Search

Lactobacillus-deficient cervicovaginal microbiota, including Gardnerella vaginalis, are implicated in cervical remodeling and preterm birth. Mechanisms by which microbes drives outcomes are not fully elucidated. We hypothesize that Gardnerella vaginalis induces matrix metalloproteinases through TLR-2, leading to epithelial barrier dysfunction and premature cervical remodeling. Cervicovaginal cells were treated with live Gardnerella vaginalis or Lactobacillus crispatus or their bacteria-free supernatants for 24 h. For TLR-2 experiments, cells were pretreated with TLR-2 blocking antibody. A Luminex panel was run on cell media. For human data, we conducted a case-control study from a prospective pregnancy cohort of Black individuals with spontaneous preterm (sPTB) (n = 40) or term (n = 40) births whose vaginal microbiota had already been characterized. Cervicovaginal fluid was obtained between 20 and 24 weeks' gestation. Short cervix was defined as < 25 mm by second trimester transvaginal ultrasound. MMP-9 was quantified by ELISA. Standard analytical approaches were used to determine differences across in vitro conditions, as well as MMP-9 and associations with clinical outcomes. Gardnerella vaginalis induced MMP-1 in cervical cells (p = 0.01) and MMP-9 in cervical and vaginal (VK2) cells (p ≤ 0.001 for all). TLR-2 blockade mitigated MMP-9 induction by Gardnerella vaginalis. MMP-9 in cervicovaginal fluid is higher among pregnant individuals with preterm birth, short cervix, and Lactobacillus-deficient microbiota (p < 0.05 for all). MMP-9 is increased in the cervicovaginal fluid of pregnant individuals with subsequent sPTB. Our in vitro work ascribes a potential mechanism by which a cervicovaginal microbe, commonly associated with adverse pregnancy outcomes, may disrupt the cervicovaginal epithelial barrier and promote premature cervical remodeling in spontaneous preterm birth., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Specific bacteria of the human microbiome influence carcinogenesis at diverse anatomical sites. Bacterial vaginosis (BV) is the most common vaginal disorder in premenopausal women that is associated with gynecologic sequelae, including cervical cancer. BV-associated microorganisms, such as Fusobacterium, Lancefieldella, Peptoniphilus, and Porphyromonas have been associated with gynecologic and other cancers, though the pro-oncogenic mechanisms employed by these bacteria are poorly understood. Here, we integrated a multi-omics approach with our three-dimensional (3-D) cervical epithelial cell culture model to investigate how understudied BV-associated bacteria linked to gynecologic neoplasia influence hallmarks of cancer in vitro. Lancefieldella parvulum and Peptoniphilus lacrimalis elicited robust proinflammatory responses in 3-D cervical cells. Fusobacterium nucleatum and Fusobacterium gonidiaformans modulated metabolic hallmarks of cancer corresponding to accumulation of 2-hydroxyglutarate, pro-inflammatory lipids, and signs of oxidative stress and genotoxic hydrogen sulfide. This study provides mechanistic insights into how gynecologic cancer-associated bacteria might facilitate a tumor-promoting microenvironment in the human cervix., (© 2022. The Author(s).)

Introduction: Gardnerella vaginalis (GV)-associated bacterial vaginosis is recognised for its detrimental effects on pregnancy resulting in poor obstetric and neonatal outcomes. There is limited knowledge of the effects on placental histomorphology following GV infection in pregnancy. We investigated the effects of GV infection on the placenta, particularly with regards to the syncytiotrophoblasts and vascular development, and related these to neonatal outcomes., Methods: A prospective cohort study involving GV-positive pregnant women presented with abnormal vaginal discharge, with gestational age-matched healthy pregnant women controls. Placental sampling was performed upon delivery and examined histologically. Vascular endothelial growth factor-A (VEGF-A) and hypoxia-inducible factor-1α (HIF-1α) mRNA and protein expression were analysed by real-time PCR and immunohistochemistry respectively. The standard measures in neonatal outcomes were recorded., Results: Placentas from GV-positive mothers were found to have significant histological evidence of maternal and/or fetal inflammatory response compared with the controls (17/28: 60.7% vs 2/20: 10%) (p = 0.0011). There was an increase in the percentage of syncytial nuclear aggregates (SNAs) per villus (47.4 ± 11.09%) in placentas from GV-positive mothers (p < 0.0001). VEGF-A was significantly increased in specifically, the villous endothelial cells of placentas with GV infection, but no difference in the immunoexpression of HIF-1α in these cells between groups. However, these were not associated with adverse neonatal outcomes., Discussion: Increased placental VEGF-A expression associated with increased SNAs in pregnant women with GV infection of the genital tract may be an intrauterine response towards placental vascular remodeling, that may also serve as a protective role in moderating birth outcomes., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Aims: To compare the cervicovaginal levels of human beta defensin (hBD)-1, 2 and 3 of women according to the status of Nugent-defined bacterial vaginosis (BV)., Methods: A total of 634 women of reproductive age were included in the study. Participants were equally distributed in two groups: according to the classification of vaginal smears according to Nugent criteria in normal (scores 0 to 3) and BV (scores ≥7). Cervicovaginal fluid samples were used for measurements of hBDs1, 2 and 3 levels by enzyme-linked immunosorbent assay (ELISA). Levels of each hBD were compared between the two study groups using Mann-Whitney test, with p-value <0.05 considered as significant. Odds ratio (OR) and 95% confidence interval (95% CI) were calculated for sociodemographic variables and hBD1-3 levels associated with BV a multivariable analysis. Correlation between Nugent score and measured levels of hBDs1-3 were calculated using Spearman's test., Results: Cervicovaginal fluids from women with BV showed lower levels of hBD-1 [median 2,400.00 pg/mL (0-27,800.00); p<0.0001], hBD-2 [5,600.00 pg/mL (0-45,800.00); p<0.0001] and hBD-3 [1,600.00 pg/mL (0-81,700.00); p = 0.012] when compared to optimal microbiota [hBD-1: [median 3,400.00 pg/mL (0-35,600.00), hBD-2: 12,300.00 pg/mL (0-95,300.00) and hBD-3: 3,000.00 pg/mL (0-64,300.00), respectively]. Multivariable analysis showed that lower levels of hBD-1 (OR: 2.05; 95% CI: 1.46-2.87), hBD-2 (OR: 1.85; 95% CI: 1.32-2.60) and hBD-3 (OR: 1.90; 95% CI: 1.37-2.64) were independently associated BV. Significant negative correlations were observed between Nugent scores and cervicovaginal levels of hBD-1 (Spearman's rho = -0.2118; p = 0.0001) and hBD-2 (*Spearman's rho = -0.2117; p = 0.0001)., Conclusions: Bacterial vaginosis is associated with lower cervicovaginal levels of hBDs1-3 in reproductive-aged women., Competing Interests: The authors have declared that no competing interests exist.

Bacterial vaginosis (BV) is a gynecologic disorder characterized by a shift in cervicovaginal microbiota from Lactobacillus spp. dominance to a polymicrobial biofilm composed of diverse anaerobes. We utilized a well-characterized human three-dimensional cervical epithelial cell model in conjunction with untargeted metabolomics and immunoproteomics analyses to determine the immunometabolic contribution of three members of the Veillonellaceae family: Veillonella atypica, Veillonella montpellierensis and Megasphaera micronuciformis at this site. We found that Veillonella spp. infections induced significant elevation of polyamines. M. micronuciformis infections significantly increased soluble inflammatory mediators, induced moderate levels of cell cytotoxicity, and accumulation of cell membrane lipids relative to Veillonella spp. Notably, both V. atypica and V. montpellierensis infections resulted in consumption of lactate, a key metabolite linked to gynecologic and reproductive health. Collectively our approach and data provide unique insights into the specific contributions of Veillonellaceae members to the pathogenesis of BV and women's health.

