IgA coating of vaginal bacteria is reduced in the setting of bacterial vaginosis (BV) and preferentially targets BV-associated species.

Immunoglobulin (Ig) bacterial coating has been described in the gastrointestinal tract and linked to inflammatory bowel disease; however, little is known about Ig coating of vaginal bacteria and whether it plays a role in vaginal health including bacterial vaginosis (BV). We examined Ig coating in 18 women with symptomatic BV followed longitudinally before, 1 week, and 1 month after oral metronidazole treatment. Immunoglobulin A (IgA) and/or immunoglobulin G (IgG) coating of vaginal bacteria was assessed by flow cytometry, and Ig coated and uncoated bacteria were sorted and characterized using 16S rRNA sequencing. Despite higher levels of IgG compared to IgA in cervicovaginal fluid, the predominant Ig coating the bacteria was IgA. The majority of bacteria were uncoated at all visits, but IgA coating significantly increased after treatment for BV. Despite similar amounts of uncoated and IgA coated majority taxa ( >1% total) across all visits, there was preferential IgA coating of minority taxa (0.2%-1% total) associated with BV including Sneathia, several Prevotella species, and others. At the time of BV, we identified a principal component (PC) driven by proinflammatory mediators that correlated positively with an uncoated BV-associated bacterial community and negatively with an IgA coated protective Lactobacillus bacterial community. The preferential coating of BV-associated species, increase in coating following metronidazole treatment, and positive correlation between uncoated BV-associated species and inflammation suggest that coating may represent a host mechanism designed to limit bacterial diversity and reduce inflammatory responses. Elucidating the role of Ig coating in vaginal mucosal immunity may promote new strategies to prevent recurrent BV.

Genetic Variation in Toll-Like Receptor 5 and Colonization with Flagellated Bacterial Vaginosis-Associated Bacteria.

Bacterial vaginosis (BV) is a vaginal dysbiotic condition linked to negative gynecological and reproductive sequelae. Flagellated bacteria have been identified in women with BV, including Mobiluncus spp. and BV-associated bacterium-1 (BVAB1), an uncultivated, putatively flagellated species. The host response to flagellin mediated through Toll-like receptor 5 (TLR5) has not been explored in BV. Using independent discovery and validation cohorts, we examined the hypothesis that TLR5 deficiency-defined by a dominant negative stop codon polymorphism, rs5744168-is associated with an increased risk for BV and increased colonization with flagellated bacteria associated with BV (BVAB1, Mobiluncus curtisii, and Mobiluncus mulieris). TLR5 deficiency was not associated with BV status, and TLR5-deficient women had decreased colonization with BVAB1 in both cohorts. We stimulated HEK-hTLR5-overexpressing NF-κB reporter cells with whole, heat-killed M. mulieris or M. curtisii and with partially purified flagellin from these species; as BVAB1 is uncultivated, we used cervicovaginal lavage (CVL) fluid supernatant from women colonized with BVAB1 for stimulation. While heat-killed M. mulieris and CVL fluid from women colonized with BVAB1 stimulate a TLR5-mediated response, heat-killed M. curtisii did not. In contrast, partially purified flagellin from both Mobiluncus species stimulated a TLR5-mediated response in vitro We observed no correlation between vaginal interleukin 8 (IL-8) and flagellated BVAB concentrations among TLR5-sufficient women. Interspecies variation in accessibility of flagellin recognition domains may be responsible for these observations, as reflected in the potentially novel flagellin products encoded by Mobiluncus species versus those encoded by BVAB1.

Influence of Intramuscular Depot Medroxyprogesterone Acetate Initiation on Vaginal Microbiota in the Postpartum Period.

