Cervicovaginal fluid acetate: a metabolite marker of preterm birth in symptomatic pregnant women

Changes in vaginal microbiota that is associated with preterm birth (PTB) leave specific metabolite fingerprints that can be detected in the cervicovaginal fluid (CVF) using metabolomics techniques. In this study, we characterize and validate the CVF metabolite profile of pregnant women presenting with symptoms of threatened preterm labor (PTL) by both 1H-nuclear magnetic resonance spectroscopy (NMR) and enzyme-based spectrophotometry. We also determine their predictive capacity for PTB, singly, and in combination, with current clinical screening tools – cervicovaginal fetal fibronectin (FFN) and ultrasound cervical length (CL). CVF was obtained by high-vaginal swabs from 82 pregnant women with intact fetal membranes presenting between 24 and 36 weeks gestation with symptoms of threatened, but not established, PTL. Dissolved CVF samples were scanned with a 400 MHz NMR spectrometer. Acetate and other metabolites were identified in the NMR spectrum, integrated for peak area, and normalized to the total spectrum integral. To confirm and validate our observations, acetate concentrations (AceConc) were also determined from a randomly-selected subset of the same samples (n = 57), by spectrophotometric absorption of NADH using an acetic acid assay kit. CVF FFN level, transvaginal ultrasound CL, and vaginal pH were also ascertained. Acetate normalized integral and AceConc were significantly higher in the women who delivered preterm compared to their term counterparts (P = 0.002 and P = 0.006, respectively). The 1H-NMR-derived acetate integrals were strongly correlated with the AceConc estimated by spectrophotometry (r = 0.69; P 0.53 g/l), and of delivery within 2 weeks of the index assessment (acetate integral: AUC = 0.77, 95% CI = 0.58–0.96; AceConc: AUC = 0.68, 95% CI = 0.5–0.9). The predictive accuracy of CVF acetate was similar to CL and FFN. The combination of CVF acetate, FFN, and ultrasound CL in a binary logistic regression model improved the prediction of PTB compared to the three markers individually, but CVF acetate offered no predictive improvement over ultrasound CL combined with CVF FFN. Elevated CVF acetate in women with symptoms of PTL appears predictive of preterm delivery, as well as delivery within 2 weeks of presentation. An assay of acetate in CVF may prove of clinical utility for predicting PTB

Comparison of Storage Conditions for Human Vaginal Microbiome Studies

BACKGROUND: The effect of storage conditions on the microbiome and metabolite composition of human biological samples has not been thoroughly investigated as a potential source of bias. We evaluated the effect of two common storage conditions used in clinical trials on the bacterial and metabolite composition of the vaginal microbiota using pyrosequencing of barcoded 16S rRNA gene sequencing and (1)H-NMR analyses. METHODOLOGY/PRINCIPAL FINDINGS: Eight women were enrolled and four mid-vaginal swabs were collected by a physician from each woman. The samples were either processed immediately, stored at -80°C for 4 weeks or at -20°C for 1 week followed by transfer to -80°C for another 4 weeks prior to analysis. Statistical methods, including Kolmogorovo-Smirnov and Wilcoxon tests, were performed to evaluate the differences in vaginal bacterial community composition and metabolites between samples stored under different conditions. The results showed that there were no significant differences between samples processed immediately after collection or stored for varying durations. (1)H-NMR analysis of the small molecule metabolites in vaginal secretions indicated that high levels of lactic acid were associated with Lactobacillus-dominated communities. Relative abundance of lactic acid did not appear to correlate with relative abundance of individual Lactobacillus sp. in this limited sample, although lower levels of lactic acid were observed when L. gasseri was dominant, indicating differences in metabolic output of seemingly similar communities. CONCLUSIONS/SIGNIFICANCE: These findings benefit large-scale, field-based microbiome and metabolomic studies of the vaginal microbiota

Analyses of the Microbial Diversity across the Human Microbiome

Analysis of human body microbial diversity is fundamental to understanding community structure, biology and ecology. The National Institutes of Health Human Microbiome Project (HMP) has provided an unprecedented opportunity to examine microbial diversity within and across body habitats and individuals through pyrosequencing-based profiling of 16 S rRNA gene sequences (16 S) from habits of the oral, skin, distal gut, and vaginal body regions from over 200 healthy individuals enabling the application of statistical techniques. In this study, two approaches were applied to elucidate the nature and extent of human microbiome diversity. First, bootstrap and parametric curve fitting techniques were evaluated to estimate the maximum number of unique taxa, Smax, and taxa discovery rate for habitats across individuals. Next, our results demonstrated that the variation of diversity within low abundant taxa across habitats and individuals was not sufficiently quantified with standard ecological diversity indices. This impact from low abundant taxa motivated us to introduce a novel rank-based diversity measure, the Tail statistic, (“τ”), based on the standard deviation of the rank abundance curve if made symmetric by reflection around the most abundant taxon. Due to τ’s greater sensitivity to low abundant taxa, its application to diversity estimation of taxonomic units using taxonomic dependent and independent methods revealed a greater range of values recovered between individuals versus body habitats, and different patterns of diversity within habitats. The greatest range of τ values within and across individuals was found in stool, which also exhibited the most undiscovered taxa. Oral and skin habitats revealed variable diversity patterns, while vaginal habitats were consistently the least diverse. Collectively, these results demonstrate the importance, and motivate the introduction, of several visualization and analysis methods tuned specifically for next-generation sequence data, further revealing that low abundant taxa serve as an important reservoir of genetic diversity in the human microbiome