Background: The presence of semen in the vagina from unprotected sex may influence the immune and microbial environment of the female genital tract. Inflammatory cytokine concentrations and BV-associated bacteria in female genital secretions may influence HIV risk, although the effect of recent sexual intercourse on incident BV and the cytokine milieu of cervicovaginal secretions has rarely been measured in previous studies. Here, we investigated the extent to which partner semen impacts the cytokine response and incident BV., Methods: At baseline, we assessed the recency of semen exposure in menstrual cup supernatants by quantifying prostate specific antigen (PSA) levels using ELISA in 248 HIV-uninfected women at high risk for HIV infection. Luminex was used to measure 48 cytokines in menstrual cup supernatants and vaginal swabs to diagnose BV by Nugent score. Point-of-care screening for Chlamydia trachomatis and Neisseria gonorrhoeae was conducted using GeneXpert while OSOM was used for Trichomonas vaginalis detection. Multivariable models, adjusted for age, sexually transmitted infections, BV, current contraception use and condom use, were used to assess the impact of semen exposure on biomarkers of inflammation and BV., Results: Presence of PSA, indicating recent semen exposure within 48 hours prior to sampling, was observed in menstrual cup supernatants of 17% (43/248) of women. Of these women, 70% (30/43) had self-reported condom use at their last sex act and 84% (36/43) had BV (Nugent score >7). PSA presence was significantly associated with prevalent BV (Relative Risk (RR), 2.609; 95% Confidence Interval (CI), 1.104 - 6.165; p = 0.029). Furthermore, women with detectable PSA had high median concentrations of macrophage inflammatory protein- beta (MIP-1α, p=0.047) and low median concentration of the stem cell growth factor beta (SCGF-β, p=0.038) compared to those without PSA., Conclusion: A degree of discordance between self-reports of consistent condom use and PSA positivity was observed. There was also evidence of a relationship between recent semen exposure, BV prevalence and altered cytokine concentrations. These findings suggest that PSA, as a semen biomarker, should be taken into consideration when investigating biological markers in the female genital tract and self-reported condom use in studies on reproductive and sexual health., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mngomezulu, Mzobe, Mtshali, Osman, Liebenberg, Garrett, Singh, Rompalo, Mindel, Karim, Karim, Baxter and Ngcapu.)

Bacterial vaginosis (BV) is a common type of vaginal inflammation caused by a proliferation of pathogenic bacteria, among which Mobiluncus curtisii. In our previous studies on M. curtisii genome, we identified the presence of a genomic fragment encoding a 25 kDa pore-forming toxin, the CAMP factor, which is known to be involved in the synergistic lysis of erythrocytes namely CAMP reaction. However, whether this hypothetical gene product has hemolytic activity is unknown. Moreover, its relative structure and function are not yet solved. Here we found that the M. curtisii CAMP factor is a monomer at pH 4.4 and oligomer at pH > 4.6. Hemolysis assays showed that M. curtisii CAMP factor could lyse sheep red blood cells efficiently in pH 5.4-7.4. Negative staining electron microscope analysis of the CAMP factor revealed ring-like structures at pH above 4.6. Additionally, the crystal structure of M. curtisii CAMP factor, determineded at 1.85 Å resolution, reveals a 5 + 3 helix motif. Further functional analysis suggested that the structural rearrangement of the N-terminal domain might be required for protein function. In conclusion, this structure-function relationship study of CAMP factor provides a new perspective of the M. curtisii role in BV development., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Up to 30% of women with vaginal symptoms are not assigned a diagnosis after standard diagnostic assessment., Methods: We compared premenopausal women with idiopathic vaginitis (IV) or vulvodynia (VVD) to healthy controls. Microbiota were characterized using rRNA sequencing. Cytokines/chemokines (IL-10, IL-1α, IL-1β, IL-6, IL-8, IL-2, IL-18, IL-4, IL-9, and IL-13) were measured in vaginal lavage fluid using the Meso Scale Discovery platform or ELISA (IL-1ra). Immunoglobulins were measured in vaginal lavage fluid using a bead-based immunoassay (Millipore). Cases and controls were compared using Kruskal-Wallis, analysis of variance, and linear regression or (for microbiome composition) the Bray-Curtis dissimilarity statistic., Results: We compared 20 women with IV, 30 with VVD, and 52 controls. Most (80%) had greater than 90% 16S rRNA gene sequences from Lactobacillus crispatus, L. jensenii, L. gasseri, or L. iners. In analyses adjusted for age and hormonal contraception (HC), Gardnerella vaginalis was less prevalent and abundant in women with VVD (2/30, 7%) versus controls (16/52, 31%) or IV (5/20, 25%) (P = 0.030). Bray-Curtis dissimilarity was not significantly different between IV and controls or VVD. Fungal sequences were only detected in 5 participants: 2 control, 1 IV, 2 VVD. In univariate analysis, cytokines were not associated with diagnosis. Median vaginal concentration of IgE (but not other immunoglobulins) was lower in women with VVD (P = 0.006)., Conclusions: Minimal differences in vaginal microbiota and inflammatory markers between women with IV, VVD or controls suggest no striking association between vaginal bacteria, fungi or inflammation and diagnosis in these women.

In recent studies, the interleukin (IL)-36 cytokines were shown to be elevated in women with non-Lactobacillus-dominated vaginal microbiomes. In this study, we evaluated IL36G expression in clinical samples from women with and without bacterial vaginosis (BV) and a human 3-dimensional cervical epithelial cell model. IL36G expression was significantly elevated in cervicovaginal epithelial cells isolated from BV-positive women and corresponded with increased neutrophil counts relative to BV-negative women. In addition, specific BV-associated bacterial species as well as a polymicrobial cocktail significantly induced IL36G expression in vitro. These findings suggest that IL-36γ may exhibit an important function in the host response to BV and other sexually transmitted infections., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Chlamydia trachomatis (CT) and Mycoplasma genitalium (MG) are two highly prevalent bacterial sexually transmitted infections (STIs) with a significant rate of co-infection in some populations. Vaginal metabolites are influenced by resident vaginal microbiota, affect susceptibility to sexually transmitted infections (STIs), and may impact local inflammation and patient symptoms. Examining the vaginal metabolome in the context of CT mono (CT+) and CT/MG co-infection (CT+/MG+) may identify biomarkers for infection or provide new insights into disease etiology and pathogenesis. Yet, the vaginal metabolome in the setting of CT infection is understudied and the composition of the vaginal metabolome in CT/MG co-infected women is unknown. Therefore, in this analysis, we used an untargeted metabolomic approach combined with 16S rRNA gene amplicon sequencing to characterize the vaginal microbiota and metabolomes of CT+, CT+/MG+, and uninfected women. We found that CT+ and CT+/MG+ women had distinct vaginal metabolomic profiles as compared to uninfected women both before and after adjustment for the vaginal microbiota. This study provides important foundational data documenting differences in the vaginal metabolome between CT+, CT+/MG+ and uninfected women. These data may guide future mechanistic studies that seek to provide insight into the pathogenesis of CT and CT/MG infections.

Bacterial vaginosis (BV) is a vaginal anaerobic dysbiosis that affects women of reproductive age worldwide. BV is microbiologically characterized by the depletion of vaginal lactobacilli and the overgrowth of anaerobic bacterial species. Accumulated evidence suggests that Gardnerella spp. have a pivotal role among BV-associated bacteria in the initiation and development of BV. However, Gardnerella spp. often colonize healthy women. Lactobacillus iners is considered as a prevalent constituent of healthy vaginal microbiota, and is abundant in BV. Gardnerella spp. and L. iners secrete the toxins vaginolysin (VLY) and inerolysin (INY), which have structural and activity features attributed to cholesterol-dependent cytolysins (CDCs). CDCs are produced by many pathogenic bacteria as virulence factors that participate in various stages of disease progression by forming lytic and non-lytic pores in cell membranes or via pore-independent pathways. VLY is expressed in the majority of Gardnerella spp. isolates; less is known about the prevalence of the gene that encodes INY. INY is a classical CDC; membrane cholesterol acts a receptor for INY. VLY uses human CD59 as its receptor, although cholesterol remains indispensable for VLY pore-forming activity. INY-induced damage of artificial membranes is directly dependent on cholesterol concentration in the bilayer, whereas VLY-induced damage occurs with high levels of membrane cholesterol (>40 mol%). VLY primarily forms membrane-embedded complete rings in the synthetic bilayer, whereas INY forms arciform structures with smaller pore sizes. VLY activity is high at elevated pH, which is characteristic of BV, whereas INY activity is high at more acidic pH, which is specific for a healthy vagina. Increased VLY levels in vaginal mucosa in vivo were associated with clinical indicators of BV. However, experimental evidence is lacking for the specific roles of VLY and INY in BV. The interplay between vaginal bacterial species affects the expression of the gene encoding VLY, thereby modulating the virulence of Gardnerella spp. This review discusses the current evidence for VLY and INY cytolysins, including their structures and activities, factors affecting their expression, and their potential impacts on the progression of anaerobic dysbiosis., (Copyright © 2020 Pleckaityte.)