BACKGROUND: The vaginal microbiome plays a key role in women's reproductive health. Use of exogenous hormones, such as intramuscular depot medroxyprogesterone acetate (DMPA-IM), may alter the composition of vaginal bacterial community. METHODS: Vaginal swab samples were collected from postpartum Kenyan women initiating DMPA-IM or nonhormonal contraception (non-HC). Bacterial vaginosis was assessed by Nugent score (Nugent-BV) and bacterial community composition was evaluated using broad-range 16S ribosomal RNA gene polymerase chain reaction with high-throughput sequencing. Changes in Nugent score, alpha diversity (Shannon diversity index), and total bacterial load between contraceptive groups from enrollment to 3 months after initiation were estimated using multivariable linear mixed effects regression. RESULTS: Among 54 human immunodeficiency virus-negative women, 33 choosing DMPA-IM and 21 choosing non-HC, Nugent-BV was more common among DMPA-IM users at enrollment. At follow-up, Nugent score had decreased significantly among DMPA-IM users (change, -1.89; 95% confidence interval [CI], -3.53 to -.25; P = .02) while alpha diversity remained stable (0.03; -.24 to .30; P = .83). Conversely, Nugent score remained relatively stable among non-HC users (change, -0.73; 95% CI, -2.18 to .73; P = .33) while alpha diversity decreased (-0.34; -.67 to -.001; P = .05). The total bacterial load decreased slightly in DMPA-IM users and increased slightly among non-HC users, resulting in a significant difference in change between the contraceptive groups (difference, -0.64 log10 gene copies per swab sample; 95% CI, -1.19 to -.08; P = .02). While significant changes in Nugent score and alpha diversity were observed within contraceptive groups, changes between groups were not significantly different. CONCLUSIONS: Postpartum vaginal bacterial diversity did not change in DMPA-IM users despite a reduction in Nugent-BV, but it decreased significantly among women using non-HC. Choice of contraception may influence Lactobacillus recovery in postpartum women.

Stool Microbiota at Neutrophil Recovery Is Predictive for Severe Acute Graft vs Host Disease After Hematopoietic Cell Transplantation.

BACKGROUND: Graft-versus-host disease (GVHD) is common after allogeneic hematopoietic cell transplantation (HCT). Risk for death from GVHD has been associated with low bacterial diversity in the stool microbiota early after transplant; however, the specific species associated with GVHD risk remain poorly defined. METHODS: We prospectively collected serial weekly stool samples from 66 patients who underwent HCT, starting pre-transplantation and continuing weekly until 100 days post-transplant, a total of 694 observations in HCT recipients. We used 16S rRNA gene polymerase chain reaction with degenerate primers, followed by high-throughput sequencing to assess the relative abundance of sequence reads from bacterial taxa in stool samples over time. RESULTS: The gut microbiota was highly dynamic in HCT recipients, with loss and appearance of taxa common on short time scales. As in prior studies, GVHD was associated with lower alpha diversity of the stool microbiota. At neutrophil recovery post-HCT, the presence of oral Actinobacteria and oral Firmicutes in stool was positively correlated with subsequent GVHD; Lachnospiraceae were negatively correlated. A gradient of bacterial species (difference of the sum of the relative abundance of positive correlates minus the sum of the relative abundance of negative correlates) was most predictive (receiver operator characteristic area under the curve of 0.83) of subsequent severe acute GVHD. CONCLUSIONS: The stool microbiota around the time of neutrophil recovery post-HCT is predictive of subsequent development of severe acute GVHD in this study.

More Easily Cultivated Than Identified: Classical Isolation With Molecular Identification of Vaginal Bacteria.

BACKGROUND: Women with bacterial vaginosis (BV) have complex communities of anaerobic bacteria. There are no cultivated isolates of several bacteria identified using molecular methods and associated with BV. It is unclear whether this is due to the inability to adequately propagate these bacteria or to correctly identify them in culture. METHODS: Vaginal fluid from 15 women was plated on 6 different media using classical cultivation approaches. Individual isolates were identified by 16S ribosomal RNA (rRNA) gene sequencing and compared with validly described species. Bacterial community profiles in vaginal samples were determined using broad-range 16S rRNA gene polymerase chain reaction and pyrosequencing. RESULTS: We isolated and identified 101 distinct bacterial strains spanning 6 phyla including (1) novel strains with <98% 16S rRNA sequence identity to validly described species, (2) closely related species within a genus, (3) bacteria previously isolated from body sites other than the vagina, and (4) known bacteria formerly isolated from the vagina. Pyrosequencing showed that novel strains Peptoniphilaceae DNF01163 and Prevotellaceae DNF00733 were prevalent in women with BV. CONCLUSIONS: We isolated a diverse set of novel and clinically significant anaerobes from the human vagina using conventional approaches with systematic molecular identification. Several previously "uncultivated" bacteria are amenable to conventional cultivation.

First Trimester Levels of BV-Associated Bacteria and Risk of Miscarriage Among Women Early in Pregnancy.