Differential CD4+ cell count increase and CD4+ : CD8+ ratio normalization with maraviroc compared with tenofovir

Studies exploring the immunologic effects of maraviroc (MVC) have produced mixed results; hence it remains unclear whether MVC has unique immunologic effects in comparison to other antiretroviral drugs. We sought to determine whether MVC has differential effects compared to tenofovir disoproxil fumarate (TDF) during initial antiretroviral therapy

Bacterial Communities of the Coronal Sulcus and Distal Urethra of Adolescent Males

Lactobacillus-dominated vaginal microbiotas are associated with reproductive health and STI resistance in women, whereas altered microbiotas are associated with bacterial vaginosis (BV), STI risk and poor reproductive outcomes. Putative vaginal taxa have been observed in male first-catch urine, urethral swab and coronal sulcus (CS) specimens but the significance of these observations is unclear. We used 16 S rRNA sequencing to characterize the microbiota of the CS and urine collected from 18 adolescent men over three consecutive months. CS microbiotas of most participants were more stable than their urine microbiotas and the composition of CS microbiotas were strongly influenced by circumcision. BV-associated taxa, including Atopobium, Megasphaera, Mobiluncus, Prevotella and Gemella, were detected in CS specimens from sexually experienced and inexperienced participants. In contrast, urine primarily contained taxa that were not abundant in CS specimens. Lactobacilllus and Streptococcus were major urine taxa but their abundance was inversely correlated. In contrast, Sneathia, Mycoplasma and Ureaplasma were only found in urine from sexually active participants. Thus, the CS and urine support stable and distinct bacterial communities. Finally, our results suggest that the penis and the urethra can be colonized by a variety of BV-associated taxa and that some of these colonizations result from partnered sexual activity

High Level of Soluble HLA-G in the Female Genital Tract of Beninese Commercial Sex Workers Is Associated with HIV-1 Infection

Most HIV infections are transmitted across mucosal epithelium. Understanding the role of innate and specific mucosal immunity in susceptibility or protection against HIV infection, as well as the effect of HIV infection on mucosal immunity, are of fundamental importance. HLA-G is a powerful modulator of the immune response. The aim of this study was to investigate whether soluble HLA-G (sHLA-G) expression in the female genital tract is associated with HIV-1 infection.Genital levels of sHLA-G were determined in 52 HIV-1-uninfected and 44 antiretroviral naïve HIV-1-infected female commercial sex workers (CSWs), as well as 71 HIV-1-uninfected non-CSW women at low risk of exposure, recruited in Cotonou, Benin. HIV-1-infected CSWs had higher genital levels of sHLA-G compared with those in both the HIV-1-uninfected CSW (P = 0.009) and non-CSW groups (P = 0.0006). The presence of bacterial vaginosis (P = 0.008), and HLA-G*01:01:02 genotype (P = 0.002) were associated with higher genital levels of sHLA-G in the HIV-1-infected CSWs, whereas the HLA-G*01:04:04 genotype was also associated with higher genital level of sHLA-G in the overall population (P = 0.038). When adjustment was made for all significant variables, the increased expression of sHLA-G in the genital mucosa remained significantly associated with both HIV-1 infection (P = 0.02) and bacterial vaginosis (P = 0.03).This study demonstrates that high level of sHLA-G in the genital mucosa is independently associated with both HIV-1 infection and bacterial vaginosis

Psychosocial Stress, Cortisol Levels, and Maintenance of Vaginal Health

Stress stimuli are ubiquitous and women do not enjoy any exemptions. The physiologic “fight-or-flight” response may be deleterious to the female lower genital tract microbiome if the stress stimuli persist for longer than necessary. Persistent exposure to psychosocial stress and stimulation of the hypothalamic-pituitary-adrenal (HPA) and sympathetic-adrenal-medullary (SAM) axes, and associated hormones are risk factors for several infections including genitourinary tract infections. Though this could be due to a dysregulated immune response, a cortisol-induced inhibition of vaginal glycogen deposition may be involved especially in the instance of vaginal infection. The estrogen-related increased vaginal glycogen and epithelial maturation are required for the maintenance of a healthy vaginal ecosystem (eubiosis). The ability of cortisol to disrupt this process as indicated in animal models is important in the pathogenesis of vaginal dysbiosis and the subsequent development of infection and inflammation. This phenomenon may be more crucial in pregnancy where a healthy Lactobacillus-dominated vaginal microbiota is sacrosanct, and there is local production of more corticotropin-releasing hormone (CRH) from the decidua, fetal membranes and placenta. To highlight the relationship between the stress hormone cortisol and the vaginal microbiomial architecture and function, the potential role of cortisol in the maintenance of vaginal health is examined