Aims: To observe the therapeutic effects of vaginal infusion of probiotic Clostridium butyricum WZ001 on bacterial vaginosis (BV) in mice., Methods and Results: Female ICR mice were used to establish the model of BV by infecting oestrogen-treated mice with Escherichia coli, and then treated with high- and low dose of C. butyricum. Clinical indexes of mice in the C. butyricum-treated groups were significantly improved and comparable to those in the antibiotic group. Pap staining showed that neutrophil count was significantly increased after modelling and largely decreased after C. butyricum treatment (P < 0·01). Dynamic observation of E. coli and Lactobacillus showed that the number of E. coli significantly decreased in the C. butyricum-treated groups or in the antibiotic group with prolonged treatment (P < 0·01). Besides, the number of E. coli in the low-dose C. butyricum group was higher than that in either its high-dose counterpart or the antibiotic group respectively (P < 0·01). The number of Lactobacillus decreased evidently in the antibiotic group (P < 0·01), while that in the C. butyricum groups remained consistent. Moreover, C. butyricum inhibited the proliferation of E. coli by the experiment in vitro. The phosphorylation of nuclear factor-kappa B (NF-κB) p65 in vaginal tissue and the serum levels of inflammatory cytokines, IL-1β, TNF-α and IL-6, increased after modelling and significantly decreased after treated with C. butyricum (P < 0·01), with no difference found when compared with the antibiotic group., Conclusion: Clostridium butyricum inhibits the growth of pathogenic bacteria as well as the inflammatory response induced by E. coli and promotes the growth of Lactobacillus to maintain the vaginal micro-ecological balance., Significance and Impact of the Study: Our results suggest that probiobitc C. butyricum WZ001 has a great potential in the clinical treatment of BV., (© 2019 The Society for Applied Microbiology.)

Studies have implicated Gardnerella vaginalis as an important etiological agent in bacterial vaginosis (BV). It produces a cholesterol-dependent cytolysin, vaginolysin (VLY). In this study, we sought to characterize the interaction between vaginal epithelium, G. vaginalis , and VLY using EpiVaginal tissues from MatTek. These tissues are three-dimensional and have distinct apical and basolateral sides, enabling comparison of the effects of G. vaginalis and VLY following exposure to either side. We measured cytotoxicity, cytokine production, and bacterial growth, following apical versus basolateral exposure. G. vaginalis exhibited more-rapid growth in coculture with the tissue model when it was exposed to the apical side. VLY permeabilized cells on the basolateral side of the tissues but failed to permeabilize apical epithelial cells. Cytokine secretion in response to VLY and G. vaginalis also depended on the polarity of exposure. VLY did not cause significant changes in cytokine levels when exposed apically. Apical tissue challenge by G. vaginalis appeared to dampen the inflammatory response, as decreases in granulocyte-macrophage colony-stimulating factor (GM-CSF) (6.6-fold), RANTES (14.8-fold), and interferon gamma inducible protein 10 kDa (IP-10) (53-fold) and an increase in interleukin-1 receptor antagonist (IL-1ra) (5-fold) were observed. In vivo , G. vaginalis normally colonizes the apical face of the vaginal epithelium. Results from this study suggest that while G. vaginalis may grow on the apical face of the vaginal epithelium, its VLY toxin does not target these cells in this model. This phenomenon could have important implications regarding colonization of the vagina by G. vaginalis and may suggest an explanation for the lack of an overt immune response to this organism., (Copyright © 2019 American Society for Microbiology.)

Objectives: Vaginal dysbiosis and STIs are important drivers of the HIV epidemic and reproductive complications. These conditions remain prevalent, partly because most cases are asymptomatic. We have shown that inflammatory cytokines interleukin (IL)-1α, IL-1β and interferon-γ-induced protein (IP)-10 are biomarkers for detecting asymptomatic STIs and vaginal dysbiosis (bacterial vaginosis (BV) or intermediate microbiota). This study aimed to validate the performance of these biomarkers in African women recruited regardless of symptoms., Methods: IL-1α, IL-1β and IP-10 were measured in menstrual cup secretions, endocervical, lateral vaginal wall and vulvovaginal swabs from 550 women from Pretoria, Soweto and Cape Town, South Africa and Bondo, Kenya using Luminex and ELISA. STIs were assessed by PCR, BV by Nugent scoring and vaginal microbiota by 16S rRNA sequencing., Results: Across four study populations and four types of genital specimens, the performance of IL-1α, IL-1β and IP-10 for identification of women with STIs, BV or intermediate microbiota was consistent. Of the genital samples assessed, biomarkers measured in lateral vaginal wall swabs performed best, correctly classifying 76%(95% CI 70% to 81%) of women according to STI, BV or intermediate microbiota status (sensitivity 77%, specificity 71%) and were more accurate than clinical symptoms (sensitivity 41%, specificity 57%) (p=0.0003). Women incorrectly classified as STI/BV positive using the biomarkers had more abundant dysbiosis-associated bacteria, including Prevotella bivia and Gardnerella sp, detected by 16S rRNA sequencing, but not Nugent scoring. Including vaginal pH with the cytokine biomarkers improved the accuracy of the test (82% (95% CI 75% to 88%) correctly classified), although pH alone had poor specificity (61%)., Conclusions: An inexpensive, point-of-care screening test including IL-1α, IL-1β and IP-10 (and potentially pH) could be used in resource-limited settings to identify women with asymptomatic STIs and dysbiosis. These women could then be referred for aetiological testing, followed by specific treatment., Competing Interests: Competing interests: JASP and LM, together with the University of Cape Town, have submitted a Patent application for IP-10 and IL-1α/β use for diagnosing an inflammatory condition in the female genital tract likely caused by an STI or BV., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: This study sought to investigate associations between serum total and free 25(OH)D and bacterial vaginosis (BV) in early and later pregnancy among US black women to provide insight into the most clinically relevant measure of vitamin D status among pregnant black women with respect to risk for BV as well as insights into critical time points for measuring and/or addressing vitamin D status in pregnancy., Methods: Data and biospecimens were derived from a subsample (N = 137) of women from the Emory University African American Vaginal, Oral, and Gut Microbiome in Pregnancy Cohort, for whom data related to vitamin D status (serum assays for total and free 25(OH)D) and Nugent score of Gram stained vaginal specimens in early (8-14 weeks) and later (24-30 weeks) were available. We compared total and free 25(OH)D concentrations for women according to Nugent score category (normal flora, intermediate flora, and BV) and assessed the odds of BV according to measures of vitamin D status., Results: Thirty-seven (27%) women had adequate vitamin D status at baseline, whereas 70 (51%) had insufficient vitamin D and 30 (22%) were vitamin D deficient; there were not significant differences in the proportion of women with adequate, insufficient, or deficient vitamin D according to Nugent score category. However, the odds of BV later in pregnancy were significantly higher for women who experienced a smaller rise in total 25(OH)D and free 25(OH)D from 8-14 through 24-30 weeks gestation., Conclusion: The change in measures of vitamin D status from early to later pregnancy is associated with the occurrence of BV in pregnancy. Further research is needed to examine the association between the change in vitamin D status over pregnancy and the occurrence of BV and other measures of vaginal microbial composition as well as to identify factors that influence change in vitamin D status over pregnancy.

Objectives: The aim of the study was to compare, using a proteomic approach, cervicovaginal fluid (CVF) proteins of women with bacterial vaginosis (BV) with those presenting normal microbiota., Materials and Methods: A total of 309 reproductive-aged women were cross-sectionally enrolled. Participants were tested for vaginal candidosis, Trichomonas vaginalis, Chlamydia trachomatis, and Neisseria gonorrhoeae and excluded if positive. Vaginal microbiota was classified microscopically according to Nugent criteria in normal, intermediate, and BV. Randomly selected CVF samples of 29 women with BV and an equal number with normal microbiota were subjected to proteomic analysis. Thus, a total of 58 CVF samples were evaluated using shotgun liquid chromatography-tandem mass spectrometry in a Q-Tof PREMIER API mass spectrometer (MicroMass/Waters) for peptide detection and relative quantification., Results: Of the 309 women enrolled, 63 (20.4%) were excluded after testing positive for at least one of the tested co-infections or because of low-quality samples. Microscopic classification of vaginal microbiota on the remaining 246 samples revealed that 132 women (53.6%) had normal microbiota, 33 (13.4%) had intermediate microbiota, and 81 (33.0%) had BV. Proteomic analysis of CVF of 58 randomly selected women with normal microbiota (n = 29) or BV (n = 29) successfully identified 74 proteins. In addition, the comparison of abundance of those proteins between the groups showed that the following five (6.7%) were enriched in BV: neutrophil elastase, kaliocin-1, neutrophil defensin-1, Ig lambda-2 chain C regions, and protein S100-A7. All of which have a recognized role in host's immunity., Conclusions: Exclusive finding of BV affects immunity-related CVF components of reproductive-aged women.