OBJECTIVES: Prior studies have examined the role of bacterial vaginosis (BV) and increased risk of miscarriage; however the risk has been modest and many BV positive pregnant women deliver at term. BV is microbiologically heterogeneous, and thus the identification of specific BV-associated bacteria associated with miscarriage is warranted. METHODS: We measured the presence and level of seven BV-associated bacteria prior to 14 weeks gestation among urban pregnant women seeking routine prenatal care at five urban obstetric practices at Temple University Hospital in Philadelphia PA from July 2008 through September 2011. 418 Pregnant women were included in this assessment and 74 experienced a miscarriage. RESULTS: Mean log concentration of BVAB3 was significantly higher among women experiencing a miscarriage (4.27 vs. 3.71, p value = 0.012). Younger women with high levels of BVAB3 had the greatest risk of miscarriage. In addition, we found a significant decreased risk of miscarriage among women with higher log concentrations of Leptotrichia/Sneathia species or Megasphaera phylotype 1-like species early in pregnancy. CONCLUSIONS FOR PRACTICE: The identification of selected vaginal bacteria associated with an increased risk of miscarriage could support screening programs early in pregnancy and promote early therapies to reduce early pregnancy loss.

Early pregnancy changes in bacterial vaginosis-associated bacteria and preterm delivery.

BACKGROUND: We evaluated the importance of measuring early vaginal levels of eight bacterial vaginosis (BV)-associated bacteria, at two points in pregnancy, and the risk of spontaneous preterm delivery (SPTD) among pregnant women and the subgroup of pregnant women with a history of preterm delivery (PTD). METHODS: This prospective cohort study enrolled women at five urban obstetric practices at Temple University Hospital in Philadelphia PA. Women with singleton pregnancies less than 16 weeks gestation self-collected vaginal swabs at two points in pregnancy, prior to 16 weeks gestation and between 20-24 weeks gestation, to measure the presence and level of eight BV-associated bacteria. Women were followed-up for gestational age at delivery via medical records. RESULTS: Among women reporting a prior PTD, women with higher levels of Leptotrichia/Sneathia species, BVAB1 and Mobiluncus spp., prior to 16 weeks gestation, were significantly more likely to experience a SPTD. In addition, pregnant women with a prior PTD and increasing levels of Leptotrichia/Sneathia species (aOR: 9.1, 95% CI 1.9, 42.9), BVAB1 (aOR: 16.4, 95% CI 4.3, 62.7) or Megasphaera phylotype 1 (aOR: 6.2, 95% CI 1.9, 20.6), through 24 weeks gestation, were significantly more likely to experience an SPTD. Among the overall group of pregnant women, the levels of BV-associated bacteria were not related to SPTD. CONCLUSION: Among the group of women reporting a prior PTD, increasing levels of BVAB1, Leptotrichia/Sneathia species, and Megasphaera phylotype 1, through mid-pregnancy were related to an increased risk of SPTD.

Vaginal Bacteria Elicit Acute Inflammatory Response in Fallopian Tube Organoids.

To facilitate in vitro mechanistic studies in pelvic inflammatory disease and subsequent tubal factor infertility, we sought to establish patient tissue derived fallopian tube (FT) organoids and to study their inflammatory response to acute vaginal bacterial infection. FT tissues were obtained from four patients after salpingectomy for benign gynecological diseases. We introduced acute infection in the FT organoid culture system by inoculating the organoid culture media with two common vaginal bacterial species, Lactobacillus crispatus and Fannyhessea vaginae. The inflammatory response elicited in the organoids after acute bacterial infection was analyzed by the expression profile of 249 inflammatory genes. Compared to the negative controls that were not cultured with any bacteria, the organoids cultured with either bacterial species showed multiple differentially expressed inflammatory genes. Marked differences were noted between the Lactobacillus crispatus infected organoids and those infected by Fannyhessea vaginae. Genes from the C-X-C motif chemokine ligand (CXCL) family were highly upregulated in Fannyhessea vaginae infected organoids. Flow cytometry showed that immune cells quickly disappeared during the organoid culture, indicating the inflammatory response observed with bacterial culture was generated by the epithelial cells in the organoids. In summary, we have shown that patient tissue derived FT organoids respond to acute bacterial infection with upregulation of inflammatory genes specific to different vaginal bacterial species. FT organoids is a useful in vitro model system to study the host-pathogen interaction during bacterial infection.

