution of subgroups within G. vaginalis has potentially significant implications for women's health diagnostics, requiring a shift away from considering G. vaginalis as a single entity. The PPROM study provides foundational information that may lead to the identification of informative sequence patterns, providing clinicians with better tools for expectant management following PPROM

Changes in Cord Blood Dendritic Cells as Biomarkers of Fetal Exposure to Stressor Stimuli

DCs are central to fetal defences and it was postulated that phenotypic changes on CBDCs in response to infectious/stressor stimuli could identify compromised fetuses. Investigations were performed on whole blood using monoclonal antibody labelling and flow cytometry. Functional studies included endocytosis of Dextran particles and MLR. Both plasmacytoid (HLA-DR⁺CD11c⁻) and myeloid (HLA-DR⁺CD11c⁺) DCs were identified in CB. Additionally CB contained a DC subset with a HLA-DR⁺CD11c⁻CD45^intermediate(inm) phenotype. This population expressed lower levels of CD45 and HLA-DR and did not express plasmacytoid (CD123, BDCA2, and CD45RA) or myeloid (CD33 and CD13) markers. All subsets exhibited endocytosis and unlabelled CBDCs exhibited lymphocytic stimulatory capacity. Both myeloid and plasmacytoid CBDC subsets showed no change with gestation. The CD11c⁻ CD45^inm subset decreased with increasing gestation representing 31.33% of total DCs in preterm, 21.26% in term CB and 1.54% in adult PB. CD11c⁻CD45inm DC numbers expressing CD40, CD86 and production of IL-12 increased significantly with stressors. The myeloid and the plasmacytoid subsets showed no upregulation of CD40 and CD86 with stressors. The myeloid subset decreased while the plasmacytoid subset increased IL-12 production with stressors. Neutrophilic activation markers of CD11b and CD16 showed significant correlation with the stressed CB samples which exhibited proinflammatory DC responses, thus validating the clinical classification. These data indicate that CB contains plasmacytoid and myeloid DC populations as seen in adult PB. Additionally this study has identified a hitherto unreported CBDC subset with an immature phenotype, exhibiting endocytosis and phenotypically distinct from plasmacytoid DCs. Of the three subsets, only the CD11c⁻CD45^inm subset showed a costimulatory response to stressors suggesting this subset to be the most kinetic; changing with advancing gestation as well as exposure to stressors. Thus investigating phenotypic changes on CBDC subsets, especially on the CD11c⁻CD45^inm subset, could serve to identify fetuses exposed to stressor stimuli and at risk of adverse sequelae.Open Acces

The reproductive tract microbiota and miscarriage

Early miscarriage (pregnancy loss before 12 weeks) occurs in 20 % of pregnancies of which half are due to aneuploidy. However, the mechanisms that drive euploid miscarriage are poorly understood and, despite its prevalence, there are no interventions that prevent sporadic miscarriage. Emerging evidence supports the role of the vaginal microbiota in adverse pregnancy outcome. Our group has previously shown that miscarriage is associated with vaginal dysbiosis but the cytogenetic status of those miscarriages was unknown. The existence and origin of an early pregnancy placental niche and the interaction between the rectal and vaginal microbiota in early pregnancy loss has not been addressed. Bacterial 16S rRNA gene based metataxonomic analysis was used to interrogate the vaginal microbiota in women who miscarried compared to term healthy pregnancies. The same approach was used to compare the trophoblast and rectal microbiota of miscarriage patients with termination of pregnancy patient controls. I have shown that, compared to aneuploid miscarriage, euploid miscarriage is associated with a significantly higher prevalence of Lactobacillus spp. deplete vaginal microbial communities. In women with Lactobacillus spp. deplete vaginal microbial communities, euploid miscarriage associates with higher concentrations of pro-inflammatory cytokines (measured using the Human Magnetic Luminex Screening 8-plex Assay) when compared to viable term pregnancy. Prevotella bivia and Streptococcus were identified as particularly common in euploid miscarriage and as drivers of proinflammatory cytokines. This shows that it is a combination of an adverse vagina microbiota and the maternal local immune response to it that predicts miscarriage. I have also demonstrated that there is a bacterial signal in trophoblast above the background contamination control, but it was difficult to account for cross contamination via the vaginal niche due to sample collection methods. In those that had an adverse vaginal microbiota significant rectal dysbiosis was seen in euploid compared to aneuploid miscarriage. Furthermore, a healthy gut microbiome appears to dampen the cervicovaginal immune response to Lactobacillus spp. deplete vaginal microbiota. Overall the findings presented in this thesis support the hypothesis that the vaginal microbiota plays an important aetiological role in euploid miscarriage and that the rectal or gut microbiota may modulate the inflammatory reaction to vaginal dysbiosis. These findings suggest that modulation of the vaginal and or rectal/gut microbiota, using prebiotics or live biotherapeutics may reduce the risk of miscarriage.Open Acces