Aim: The main goal of this work was to highlight the significance of redox imbalance in the pathophysiology of bacterial vaginosis (BV). We studied the pro-oxidant (malondialdehyde) and antioxidants (glutathione, total antioxidant capacity) in the vaginal fluids of women and compared them on the basis of their Nugent score (NS)., Methods: Women were clinically screened using Amsel criteria (≥2 were regarded as positive) and were further screened for NS on the basis of microscopic examination. Subjects were classified into one of three groups - healthy controls, intermediate, and BV - on the basis of NS (0-3, 4-6, and 7-10, respectively). High vaginal swabs were collected from the study participants in order to estimate the levels of pro and antioxidants in the vaginal fluids., Results: Our results indicated that levels of both pro- and antioxidants were elevated in high vaginal swabs of women in the intermediate (NS: 4-6) and BV (NS: 7-10) groups as compared to those of healthy control women. The difference in mean values for total antioxidant capacity and glutathione was found to be statistically significant. Furthermore, in the BV group (NS: ≥7) both antioxidants (glutathione and total antioxidant capacity) and the pro-oxidant, malondialdehyde, were found to be negatively correlated to NS. Interestingly, the correlation between NS and malondialdehyde was statistically significant., Conclusion: Our results suggest a significant correlation between redox imbalance and NS, which signifies changes in vaginal ecology from normal flora (Lactobacillus spp.) towards a more mixed bacterial population representing BV., (© 2017 Japan Society of Obstetrics and Gynecology.)

The innate defense system of the female mucosal genital tract involves a close and complex interaction among the healthy vaginal microbiota, different cells, and various proteins that protect the host from pathogens. Vaginal lactobacilli and lactoferrin represent two essential actors in the vaginal environment. Lactobacilli represent the dominant bacterial species able to prevent facultative and obligate anaerobes outnumber in vaginal microbiota maintaining healthy microbial homeostasis. Several mechanisms underlie the protection exerted by lactobacilli: competition for nutrients and tissue adherence, reduction of the vaginal pH, modulation of immunity, and production of bioactive compounds. Among bioactive factors of cervicovaginal mucosa, lactoferrin, an iron-binding cationic glycoprotein, is a multifunctional glycoprotein with antibacterial, antifungal, antiviral, and antiparasitic activities, recently emerging as an important modulator of inflammation. Lactobacilli and lactoferrin are largely under the influence of female hormones and of paracrine production of various cytokines. Lactoferrin is strongly increased in lower genital tract mucosal fluid of women affected by Neisseria gonorrheae, Chlamydia trachomatis , and Trichomonas vaginalis infections promoting both innate and adaptive immune responses. In vaginal dysbiosis characterized by low amounts of vaginal lactobacilli and increased levels of endogenous anaerobic bacteria, the increase in lactoferrin could act as an immune modulator assuming the role normally played by the healthy microbiota in vaginal mucosa. Then lactoferrin and lactobacilli may be considered as biomarkers of altered microbial homeostasis at vaginal level. Considering the shortage of effective treatments to counteract recurrent and/or antibiotic-resistant bacterial infections, the intravaginal administration of lactobacilli and lactoferrin could be a novel efficient therapeutic strategy and a valuable tool to restore mucosal immune homeostasis.

Objective: To investigate the association of sexual debut and vaginal, anorectal, and oral microbiota and vaginal inflammatory markers in female adolescents., Methods: We conducted a school-based study in adolescents in Antwerp, Belgium. During three visits over 8 months, participants answered questionnaires and self-collected vaginal, anorectal, and oral swabs. Five Lactobacillus species, Lactobacillus genus, Gardnerella vaginalis, and Atopobium vaginae were quantified; and seven inflammatory markers were measured in the vaginal specimens. In the oral and anorectal specimens, Lactobacillus genus, G vaginalis, and A vaginae were ascertained., Results: Of the 93 adolescents (mean age 16.2 years) at the first visit, 41 (44.1%) had passed sexual debut (penile-vaginal intercourse) and five (5.4%) had sexual experience without passing sexual debut. Having sexual experience at the first visit was not found to be associated with species presence or concentrations (acknowledging an underpowered study because the required sample size was not attained). Modeling the longitudinal data on all girls showed that sexual debut was associated with increased odds of vaginal and anorectal G vaginalis (P=.021; P=.030) and A vaginae (P=.041; P=.012) with increments of interleukins (interleukin [IL]-1α P<.001, IL-1β P=.046, IL-8 P=.033) and chemokines (regulated on activation, normal T cell expressed and secreted P<.001; macrophage inflammatory protein-1β P=.040), whereas no difference was seen when modeling (before-after) the girls initiating and girls staying without sexual intercourse. The association of sexual intercourse with IL-1α (P<.001), IL-1β (P=.030), and IL-8 (P=.002) at the first visit was (greater than 70%) mediated by vaginal G vaginalis and A vaginae concentrations., Conclusion: Sexual debut in adolescents is associated with an inflammatory vaginal reaction and with the presence of bacterial vaginosis-related species. Strategies preventing the colonization of bacterial vaginosis-related organisms during early sexual debut are urgently needed and may prevent acquisition of sexually transmitted infections including human immunodeficiency virus in early life.

Efforts to develop vaccines that can elicit mucosal immune responses in the female genital tract against sexually transmitted infections have been hampered by an inability to measure immune responses in these tissues. The differential expression of adhesion molecules is known to confer site-dependent homing of circulating effector T cells to mucosal tissues. Specific homing molecules have been defined that can be measured in blood as surrogate markers of local immunity (e.g. α4β7 for gut). Here we analyzed the expression pattern of adhesion molecules by circulating effector T cells following mucosal infection of the female genital tract in mice and during a symptomatic episode of vaginosis in women. While CCR2, CCR5, CXCR6 and CD11c were preferentially expressed in a mouse model of Chlamydia infection, only CCR5 and CD11c were clearly expressed by effector T cells during bacterial vaginosis in women. Other homing molecules previously suggested as required for homing to the genital mucosa such as α4β1 and α4β7 were also differentially expressed in these patients. However, CD11c expression, an integrin chain rarely analyzed in the context of T cell immunity, was the most consistently elevated in all activated effector CD8+ T cell subsets analyzed. This molecule was also induced after systemic infection in mice, suggesting that CD11c is not exclusive of genital tract infection. Still, its increase in response to genital tract disorders may represent a novel surrogate marker of mucosal immunity in women, and warrants further exploration for diagnostic and therapeutic purposes.

ITALIC! Staphylococcus aureusbiofilms can be found on vaginal epithelia, secreting toxins and causing inflammation. The co-vaginal species ITALIC! Lactobacilluscan alter staphylococcal-induced epithelial secretion of inflammatory cytokines and quench staphylococcal toxic shock syndrome toxin-1 secretion. It is hypothesized that these effects of lactobacilli require direct physical contact between lactobacilli, staphylococci and the epithelium. Indeed, lactobacilli only reduced ITALIC! S. aureus-induced inflammatory cytokine expression when allowed physical contact with vaginal epithelial cells. Furthermore, a reduction in toxic shock syndrome toxin-1 secretion only occurred when a probiotic ITALIC! Lactobacillusstrain was allowed contact, but not when being physically separated from ITALIC! S. aureus Bacterial-probe atomic force microscopy demonstrated that lactobacilli and staphylococci strongly adhere to epithelial cells, while lactobacilli adhere stronger to staphylococci than staphylococci to each other, giving lactobacilli opportunity to penetrate and reside in staphylococcal biofilms, as visualized using confocal laser scanning microscopy with fluorescence ITALIC! in situhybridization probes. These results identify that physical contact and biochemical signaling by lactobacilli are intrinsically linked mechanisms that reduce virulence of ITALIC! S. aureusbiofilm., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: Do metabolites in vaginal samples vary between women with different vaginal disorders., Design: Cross-sectional study., Setting: Campinas, Brazil., Sample: Seventy-seven women (39.9%) with no vaginal disorder, 52 women (26.9%) with vulvovaginal candidiasis (VVC), 43 women (22.3%) with bacterial vaginosis (BV), and 21 women (10.9%) with cytolytic vaginosis (CTV)., Method: Concentrations of D- and L-lactic acid, extracellular matrix metalloproteinase inducer (EMMPRIN), and matrix metalloproteinase-8 (MMP-8), and the influence of Candida albicans on EMMPRIN production by cultured vaginal epithelial cells, were determined by enzyme-linked immunosorbent assay (ELISA). Associations were determined by the Mann-Whitney U-test and by Spearman's rank correlation test., Main Outcome Measures: Metabolite levels and their correlation with diagnoses., Results: Vaginal concentrations of D- and L-lactic acid were reduced from control levels in BV (P < 0.0001); L-lactic acid levels were elevated in CTV (P = 0.0116). EMMPRIN and MMP-8 concentrations were elevated in VVC (P < 0.0001). EMMPRIN and L-lactic acid concentrations (P ≤ 0.008), but not EMMPRIN and D-lactic acid, were correlated in all groups. EMMPRIN also increased in proportion with the ratio of L- to D-lactic acid in controls and in women with BV (P ≤ 0.009). Concentrations of EMMPRIN and MMP-8 were correlated in controls and women with VVC (P ≤ 0.0002). Candida albicans induced EMMPRIN release from vaginal epithelial cells., Conclusions: Vaginal secretions from women with BV are deficient in D- and L-lactic acid, women with VVC have elevated EMMPRIN and MMP-8 levels, and women with CTV have elevated L-lactic acid levels. These deviations may contribute to the clinical signs, symptoms, and sequelae that are characteristic of these disorders., (© 2014 Royal College of Obstetricians and Gynaecologists.)