Effect of Metronidazole on Concentrations of Vaginal Bacteria Associated with Risk of HIV Acquisition.

Several bacterial vaginosis (BV)-associated bacteria have been associated with elevated risk of HIV acquisition, however susceptibility of these bacteria to antibiotics is poorly understood. Vaginal samples were collected from 22 persons daily for two weeks following BV diagnosis. Metronidazole treatment was prescribed for 5-7 days. Changes in bacterial concentrations were measured with taxon-specific 16S rRNA gene quantitative PCR (qPCR) assays. A culture-based antimicrobial assay confirmed presence of antibiotics in vaginal swab samples. Bacterial DNA concentrations decreased during antibiotic administration for all thirteen bacterial taxa tested. Comparison of bacterial DNA concentrations in samples before administration of antibiotics to samples taken on the last day of antimicrobial assay-confirmed antibiotic presence showed a 2.3-4.5 log10-fold decrease across all taxa. Concentrations were frequently reduced to the qPCR assay's limit of detection, suggesting eradication of bacteria. Mean clearance time varied across taxa (1.2-8.6 days), with several bacteria (e.g., Gemella asaccharolytica, Sneathia spp., Eggerthella-like sp.) taking >7 days to suppress. Metronidazole reduces quantities of bacterial taxa associated with increased HIV acquisition risk. Eradication of high-risk vaginal bacteria using metronidazole is one promising avenue for reducing HIV acquisition risk. A 5-7-day treatment course may not be sufficient to suppress all bacteria.

Identification of fallopian tube microbiota and its association with ovarian cancer.

Investigating the human fallopian tube (FT) microbiota has significant implications for understanding the pathogenesis of ovarian cancer (OC). In this large prospective study, we collected swabs intraoperatively from the FT and other surgical sites as controls to profile the microbiota in the FT and to assess its relationship with OC. Eighty-one OC and 106 non-cancer patients were enrolled and 1001 swabs were processed for 16S rRNA gene PCR and sequencing. We identified 84 bacterial species that may represent the FT microbiota and found a clear shift in the microbiota of the OC patients when compared to the non-cancer patients. Of the top 20 species that were most prevalent in the FT of OC patients, 60% were bacteria that predominantly reside in the gastrointestinal tract, while 30% normally reside in the mouth. Serous carcinoma had higher prevalence of almost all 84 FT bacterial species compared to the other OC subtypes. The clear shift in the FT microbiota in OC patients establishes the scientific foundation for future investigation into the role of these bacteria in the pathogenesis of OC.

Relationship of selected bacterial vaginosis-associated bacteria to Nugent score bacterial vaginosis among urban women early in pregnancy.

Among urban, primarily African American pregnant women, 74% were identified with Nugent score bacterial vaginosis (BV). All BV-associated bacteria were more prevalent among women with Nugent score BV. Bacterial vaginosis-associated bacteria 3 (BVAB3) had the highest positive predictive value, whereas Gardnerella vaginalis and Atopobium spp. had the highest sensitivity. Atopobium spp. levels had the most significant area under the curve.

Associations between vaginal bacteria and levels of vaginal defensins in pregnant women.

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.

Extravaginal reservoirs of vaginal bacteria as risk factors for incident bacterial vaginosis.

BACKGROUND: Bacterial vaginosis (BV) represents shifts in microbiota from Lactobacillus spp. to diverse anaerobes. Although antibiotics relieve symptoms and temporarily eradicate BV-associated bacteria (BVAB), BV usually recurs. We investigated the role of extravaginal BVAB reservoirs in recurrence. METHODS: Risks for BV acquisition over the course of 1 year were defined. DNA in vaginal, anal, and oral swab samples from enrollment was subjected to quantitative polymerase chain reaction assays targeting 16S ribosomal RNA genes of Gardnerella vaginalis, Lactobacillus crispatus, BVAB1, BVAB2, BVAB3, Megasphaera spp., Lactobacillus jensenii, and Leptotrichia/Sneathia spp. A case-control approach analyzed BVAB detection at enrollment for case patients (BV acquisition) versus controls (none). RESULTS: Of 239 women enrolled without BV, 199 were seen in follow-up, and 40 experienced BV; 15 had all samples for analysis. Detection of G. vaginalis in oral cavity or anal samples and Leptotrichia/Sneathia spp. in anal samples was more common at enrollment among case patients, who also had higher concentrations of these bacteria and Megasphaera relative to 30 controls at each site. In contrast, L. crispatus was detected more frequently in anal samples among controls. CONCLUSIONS: Women who acquire BV are more likely have previous colonization of extravaginal reservoirs with some BVAB, and less likely to have L. crispatus, suggesting that BVAB may be acquired vaginally from extravaginal reservoirs.

