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.
Bacterial vaginosis (BV) is a vaginal syndrome that is characterized by a vaginal microbiota with a diverse community of anaerobic species. BV in pregnancy has been associated with a 2-fold increase in preterm birth, presumably because of ascending infection and inflammation. BV is characterized by complex and heterogeneous bacterial communities, and it is unclear whether particular bacterial species are associated with an increased risk of preterm birth. Current therapy for BV has been unsuccessful in reducing rates of preterm birth. If a particular pathogen could be identified as more inflammatory or more pathogenic, it might allow more targeted therapy to reduce preterm deliveries.
Defensins are cationic antimicrobial peptides that are produced as part of the innate immune response at mucosal surfaces. The human beta defensins (HBD) are primarily produced by epithelial cells and have been shown to be elevated in the amniotic fluid of women with intrauterine infection and preterm labor. However, there are mixed data about the response of HBD to BV; some studies have shown an increase, and other studies have shown no change. Alpha-defensins (human neutrophil peptides; HNP) are primarily produced by neutrophils and have also been shown to be elevated in the amniotic fluid of women with intrauterine infection and preterm labor; however, data about levels in women with and without BV are mixed.
We hypothesized that bacterial species that are associated both with BV and with preterm birth in a pregnant cohort, such as Gardnerella vaginalis and Megasphaera species, would increase beta-defensin levels, but not alpha-defensin levels, because neutrophils usually are absent in women with BV. We evaluated this association in a cohort study of pregnant women with a high prevalence of BV.
Materials and Methods
This analysis was conducted on a subset of pregnant women who were enrolled in a prospective cohort study of vaginal bacteria and preterm birth in Philadelphia, PA (ProjectBABIES). English- or Spanish-speaking pregnant women were eligible for enrollment in the parent study if they were at <16 weeks’ gestation, based on self-reported last menstrual period, if they lived in Philadelphia, and if they contributed multiple vaginal swabs to measure various aspects of BV. At enrollment, women completed an interview and self-collected vaginal swabs, which then were stored at −80°C until processing. Vaginal fluid self-collected were spread on a glass slide and transported, in batches, to the clinical microbiology laboratory at the University of Pennsylvania for gram staining and BV identification with the Nugent criteria. All slides were examined and interpreted by a single individual during the course of the study. BV was diagnosed for the study by Nugent score of 7-10. Women were not treated for BV as part of this study. These procedures were repeated at a follow-up visit scheduled at <28 weeks’ gestation. At the follow-up visit, the women were asked about interim diagnoses of sexually transmitted infections, BV, and/or any antibiotic treatment. Women were included in the substudy if they were enrolled between June 2008 and January 2010 and had both an enrollment and a follow-up swab available for analysis.
Swabs were thawed and then eluted into 1-mL phosphate-buffered saline solution and then centrifuged at 10,000 g for 10 minutes. The resulting cell pellet underwent DNA extraction with the MoBio BiOstic Bacteremia kit (MoBio Laboratories Inc, Carlsbad, CA) and testing with species-specific quantitative polymerase chain reaction (qPCR) assays for the BV-associated species G vaginalis, Atopobium vaginae, Leptotrichia spp, Sneathia spp, Mobiluncus spp, Megasphaera spp , and BV-associated bacterium (BVAB) 1, 2, and 3. As previously described, all samples also underwent qPCR testing for the human 18S gene to confirm contact with a mucosal surface and evaluation for PCR inhibition with an amplification control. Mock swabs were put through DNA extraction and PCR as a negative control to detect reagent contamination. Samples that had undetectable levels of bacteria were assigned the value of lower limit of the assay (250 copies 16S ribosomal RNA/swab).
Commercial enzyme-linked immunosorbent assay kits were used to test the swab supernatant for HBD2, HBD3 (Alpha Diagnostic International, San Antonio, TX), and HNP1-3 (Hycult Biotech, Plymouth Meeting, PA). Samples with undetectable levels of defensins were assigned the value of the lower limit of detection for each assay (HBD2, 12.5 pg/mL; HBD3, 50 pg/mL; HNP1-3, 156 pg/mL).
All comparisons of mean concentrations of defensins or bacteria were performed as a cross-sectional analysis at 1 time point: either enrollment or follow-up visit. Quantities of bacteria and defensins were log transformed for analysis. Mean quantities of bacteria at enrollment and follow-up visit were compared with the use of a paired Student t test. Mean quantities of defensins were compared between women with and without BV with the Student t test. Differences in defensin concentrations across quartiles of bacterial concentrations were compared with the use of analysis of variance. Associations between defensin concentrations and demographic factors were assessed with the use of linear regression with robust standard errors. We also conducted a longitudinal analysis using a multivariable linear regression model that was controlled for race. Percentage of change in quantity of bacteria between enrollment and follow-up visit was used as the independent variable; percentage of change in defensin concentration was used as the dependent variable.