Objective: Neutrophil gelatinase-associated lipocalin (NGAL) is a component of innate immunity that prevents iron uptake by microorganisms. We evaluated whether NGAL was present in vaginal fluid and whether concentrations were altered in women with bacterial vaginosis (BV) or vulvovaginal candidiasis (VVC)., Methods: Vaginal secretions from 52 women with VVC, 43 with BV, and 77 healthy controls were assayed by enzyme-linked immunosorbent assay for NGAL and for concentrations of L-lactic acid., Results: The median concentration of NGAL in vaginal fluid was significantly higher in control women (561 pg/mL) than in women with BV (402 pg/mL; P = .0116) and lower in women with VVC (741 pg/mL; P = .0017). Median lactic acid levels were similar in controls (0.11 mmol/L) and women with VVC (0.13 mmol/L) and were lower in women with BV (0.02 mmol/L; P < .0001). The NGAL and lactic acid concentrations were highly correlated (P < .0001)., Conclusion: A decrease in Lactobacilli and/or lactic acid plus the absence of leukocytes results in lower vaginal NGAL levels that might facilitate the growth of bacteria associated with BV., (© The Author(s) 2015.)

Objectives: Treatment for lower genital tract infections is the major demand for gynecological services in public and private health centers in Brazil. The aims of this study were to evaluate the diagnostic resources proposed by Amsel, comparing them with the microflora evaluation by the Nugent score and thus propose a new cutoff point in this rating score, showing the complementarity of both diagnostic criteria., Methods: A total of 136 female patients aged between 18 and 69years were evaluated and had their vaginal discharge samples collected., Results: Diagnosis based on the isolated analysis of the Amsel's criteria may lead clinicians to apply inadequate treatment techniques. When patients were evaluated according to the Amsel criteria, it was seen that the presence of clue cells had a higher Kappa index in the vaginosis diagnosis; when patients were distributed according to the Nugent criteria in relation to each Amsel criterion, it was observed that clue cells differentiate positive patients more efficiently than the Nugent criteria. In the proposed cutoff point, the identification of clue cells complied with pH alterations. It also complied with the positive Nugent score (≥7). However, when clue cells were analyzed by both Amsel and Nugent methods, the diagnostic conclusion was reached once this was the parameter with a higher Kappa value., Conclusion: The Amsel method could be used as a screening tool whereas the Nugent score could serve as a confirmatory resource of diagnosis, considering a new assessment cutoff point (negative 0-6 and positive ≥7)., (Copyright © 2015. Published by Elsevier B.V.)

The objective of this study was to evaluate the impact of hormonal status and bacterial vaginosis (BV) on the glycosidases present and glycosylation changes as assessed by lectin binding to cervicovaginal lavage constituents. Frozen cervicovaginal lavage samples from a completed study examining the impact of reproductive hormones on the physicochemical properties of vaginal fluid were utilized for the present study. In the parent study, 165 women were characterized as having BV, intermediate or normal microflora using the Nugent criteria. The presence of glycosidases in the samples was determined using quantitative 4-methyl-umbelliferone based assays, and glycosylation was assessed using enzyme linked lectin assays (ELLA). Women with BV had elevated sialidase, α-galactosidase, β-galactosidase and α-glucosidase activities compared to intermediate or normal women (P<0.001, 0.003, 0.006 and 0.042 respectively). The amount of sialic acid (Sambucus nigra, P = 0.003) and high mannose (griffithsin, P<0.001) were reduced, as evaluated by lectin binding, in women with BV. When the data were stratified according to hormonal status, α-glucosidase and griffithsin binding were decreased among postmenopausal women (P<0.02) when compared to premenopausal groups. These data suggest that both hormonal status and BV impact the glycosidases and lectin binding sites present in vaginal fluid. The sialidases present at increased levels in women with BV likely reduce the number of sialic acid binding sites. Other enzymes likely reduce griffithsin binding. The alterations in the glycosidase content, high mannose and sialic acid binding sites in the cervicovaginal fluid associated with bacterial vaginosis may impact susceptibility to viruses, such as HIV, that utilize glycans as a portal of entry.

The cervicovaginal fluid (CVF) coating the vaginal epithelium is an important immunological mediator, providing a barrier to infection. Glycosylation of CVF proteins, such as mucins, IgG and S-IgA, plays a critical role in their immunological functions. Although multiple factors, such as hormones and microflora, may influence glycosylation of the CVF, few studies have examined their impact on this important immunological fluid. Herein we analyzed the glycosylation of cervicovaginal lavage (CVL) samples collected from 165 women under different hormonal conditions including: (1) no contraceptive, post-menopausal, (2) no contraceptive, days 1-14 of the menstrual cycle, (3) no contraceptive, days 15-28 of the menstrual cycle, (4) combined-oral contraceptive pills for at least 6 months, (5) depo-medroxyprogesterone acetate (Depo-Provera) injections for at least 6 months, (6) levonorgestrel IUD for at least 1 month. Glycomic profiling was obtained using our lectin microarray system, a rapid method to analyze carbohydrate composition. Although some small effects were observed due to hormone levels, the major influence on the glycome was the presence of an altered bacterial cohort due to bacterial vaginosis (BV). Compared to normal women, samples from women with BV contained lower levels of sialic acid and high-mannose glycans in their CVL. The change in high mannose levels was unexpected and may be related to the increased risk of HIV-infection observed in women with BV, as high mannose receptors are a viral entry pathway. Changes in the glycome were also observed with hormonal contraceptive use, in a contraceptive-dependent manner. Overall, microflora had a greater impact on the glycome than hormonal levels, and both of these effects should be more closely examined in future studies given the importance of glycans in the innate immune system.

Objective: The purpose of this study was to determine the impact of contraception, menopause, and vaginal flora on the physical and biochemical properties of cervicovaginal fluid (CVF)., Study Design: Vaginal swabs, CVF, and cervicovaginal lavage (CVL) were collected from a total of 165 healthy asymptomatic women including: postmenopausal women (n = 29), women in the proliferative (n = 26) or follicular (n = 27) phase, and women using the levonogestrel intrauterine device (n = 28), depomedroxyprogesterone acetate (n = 28) or combined oral contraceptives (n = 27). Vaginal smears were evaluated using the Nugent score. The osmolality, viscosity, density, and pH of CVL samples were measured., Results: CVL from postmenopausal women and women with abnormal vaginal flora was less viscous and had higher pH than premenopausal women and women with normal flora, respectively. Women using hormonal contraceptives had more viscous CVL as compared with premenopausal women not using hormonal contraceptives, but this increase in viscosity was mitigated in the presence of bacterial vaginosis. Women using depomedroxyprogesterone acetate had less total protein in the CVL as compared with women using the levonogestrel intrauterine device, and had similar protein content when compared with postmenopausal women., Conclusion: The differences in CVL protein content between depomedroxyprogesterone acetate and levonogestrel intrauterine device suggest that type of progesterone and route of delivery impact the vaginal environment. Contraceptive hormone users had more viscous CVL than women not using contraceptives. However, the presence of bacterial vaginosis impacted both the pH and viscosity (regardless of hormonal contraceptive use), demonstrating that vaginal flora has a greater impact on the physical properties of CVF than reproductive hormones., (Copyright © 2014 Mosby, Inc. All rights reserved.)

hCAP18/LL-37 is the sole human cathelicidin; a family of host defence peptides with key roles in innate host defence. hCAP18/LL-37 is expressed primarily by neutrophils and epithelial cells, but its production and function in the lower genital tract is largely uncharacterised. Despite the significant roles for cathelicidin in multiple organs and inflammatory processes, its impact on infections that could compromise fertility and pregnancy is unknown. The aim of this study was to investigate cathelicidin production, regulation and function in the cervix. hCAP18/LL-37 was found to be present in cervicovaginal secretions collected from women in the first trimester of pregnancy and to be expressed at significantly higher levels in samples from women with alterations in vaginal bacterial flora characteristic of bacterial vaginosis. In endocervical epithelial cell lines, expression of the gene encoding hCAP18/LL-37 (CAMP) was not affected by TLR agonists, but was found to be up-regulated by both 1, 25 hydroxyvitamin D3 and 25 hydroxyvitamin D3. However, no association was found between serum levels of vitamin D and hCAP18/LL-37 concentrations in cervicovaginal secretions (n = 116). Exposure to synthetic LL-37 had a pro-inflammatory effect on endocervical epithelial cell lines, increasing secretion of inflammatory cytokine IL-8. Together these data demonstrate inducible expression of hCAP18/LL-37 in the female lower reproductive tract in vivo and suggest the capacity for this peptide to modulate host defence to infection in this system. Further investigation will elucidate the effects of hCAP18/LL-37 on the physiology and pathophysiology of labour, and may lead to strategies for the prevention of infection-associated preterm birth.