Vaginal bacteria elicit acute inflammatory response in fallopian tube organoids: a model for pelvic inflammatory disease.

Objective: To facilitate in vitro mechanistic studies in pelvic inflammatory disease (PID) and subsequent tubal factor infertility, as well as ovarian carcinogenesis, we sought to establish patient tissue derived fallopian tube (FT) organoids and to study their inflammatory response to acute vaginal bacterial infection. Design: Experimental study. Setting: Academic medical and researchcenter. Patients: FT tissues were obtained from four patients after salpingectomy for benign gynecological diseases. Interventions: We introduced acute infection in the FT organoid culture system by inoculating the organoid culture media with two common vaginal bacterial species, Lactobacillus crispatus and Fannyhesseavaginae . Main Outcome Measures: The inflammatory response elicited in the organoids after acute bacterial infection was analyzed by the expression profile of 249 inflammatory genes. Results: Compared to the negative controls that were not cultured with any bacteria, the organoids cultured with either bacterial species showed multiple differentially expressed inflammatory genes. Marked differences were noted between the Lactobacillus crispatus infected organoids and those infected by Fannyhessea vaginae . Genes from the C-X-C motif chemokine ligand (CXCL) family were highly upregulated in F. vaginae infected organoids. Flow cytometry showed that immune cells quickly disappeared during the organoid culture, indicating the inflammatory response observed with bacterial culture was generated by the epithelial cells in the organoids. Conclusion : Patient tissue derived FT organoids respond to acute bacterial infection with upregulation of inflammatory genes specific to different vaginal bacterial species. FT organoids is a useful model system to study the host-pathogen interaction during bacterial infection which may facilitate mechanistic investigations in PID and its contribution to tubal factor infertility and ovarian carcinogenesis.

Identification of fallopian tube microbiota and its association with ovarian cancer: a prospective study of intraoperative swab collections from 187 patients.

Investigating the human fallopian tube (FT) microbiota has significant implications for understanding the pathogenesis of ovarian cancer (OC). In this large prospective study, we collected swabs intraoperatively from the FT and other surgical sites as controls to profile the microbiota in the FT and to assess its relationship with OC. 81 OC and 106 non-cancer patients were enrolled and 1001 swabs were processed for 16S rRNA gene PCR and sequencing. We identified 84 bacterial species that may represent the FT microbiota and found a clear shift in the microbiota of the OC patients when compared to the non-cancer patients. Of the top 20 species that were most prevalent in the FT of OC patients, 60% were bacteria that predominantly reside in the gastrointestinal tract, while 30% normally reside in the mouth. Serous carcinoma had higher prevalence of almost all 84 FT bacterial species compared to the other OC subtypes. The clear shift in the FT microbiota in OC patients establishes the scientific foundation for future investigation into the role of these bacteria in the pathogenesis of ovarian cancer. .

Vaginal bacteria elicit acute inflammatory response in fallopian tube organoids: a model for pelvic inflammatory disease.

Objective: To facilitate in vitro mechanistic studies in pelvic inflammatory disease (PID) and subsequent tubal factor infertility, as well as ovarian carcinogenesis, we sought to establish patient tissue derived fallopian tube (FT) organoids and to study their inflammatory response to acute vaginal bacterial infection. Design: Experimental study. Setting: Academic medical and research center. Patients: FT tissues were obtained from four patients after salpingectomy for benign gynecological diseases. Interventions: We introduced acute infection in the FT organoid culture system by inoculating the organoid culture media with two common vaginal bacterial species, Lactobacillus crispatus and Fannyhessea vaginae . Main Outcome Measures: The inflammatory response elicited in the organoids after acute bacterial infection was analyzed by the expression profile of 249 inflammatory genes. Results: Compared to the negative controls that were not cultured with any bacteria, the organoids cultured with either bacterial species showed multiple differentially expressed inflammatory genes. Marked differences were noted between the Lactobacillus crispatus infected organoids and those infected by Fannyhessea vaginae . Genes from the C-X-C motif chemokine ligand (CXCL) family were highly upregulated in F. vaginae infected organoids. Flow cytometry showed that immune cells quickly disappeared during the organoid culture, indicating the inflammatory response observed with bacterial culture was generated by the epithelial cells in the organoids. Conclusion: Patient tissue derived FT organoids respond to acute bacterial infection with upregulation of inflammatory genes specific to different vaginal bacterial species. FT organoids is a useful model system to study the host-pathogen interaction during bacterial infection which may facilitate mechanistic investigations in PID and its contribution to tubal factor infertility and ovarian carcinogensis.