Results
A total of 1560 pregnant women were enrolled in the parent study; 126 of them were selected for this subanalysis, which gave us 252 samples for analysis. Participants were primarily young, African American women, with a high school education or less ( Table 1 ). Women were enrolled mostly in the first trimester (mean, 10 ± 3 weeks’ gestation; range, 4−19 weeks) and returned for a follow-up visit in the mid second trimester, at a mean gestational age of 25 ± 6 weeks (range, 15−38 weeks). Two women (8%) were diagnosed with twin pregnancy between their enrollment and follow-up visit. There were no significant differences in race, education, pregnancy history, or prevalence of BV between women in the subanalysis and the larger cohort (data not shown).
| Variable | Measure |
|---|---|
| Age, y a | 24 ± 6 |
| Gravidity, n b | 1 (0, 3) |
| Parity, n b | 1 (0, 2) |
| Race, n (%) | |
| Black | 76 (60) |
| White | 8 (6) |
| Asian | 2 (2) |
| Hispanic | 36 (29) |
| Other | 4 (3) |
| Education, n (%) | |
| Some high school or less | 54 (43) |
| High school graduate or general education diploma | 47 (37) |
| Some college or vocational training | 21 (17) |
| College graduate | 4 (3) |
| Marital status, n (%) | |
| Single | 97 (77) |
| Married (or living as married) | 27 (21) |
| Separated | 2 (2) |
| Smoker, n (%) | 26 (21) |
| Gestational age at enrollment, wk a | 10 ± 3 (range, 4–19) |
| Nugent score, n (%) | |
| 0-3 | 8 (6) |
| 4-6 | 20 (16) |
| 7-10 | 93 (74) |
| Diagnosed with gonorrhea or chlamydia this pregnancy before enrollment, n (%) | 7 (6) |
| Detectable bacteria present at baseline, n (%) | |
| Gardenerella vaginalis | 116 (92) |
| Atopobium vaginae | 106 (84) |
| Leptotrichia/Sneathia | 89 (71) |
| Mobiluncus | 73 (58) |
| Megashpaera | 83 (66) |
| Bacterial vaginosis–associated bacteria | |
| 1 | 50 (40) |
| 2 | 77 (61) |
| 3 | 50 (40) |
Most bacterial species were detected in most of the population at enrollment, with the exception of BVAB1 and 3, which were detected in only 40% of women ( Table 1 ). This is likely due to the high prevalence of BV in 74% of participants (94/126 women). At the follow-up visit, the prevalence of BV had decreased to 60% (75/126 women). Only 2 women reported being diagnosed with BV by their physician between enrollment and the follow-up visit, both of whom reported treatment with antibiotics. Of those women with BV at the follow-up visit, only 4 (5%) had an incident of BV; 69 women (95%) had persistent or recurrent BV by Nugent score. In keeping with the decrease in prevalence of BV, the quantity of BVAB present decreased between enrollment and the follow-up visit among women with BV at enrollment but stayed constant among women with normal or intermediate flora at baseline ( Figure 1 ). Quantities of each bacteria were significantly higher among women with BV at enrollment compared with those without BV (data not shown).
HBD2 was detectable in 110 of women (87%) at enrollment; HBD3 was detectable in 76 women (60%), and HNP1-3 were detectable in 105 women (83%). Concentrations of HBD2 and HNP1-3 were not different between women with and without BV at enrollment, although HBD3 concentrations were significantly lower in women with BV ( Figure 2 ). For each increasing quartile of bacterial concentration at enrollment, HBD3 concentrations were significantly lower ( Figure 3 ) for all species except Mobiluncus , BVAB1, and BVAB3. No significant associations were seen between concentrations of individual bacterial species and HBD2 or HNP1-3. At the follow-up visit, HBD2 was detectable in 104 women (83%); HBD3 was detectable in 80 women (63%), and HNP1-3 was detectable in 107 women (85%). At the second visit, HBD2 was significantly higher in women with BV, although HBD3 concentrations were lower in women with BV and HNP1-3 was not different between women with and without BV ( Figure 2 ). A similar trend toward lower HBD3 concentrations with increasing quartiles of bacterial concentrations was seen at the follow-up visit, but reached only statistical significance for BVAB2 (data not shown). The 4 women who experienced incident BV between enrollment and the follow-up visit had significantly lower levels of HBD3 (median, 50 vs 4855 pg/mL; P = .001) and HNP1-3 (median, 3199 vs 37582 pg/mL; P = .001) at enrollment than the 24 women who remained BV negative through the entire study.
Defensin concentrations were not associated significantly with gestational age, either at enrollment or follow-up visit ( Table 2 ) or with maternal age or smoking status. However, African American and Hispanic women had lower concentrations of HNP1-3 compared with white women ( P < .001). For this reason, in our regression model that evaluated the association between change in bacterial concentrations between visits and the change in defensin concentrations, we adjusted only for race. In that multivariate model, with change in defensin concentrations as the dependent variable, increased concentrations of A vaginae , BVAB1, and BVAB2 were associated with decreased concentrations of HBD3, but not HBD2 or HNP1-3 ( Table 3 ). Adjustment for gestational age at enrollment or time between visits did not significantly change the results of this analysis (data not shown).
| Variable | Human beta defensins | Human neutrophil peptides 1-3 | |
|---|---|---|---|
| 2 | 3 | ||
| Age at enrollment | 0.01 (−0.02 to 0.04) | 0.02 (−0.01, 0.05) | −0.01 (−0.04, 0.03) |
| Gestational age at enrollment | −0.03 (−0.07 to 0.01) | 0.002 (−0.05 to 0.06) | −0.01 (−0.07 to 0.06) |
| Gestational age at follow-up visit | −0.02 (−0.04 to 0.01) | −0.02 (−0.05 to 0.01) | 0.02 (−0.01 to 0.06) |
| Smoker at enrollment | 0.01 (−0.24 to 0.42) | 0.32 (−0.15 to 0.79) | −0.02 (−0.58 to 0.54) |
| Race | |||
| White | Reference | Reference | Reference |
| Black | −0.15 (−0.46 to 0.16) | 0.10 (−0.55 to 0.75) | −0.89 (−1.26 to −0.53) a |
| Hispanic | −0.20 (−0.55 to 0.15) | −0.04 (−0.73 to 0.64) | −1.04 (−1.49 to −0.59) a |
| Asian | 0.18 (−0.14 to 0.50) | −0.50 (−1.53 to 0.52) | 0.13 (−0.65 to 0.90) |
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