Background & Objectives: Bacterial vaginosis (BV) is highly prevalent among women in reproductive age group. Little information exists on routine vaginal p H measurement in women with BV. We undertook this study to assess the utility of vaginal p H determination for initial evaluation of bacterial vaginosis., Methods: In this cross-sectional study vaginal swabs were collected from women with complaints of white discharge, back ache and pain abdomen attending a government hospital and a community health clinic, and subjected to vaginal p H determination, Gram stain, wet mount and whiff test. Nugent score and Amsel criteria were used for BV confirmation., Results: Of the 270 women included in the analysis, 154 had BV based on Nugents' score. The mean vaginal p H in women with BV measured by p H strips and p H glove was 5 and 4.9, respectively. The vaginal p H was significantly higher in women with BV. Vaginal discharge was prevalent in 84.8 per cent women, however, only 56.8 per cent of these actually had BV by Nugent score (NS). Presence of clue cells and positive whiff test were significant for BV. Vaginal p H >4.5 by p H strips and p H Glove had a sensitivity of 72 and 79 per cent and specificity of 60 and 53 per cent, respectively to detect BV. Among the combination criteria, clue cells and glove p H >4.5 had highest sensitivity and specificity to detect BV., Interpretation & Conclusions: Vaginal p H determination is relatively sensitive, but less specific in detecting women with BV. Inclusion of whiff test along with p H test reduced the sensitivity, but improved specificity. Both, the p H strip and p H glove are equally suitable for screening women with BV on outpatient basis.

Lactobacillus iners is prominent in the human vagina and is able to persist despite development of bacterial vaginosis and treatment with antibiotics. A probable factor in its persistent survival is its ability to be retained in the vaginal epithelia. Genome sequencing of the strain showed an organism deplete of many metabolic pathways, yet equipped with fibronectin (Fn)-binding adhesins. The objective of the present study was to assess the ability of L iners AB-1 to bind immobilized Fn. Results showed that the organism superiorly bound the protein compared to other species of Lactobacillus and known binders such as Staphylococcus aureus. Treatment of L iners cells by protease rendered its binding abilities to Fn nonfunctional. The findings indicate a mechanism of vaginal persistence for a Lactobacillus species, with implications for reproductive health.

Objective: To evaluate levels of proinflammatory cytokines and sialidase activity in aerobic vaginitis (AV) in relation to normal vaginal flora and bacterial vaginosis (BV)., Study Design: In this cross-sectional study, a total of 682 consecutive non-pregnant women attending the gynecology service were assessed and 408 women were included. Vaginal rinsing samples were collected from 223 women with microscopic finding of BV (n=98), aerobic vaginitis (n=25) and normal flora (n=100). Samples were tested for interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α, and sialidase activity., Results: Compared to women with normal flora, vaginal levels of IL-1β were highly increased in both BV and AV (p<0.0001). Significantly higher vaginal IL-6 was detected in AV (p<0.0001) but not in BV, in relation to normal flora. Women with AV also presented increased IL-8 levels (p<0.001), while those with BV presented levels similar to normal flora. Sialidase was increased in BV and AV compared with the normal group (p<0.0001) but no difference in sialidase activity was observed between BV and AV., Conclusion: A more intense inflammatory host response occurs for AV than for BV when compared with normal flora. Furthermore, the increased sialidase activity in AV and BV indicates that both abnormal vaginal flora types can be harmful to the maintenance of a healthy vaginal environment., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Objective: We evaluated vaginal defensin concentrations and levels of bacterial vaginosis-associated bacterial species in pregnant women., Study Design: Self-collected vaginal swabs from 2 visits during pregnancy were tested with quantitative polymerase chain reaction for 9 bacterial species. Beta defensins 2-3 and alpha defensins 1-3 were measured by enzyme-linked immunosorbent assay., Results: Our 126 participants were primarily African American (60%), had a mean gestational age at enrollment of 10 ± 3 weeks and at follow-up visit of 25 ± 6 weeks. At enrollment, the prevalence of bacterial vaginosis was 74% (94/126 women), which decreased to 60% (75/126 specimens) at follow-up visit. At enrollment, beta defensin 3 concentrations were significantly lower in women with bacterial vaginosis (2.64 ± 0.91 vs 3.25 ± 0.99 log(10) pg/mL; P = .003). Higher concentrations of Atopobium vaginae, bacterial vaginosis-associated bacteria1 and 2 were associated with significantly lower concentrations of beta defensin 3 (P < .01)., Conclusion: Bacterial vaginosis was associated with lower vaginal concentrations of beta defensin 3, but not beta defensin 2 or alpha defensins 1-3, in pregnant women., (Copyright © 2013 Mosby, Inc. All rights reserved.)

Introduction: The objective is to analyze proinflammatory cytokines [interleukin-1β (IL-1β), interleukin-6 and tumor necrosis factor-α] and sphingomyelinase in women with bacterial vaginosis (BV), cervicitis and vaginitis., Methods: From January 2009 to June 2010, human immunodeficiency virus (HIV)-negative, nonpregnant, married women, living with husband, aged 20 to 40 years were recruited from a slum at Hyderabad, India, after taking written consent. One hundred forty-six women including 61 women with BV, 47 women with intermediate flora and 38 women with normal vaginal flora were evaluated for local proinflammatory cytokines and sphingomyelinase. Cervicitis and vaginitis were also analyzed by scoring white blood cells in the cervix and vaginal smears, respectively., Results: Of the 146 women, 50.7% had cervicitis and 19.5% had vaginitis. IL-1β, tumor necrosis factor-α and interleukin-6 levels were significantly high in women with cervicitis (P < 0.001) and vaginitis (P < 0.001) and IL-1β in BV (P < 0.005), intermediate flora (P < 0.05) when compared with normal women. High vaginal pH was associated with IL-1β. Neutral sphingomelinase showed an inverse association (P < 0.05) with cervicitis. Acid sphingomelinase directly correlated with IL-1β although not significantly., Conclusions: This study shows proinflammatory response not only in BV but also in women with vaginitis and cervicitis. These conditions are likely to be important in promoting the transmission of HIV-1 and should be the focus of HIV prevention strategies.

Purpose: To assess the prevalence of bacterial vaginosis and to compare the accuracy of testing pH and KOH with the Nugent gradient, the gold standard for the diagnosis of bacterial vaginosis (BV) in asymptomatic and symptomatic pregnant women at low risk., Methods: We conducted a cross-sectional study on 321 pregnant women with gestational age between 14 and 26 weeks, 218 of them asymptomatic and 103 with vaginal complaints suggestive of bacterial vaginosis. All women were assessed by the criteria of Nugent and subjected to the measurement of vaginal pH and to the 10% KOH test. The Kappa coefficient was used to evaluate the methods in terms of diagnostic agreement., Results: Most patients were adolescents (mean age 21.0 ± 5.6 years), nulliparous and mulattos. The prevalence of bacterial vaginosis was 33.3% as estimated by the pH and KOH method and 35.5% by the Nugent method. Excellent agreement of the methods was found among asymptomatic pregnant women, with 72.5% of them showing negative results to both tests, which resulted in a high Kappa coefficient (k=0.82). The group of symptomatic women showed 49.5% positivity to both diagnostic methods, with excellent agreement (k=0.74)., Conclusion: The prevalence of bacterial vaginosis determined by both the pH and KOH method and the Nugent score was high. The pH and KOH method can diagnose bacterial vaginosis as accurately as the Nugent criterion.

Background: Misoprostol has been shown to be an effective agent for cervical ripening and termination of early pregnancy especially when administered vaginally. Our objective was to evaluate whether bacterial vaginosis (BV) affected the pharmacokinetics of vaginally administered misoprostol during early pregnancy., Methods: Ten women with BV and 10 healthy women requesting medical abortion up to 9 weeks of pregnancy were administered 200 mg mifepristone followed 24-48 h later by a single dose of 800 µg misoprostol vaginally. Blood samples were taken before (0 h) and 0.5, 1, 2, 3 and 4 h after misoprostol administration. Misoprostol acid was determined in serum samples using liquid chromatography/tandem mass spectrometry., Results: All women with BV had a vaginal pH > 4.7. The mean bioavailability measured as the area under the curve (AUC) and maximum concentration (C(max)) appeared higher in the control than in the BV group (1458.7 versus 878.1 pg h/ml) and (630.7 versus 342.5 pg/ml), respectively, but did not achieve statistical significance and there was no other significant difference in the pharmacokinetics between the two groups. However, if two women with vaginal pH > 4.7 were excluded from the control group the difference in AUC₂₄₀ (1359 versus 878.1 pgh/ml) reached statistical significance (P = 0.048)., Conclusions: BV had an effect on pharmacokinetics of vaginally administered misoprostol in early pregnancy. However, the results should be interpreted with caution due to the small sample size and marked individual variations.