Detection of hydrogen peroxide-producing Lactobacillus species in the vagina: a comparison of culture and quantitative PCR among HIV-1 seropositive women.

BACKGROUND: The presence of hydrogen peroxide (H(2)O(2)) producing Lactobacillus in the vagina may play a role in controlling genital HIV-1 shedding. Sensitive molecular methods improve our ability to characterize the vaginal microbiota; however, they cannot characterize phenotype. We assessed the concordance of H(2)O(2)-producing Lactobacillus detected by culture with quantitative PCR (qPCR) detection of Lactobacillus species commonly assumed to be H(2)O(2)-producers. METHODS: Samples were collected as part of a prospective cohort study of HIV-1 seropositive US women. Cervicovaginal lavage specimens were tested for L. crispatus and L. jensenii using 16S rRNA gene qPCR assays. Vaginal swabs were cultured for Lactobacillus and tested for H(2)O(2)-production. We calculated a kappa statistic to assess concordance between culture and qPCR. RESULTS: Culture and qPCR results were available for 376 visits from 57 women. Lactobacilli were detected by culture at 308 (82%) visits, of which 233 of 308 (76%) produced H(2)O(2). L. crispatus and/or L. jensenii were detected at 215 (57%) visits. Concordance between detection of L. crispatus and/or L. jensenii by qPCR and H(2)O(2)-producing Lactobacillus by culture was 75% (kappa = 0.45). CONCLUSIONS: Among HIV-1 seropositive women, there was a moderate level of concordance between H(2)O(2)-producing Lactobacillus detected by culture and the presence of L. crispatus and/or L. jensenii by qPCR. However, one-quarter of samples with growth of H(2)O(2)-producing lactobacilli did not have L. crispatus or L. jensenii detected by qPCR. This discordance may be due to the presence of other H(2)O(2)-producing Lactobacillus species.

Changes in key vaginal bacteria among postpartum African women initiating intramuscular depot-medroxyprogesterone acetate.

BACKGROUND: The ECHO trial has relieved apprehension about intramuscular depot medroxyprogesterone acetate (DMPA-IM), however it is still important to understand how DMPA-IM affects the vaginal environment. We sought to describe how DMPA-IM initiation influences vaginal bacteria associated with HIV acquisition in postpartum women. METHODS: Vaginal swabs were collected for Nugent score determination and taxon-specific quantitative PCR of eight bacteria. Enrollment occurred at contraceptive initiation (DMPA-IM or non-hormonal contraception (non-HC)) and repeat vaginal swabs were collected after three months. Generalized estimating equations were used to estimate changes in Nugent score, total bacterial load, and taxa concentrations among contraceptive groups. RESULTS: Women who chose DMPA-IM (n = 33) were more likely to be married (97%vs.67%) and have resumed intercourse since delivery (52%vs.29%) compared to women who chose non-HC (n = 21). After three months, significant decreases in the concentrations of Sneathia species, Mycoplasma hominis, and Parvimonas species Type 1 were seen among non-HC users, however concentrations remained stable among DMPA-IM users; contraceptive method was associated with significantly different changes in M. hominis concentration between groups (p = 0.010). CONCLUSIONS: Our findings suggest that postpartum use of DMPA-IM and non-HC may have differential impacts on the vaginal concentrations of some bacteria that have previously been associated with HIV acquisition.

Tenofovir disoproxil fumarate intravaginal ring for HIV pre-exposure prophylaxis in sexually active women: a phase 1, single-blind, randomised, controlled trial.