Bacterial vaginosis (BV) is a common polymicrobial imbalance of the vaginal flora associated with a wide variety of obstetric and gynecologic complications including serious infections and preterm birth. As evidenced by high recurrence rates following treatment, interventions for BV are still lacking. Several hydrolytic activities, including glycosidases and proteases, have been previously correlated with BV and have been hypothesized to degrade host sialoglycoproteins that participate in mucosal immune functions. Sialidase activity is most predictive of BV status and correlates strongly with adverse health outcomes. Here we combine clinical specimens with biochemical approaches to investigate secretory immunoglobulin A (SIgA) as a substrate of BV-associated glycosidases and proteases. We show that BV clinical specimens hydrolyze sialic acid from SIgA, but not in the presence of the sialidase inhibitor dehydro-deoxy-sialic acid. The collective action of BV-associated glycosidases exposes underlying mannose residues of SIgA, most apparent on the heavily N-glycosylated secretory component of the antibody. Terminal sialic acid residues on SIgA protect underlying carbohydrate residues from exposure and hydrolysis by exoglycosidases (galactosidase and hexosaminidase). It is known that both IgG and SIgA are present in the human reproductive tract. We show that the IgG heavy chain is more susceptible to proteolysis than its IgA counterpart. Gentle partial deglycosylation of the SIgA secretory component enhanced susceptibility to proteolysis. Together, these data support a model of BV in which SIgA is subject to stepwise exodeglycosylation and enhanced proteolysis, likely compromising the ability of the reproductive mucosa to neutralize and eliminate pathogens.

Understanding factors that affect heterosexual transmission of HIV in women is of great importance. Lactobacilli in the lower genital tract of women utilize glycogen in vaginal epithelial cells as an energy source and produce lactic acid. The resultant vaginal acidity is believed to provide protection against HIV infection. Conversely, bacterial vaginosis (BV) is characterized by less lactic acid and a higher pH, and is associated with increased susceptibility to HIV infection. Because vaginal infection of macaques with simian immunodeficiency virus (SIV) or simian-human immunodeficiency virus (SHIV) is used as a model to study HIV sexual transmission, and because previous studies have shown a paucity of lactobacilli in rhesus macaques' lower genital tract, we compared lactic acid and glycogen levels in the genital fluid of rhesus and pigtail macaques with levels found in humans. The levels of lactic acid were lower in both rhesus (median=1.2 mol lactate/mg protein) and pigtail macaques (median=0.7 mol/mg) compared to women with healthy genital microbiota (median=4.2 mol/mg). Glycogen levels were significantly lower in both rhesus (median=0.004 μg glycogen/μg protein) and pigtail macaques (median=0 μg/μg) than in women (median=0.2 μg/μg). No significant differences in glycogen or lactate levels were observed comparing longitudinally collected samples from cycling pigtail macaques. These data show that the previously reported scarcity of lactobacilli in macaques correlates with low glycogen and lactic acid levels. These findings have important implications for studies of vaginal infection of macaques with SIV or SHIV and further our understanding of how the bacterial microbiota influences HIV infection.

The objective was to assess the impact of genetic variation on cervical cytokine concentrations of interleukin (IL)-1α, IL-1β, IL-6, IL-8 and tumor necrosis factor alpha (TNF-α), and first, to determine if these variants interact with polymorphisms in Toll-like receptor 4 (TLR4) that were previously shown to associate with pro-inflammatory cervical cytokine concentrations, and second, to determine if findings are affected by bacterial vaginosis (BV). We examined 183 single nucleotide polymorphisms (SNPs) in 13 cytokine genes and receptors for associations with cervical cytokine levels in 188 African American and European American women. We tested for associations of gene-gene interactions between SNPs in TLR4 and cytokine gene and receptor polymorphisms with cervical pro-inflammatory cytokines. None of the single locus associations were significant after correction for multiple testing in either European Americans or African Americans. However, there were significant gene-gene interactions between IL-1R2 rs485127 and two SNPs in TLR4 (rs1554973 and rs7856729) with IL-1β after correction for multiple testing. Our study demonstrates that interactions between TLR4 and IL-1R2 are associated with cervical pro-inflammatory cytokine concentrations. These results provide important insights into the possible regulatory mechanisms of the inflammatory response in the presence and absence of microbial disorders such as BV. Additionally, the observed differences in allele frequencies between African Americans and those of European descent may partially explain population disparity in pregnancy-related phenotypes that are cytokine concentration-dependent., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Objective: The present study was undertaken to investigate the pharmacokinetics of metronidazole in pregnant patients with bacterial vaginosis., Methods: Twenty patients received metronidazole (Flagyl ®, Pfizer, 235 East 42nd Street, NY, NY 10017) oral dose 500 mg twice a day for 3 consecutive days. Pharmacokinetic analyses of metronidazole were performed after a single oral dose on the morning of day 4., Results: Although absolute estimates of metronidazole total body exposure were highest in women during early term pregnancy, weight-corrected estimates of exposure maximum plasma drug concentration (C(max)) and the area under the plasma concentration-versus-time curve (AUC(0-12)), along with apparent oral clearance and distribution volume, were not significantly different between women at early, middle, and late stages of pregnancy and were in the range of reported values for nonpregnant patients receiving a similar dose., Conclusions: The pharmacokinetic profile of metronidazole did not change at the different time points assessed during pregnancy.

Background: 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., Methods and Findings: 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)., Conclusion: This study demonstrates that high level of sHLA-G in the genital mucosa is independently associated with both HIV-1 infection and bacterial vaginosis.

Intrauterine infection/inflammation complicates 25% to 40% of preterm births (PTB). The human vagina is normally populated by Lactobacillus species, some of which upregulate interleukin 10 (IL-10) output in lipopolysaccharide (LPS)-treated human placental trophoblast cells. We hypothesize that a probiotic strain, L rhamnosus GR-1 exerts its anti-inflammatory effect through activation of the Janus Kinases/Signal Transducers and Activators of Transcription (JAK/STAT) and mitogen-activated protein kinase (MAPK) pathways. Placental trophoblasts from term healthy pregnancies were treated with LPS in the presence or absence of pretreatments with GR-1 supernatant and/or chemical inhibitors of the intracellular signaling pathways. Phosphorylation of STAT3 and p38 was measured by Western Blot analysis, and output of IL-10 was determined by enzyme-linked immunosorbent assay (ELISA). Phosphorylation of STAT-3 and p38 was upregulated by GR-1 supernatant alone or in combination with LPS, while IL-10 output was inhibited by both JAK and p38 inhibitors. These data provide an underlying intracellular mechanism for cytokine regulation in the human placenta by L rhamnosus GR-1 and potential prevention of infection/inflammation-mediated PTB.

We compared vaginal proinflammatory cytokine and secretory leukocyte protease inhibitor (SLPI) concentrations among pregnant and nonpregnant women according to bacterial vaginosis (BV) status. One-hundred and twenty-two women at 12-20 weeks' gestation and 133 nonpregnant controls had vaginal concentrations of interleukin (IL)-1β, IL-6, IL-8, and SLPI measured by enzyme immunoassay. Multivariable linear regression was used to evaluate factors independently associated with vaginal cytokine and SLPI response. Pregnancy and BV were both independently associated with increased vaginal concentrations of IL-1β and IL-8; pregnant women had increased concentrations of SLPI, while women with BV had decreased SLPI concentrations.

Background: There has been a recent recognition on the influence of local vaginal immunity on the acquisition of vulvovaginal disorders and their adverse consequences. Variations in local immune responses seem to play an important role in susceptibility to different vaginal infections as well as to the likelihood of recurrences. Bacterial vaginosis (BV), the most frequent vaginal disorder in most populations, is enigmatic in that the etiology is unknown, recurrences are common and vaginal inflammation is absent. We investigated the influence of BV on the vaginal concentration of the pro-inflammatory cytokine interleukin (IL)-12, the pleiotropic cytokine IL-6 and the anti-inflammatory cytokine IL-10 in non-pregnant women., Methods: Vaginal lavage samples were obtained from 45 patients with BV and from 46 asymptomatic controls. The supernatant fractions were examined for IL-6, IL-10 and IL-12 by commercial ELISAs. Analysis of the cytokine levels in the two groups was by the Mann-Whitney test., Results: IL-6 concentrations varied considerably among women in the BV and control groups but the median levels were almost identical. The median concentrations of IL-10 and IL-12 were uniformly low in both groups but median levels were not statistically different., Conclusion: The marked alteration in the vaginal bacterial flora that is characteristic of BV does not result in enhancement or inhibition of the vaginal levels of IL-6, IL-10 and IL-12. Mechanisms to explain this striking lack of immune system variation remain to be determined.