BACKGROUND: An intravaginal ring that releases the tenofovir prodrug, tenofovir disoproxil fumarate, provided 100% protection in macaques against simian HIV and was safe in a 14-day clinical trial in sexually abstinent women. We aimed to assess the safety and pharmacokinetics of this intravaginal ring over 90 days in sexually active women. METHODS: We did a phase 1, single-blind, randomised, placebo-controlled trial to assess safety, pharmacokinetics, and acceptability of a tenofovir disoproxil fumarate intravaginal ring used continuously with monthly ring changes for 3 months. Sexually active women who were HIV negative were randomly assigned (3:1) to a tenofovir disoproxil fumarate ring or placebo ring. Primary safety endpoint was the proportion of women who had grade 2 or higher genitourinary adverse events judged related to study product and any grade 2 or higher adverse event as defined by the Division of AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events. We quantified tenofovir disoproxil fumarate and tenofovir concentrations in cervicovaginal fluid, tenofovir in plasma, and tenofovir diphosphate, the active metabolite, in cervical tissue and dried blood spots 1 month after each ring insertion. We compared changes over time in cervicovaginal fluid cytokine and chemokine concentrations and vaginal microbiota. The study was electively stopped early and is registered with ClinicalTrials.gov, number NCT02762617. FINDINGS: Between Feb 24 and July 20, 2017, 17 women were enrolled before study termination. 12 were assigned to receive the tenofovir disoproxil fumarate ring and five were assigned to receive the placebo ring. Two participants in the tenofovir disoproxil fumarate ring group completed 3 months of continuous ring use; eight were asked to discontinue ring use early because of ulcerations (grade 1) near the ring; in the remaining two women, rings were electively removed by study staff on day 20 and day 23. Ulcers were detected a mean of 32 days after ring use (range 23-56). Four of eight participants with ulcers were symptomatic with vaginal discharge; four had ulcers identified when examined; three had two ulcers; all ulcers resolved after ring removal. No participants in the placebo group developed ulcers. No grade 2 product-related adverse events were reported in either group and four non-product-related grade 2 adverse events were reported in the tenofovir disoproxil fumarate ring group. Cervicovaginal fluid tenofovir concentrations did not differ at day 14 (p=0·14) comparing the eight patients who did (median 1·0 × 105 ng/mL [IQR 9·1 × 104-1·1 × 105]) with the four who did not (6·0 × 104 ng/mL [5·6 × 104-1·1 × 105]) develop ulcers. No significant changes in vaginal microbiota were detected in either group. Concentrations of multiple inflammatory cytokines and chemokines were significantly higher at days 14 and 28 compared with baseline in the tenofovir disoproxil fumarate ring group but not the placebo group. INTERPRETATION: Future studies are needed to establish whether the unanticipated finding of ulcerations is specific to this tenofovir disoproxil fumarate ring or generalisable to other sustained topical release formulations of tenofovir or its prodrugs. FUNDING: National Institutes of Health.

Associations between improvement in genitourinary symptoms of menopause and changes in the vaginal ecosystem.

OBJECTIVE: The aim of the study was to identify associations between improvement in genitourinary symptoms of menopause (GSM) and vaginal microbiota, vaginal glycogen, and serum estrogen. METHODS: Thirty postmenopausal women enrolled in a hot flash treatment trial (oral estradiol vs venlafaxine vs placebo) who reported GSM and provided vaginal swabs at 0, 4, and 8 weeks were studied. Bacterial communities were characterized using deep sequencing targeting the 16S rRNA gene V3-V4 region. Participants selected a most bothersome genitourinary symptom (dryness, discharge, pain, itch/burn, or inability to have sex) and rated severity on a 10-point scale at baseline and 8 weeks. Vaginal glycogen and serum estradiol and estrone were measured at enrollment and 8 weeks. Comparisons according to improvement in most bothersome symptom (MBS) were made using χ, Wilcoxon signed-rank test, or Hotelling's t test. RESULTS: Of 30 participants, 21 (70%) had improvement in MBS over the 8-week study and 9 (30%) had no improvement or worsening of MBS. A higher proportion of women receiving estradiol or venlafaxine reported improvement in MBS (88%, 78%) compared with placebo (54%; P = 0.28). MBS improvement was associated with Lactobacillus-dominant vaginal microbiota at enrollment (57% vs 22%, P = 0.08). Vaginal glycogen, serum estradiol, and estrone significantly increased in women whose MBS improved. CONCLUSIONS: A larger proportion of women whose MBS improved had a Lactobacillus dominant microbiota at enrollment than those who had no improvement during the trial, though this difference was not statistically significant. Larger trials are needed to determine whether vaginal microbiota modify or mediate treatment responses in women with GSM.