Objectives: To examine cervicovaginal elafin production in pregnancy and determine its relationship in bacterial vaginosis., Study Design: Samples of cervicovaginal secretions were collected from women with uncomplicated singleton pregnancies (n = 112) below 20 weeks gestation. Bacterial flora was assessed using Nugent's criteria, and levels of elafin were measured by enzyme-linked immunosorbent serologic assay (ELISA). Elafin expression in the cervix was also examined by immunohistochemistry. In vitro expression of elafin was examined using cervix and vaginal cell lines., Results: Elafin is expressed in the cervical glandular epithelium. Elafin was found in all 112 samples of cervicovaginal secretions and levels were diminished in women with bacterial vaginosis (P < .05). Interleukin 1beta (IL-1beta) stimulated elafin expression in cells derived from the endocervix, but not in those derived from the vaginal epithelium., Conclusions: Elafin is a component of cervicovaginal secretions in pregnancy, and levels are diminished in bacterial vaginosis. It may be an important component of innate immunity in the lower genital tract.

Objective: The objective of the study was to determine the effect of fetal sex on the output of cytokines and prostaglandin-regulating enzymes in lipopolysaccharide (LPS) and probiotic lactobacilli-treated placental trophoblast cells., Study Design: We examined the effect of LPS and Lactobacillus rhamnosus GR-1 supernatant in placental trophoblast cells on tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-10 using enzyme-linked immunosorbent assay and on prostaglandin-endoperoxide synthase 2 (PTGS2), 15-hydroxy prostaglandin dehydrogenase (PGDH), and toll-like receptor-4 (TLR-4) using Western blotting. Comparisons were performed using one-way analysis of variance and Student t test., Results: LPS increased the output of TNF-alpha, IL-10, and PTGS2 with a greater response in male placentae. L rhamnosus GR-1 supernatant inhibited the LPS-stimulated TNF-alpha and increased IL-10. It also up-regulated expression of PGDH in female placentae and partially reduced the LPS-stimulated PTGS2 in male placentae. There was no change in IL-1beta. Expression of TLR-4 was greater in placentae of male fetuses., Conclusion: These findings suggest an underlying mechanism for the sex difference in the incidence of preterm birth and provide potential evidence for a therapeutic benefit of lactobacilli in reducing preterm labor.

Background: Fastidious bacteria have been associated with bacterial vaginosis (BV) using PCR methods. We assessed the prevalence of these bacteria in HIV-1 infected women and their relationship with vaginal pH and shedding of HIV-1 RNA., Methods: 64 cervicovaginal lavage (CVL) samples were collected from 51 women. Vaginal microbiota were characterized using 8 bacterium-specific quantitative PCR assays., Results: Women with the fastidious bacteria Bacterial Vaginosis Associated Bacterium (BVAB) 1, 2, and 3 showed a trend to increased HIV-1 shedding (OR 2.59-3.07, P = .14-.17). Absence of Lactobacillus crispatus (P < .005) and presence of BVAB2 (P < .001) were associated with elevated vaginal pH. BVAB1, 2, and 3 were highly specific indicators of BV in HIV-infected women, with specificities of 89%-93%., Conclusions: Fastidious bacteria (BVAB 1, 2, and 3) remain specific indicators of BV in HIV-infected women, and BVAB2 may contribute to the elevated vaginal pH that is a hallmark of this syndrome.

Objective: To investigate the profile of local immunity of vagina and the immune defense mechanisms against lower genital tract infections., Methods: Vaginal lavage was collected from healthy women and patients of vulvovaginal candidiasis, bacterial vaginosis, Trichomonol vaginitis, human papilloma virus infection (VVC), and chlamydia trachomatis infection. Each group included 60 cases. The level of interleukin (IL)2, 4, 5, 13, 8 and human defensin 5 (HD5) were detected by enzyme linked immunosorbent assay(ELISA)., Results: (1) Cytokine of helper T cell 1(Th1): the level of IL-2 between healthy women and VVC/ bacterial vaginosis (BV)/ trichomonol vaginitis (TV)/ chlamydia trachomatis (CT) patients had no significant difference. The IL-2 level(96 +/- 33) x 10(-3) pg/L of human papilloma virus (HPV) infection patients was significantly higher than that of healthy women (P < 0.05). (2) Cytokine of helper T cell 2 (Th2): the level of IL-4 between healthy women and VVC/CT patients had no significant difference. The level of IL-5 between healthy women and BV patients had no significant difference. The IL-13 level (42 +/- 15) x 10(-3) pg/L of TV patients was significantly higher than that of healthy women (30 +/- 29) x 10(-3) pg/L (P < 0.05). The IL-4 level (103 +/- 28) x 10(-3) pg/L of HPV infection patients was significantly higher than that of healthy women (36 +/- 22) x 10(-3) pg/L (P < 0.05). (3) IL-8: the IL-8 level (5.8 +/- 2.7) pg/L of TV infection patients was significantly higher than that of healthy women (2.6 +/- 2.4) pg/L (P < 0.05). The level of IL-8 between healthy women and BV patients had no significant difference. (4) HD5: the HD5 level of TV, BV, VVC, HPV and CT infection patients were significantly higher than that of healthy women (P < 0.05)., Conclusions: (1) HD5 plays an important role in the defence of vaginal epithelial cell. (2) Th2 may be more important than Thl in lower genital tract infections. (3) IL-8 plays an important role in extrinsic source infections.

Objective: The purpose of the study was to measure and compare concentrations of IFN-gamma, IL-8, IL-10 and IL-12 in cervical fluids of pregnant women with normal and pathologic microscopic findings of vaginal wet mount., Study Design: Vaginal samples were obtained from 100 pregnant women (22-32 weeks of gestation) without signs and symptoms of preterm labour (PTL), selected on the basis of the microscopic examination of vaginal wet mounts. Cytokine (IFN-gamma, IL-8, IL-10 and IL-12) concentrations were measured in cervical fluids by an enzyme-linked immunosorbent assay., Results: The cytokines were found in cervical fluids of the majority of pregnant women, with IFN-gamma and IL-8 detected in all samples. Concentrations of IL-8 were significantly higher in pregnant women with pathologic findings on vaginal wet mount, including elevated polymorphonuclear (PMN) cell counts., Conclusion: A significant correlation between microscopic findings and cervical fluid IL-8 concentrations may make vaginal wet mount microscopy a useful tool for identification of patients with cervicovaginal inflammation, which is considered a risk factor for spontaneous preterm delivery.

Objective: To measure the concentrations of interferon gamma (IFN-gamma), interleukin (IL)-2, IL-4, IL-5, human beta-defensin 2 (HBD-2), and human defensin 5 (HD-5) in the vaginal lavage fluid (VLF) of healthy women and women with bacterial vaginosis (BV)., Methods: VLF samples were obtained from 73 women with BV, 15 women with intermediate vaginal flora, 33 healthy women with vaginal pH greater than or equal to 4.5, and a control group of 39 healthy women with vaginal pH less than 4.5. The concentrations of IFN-gamma, IL-2, IL-4, IL-5, HBD-2, and HD-5 were measured using enzyme-linked immunosorbent assay kits., Results: Concentrations of HBD-2 and HD-5 in the VLF of women with BV were significantly higher than in the control group (P<0.05). IL-4 concentration was significantly lower in the VLF of women with BV than in the control group (P<0.05)., Conclusions: HBD-2 and HD-5 may be involved in defending against invasion by BV-related microorganisms and the decrease in IL-4 concentration may increase susceptibility to BV.

Objective: The purpose of this study was to evaluate the contribution of maternal genotype to the concentration of interleukin (IL)-4, -10, and -13 in the cervix during the first trimester in the context of bacterial vaginosis and cigarette smoking., Study Design: In this prospective cohort of 116 pregnant women, we examined 72 single nucleotide polymorphisms (SNPs) in 6 genes: IL-4, IL-4 receptor, IL-10, IL-10 receptor alpha, IL-10 receptor beta, and IL-13., Results: Among black women, an IL-10 receptor beta SNP (rs6517158) demonstrated significant association with IL-10 concentration (P = .04), adjusted for bacterial vaginosis. Among white women, there were 4 SNPs in IL-10 receptor alpha with a highly significant association with IL-10 concentration, adjusted for bacterial vaginosis and smoking., Conclusion: Our data demonstrate a significant relation between cervical concentrations of IL-10 and SNPs in the IL-10 receptor alpha and beta genes. The contribution of genotype to IL-10 concentration is significant, even in the consideration of bacterial vaginosis and smoking.