Objective
Genital tract infection accounts for approximately 25-40% of all preterm births. We sought to assess the relationship between preterm birth and selected vaginal bacterial taxa associated with preterm birth either directly or through their association with bacterial vaginosis (BV).
Study Design
Vaginal fluid for Gram stain was collected between 17 and 22 weeks’ gestation as part of a randomized trial of ultrasound-indicated cerclage for preterm birth prevention in women at high risk for recurrent spontaneous preterm birth. Bacterial deoxyribonucleic acid was extracted from the Gram stain slides and analyzed using quantitative polymerase chain reaction.
Results
Among the 499 participants, Mycoplasma was positively correlated with increased risk of preterm (risk ratio [RR], 1.83; 95% confidence interval [CI], 1.52–2.22) as was Mobiluncus (RR, 1.36; 95% CI, 1.07–1.73) and Atopobium (RR, 1.44; 95% CI, 1.1–1.87). However, there were strong interactions between the race/ethnic group and the presence of these and other individual taxa on risk of preterm birth. By contrast, bacterial vaginosis–associated bacteria (BVAB)-3 was consistently associated with a reduction in the risk of preterm birth for all racial/ethnic groups (0.55; 95% CI, 0.39–0.78).
Conclusion
BV is characterized by a reduction of Lactobacillus , and lactic acid–producing bacteria and the presence of Mobiluncus ; we found these factors and the presence of Mycoplasma to be associated with an increased risk of preterm birth. By contrast, the presence of a recently identified organism sufficient to cause BV, BVAB3, decreased the risk of preterm birth. These findings give insight into why treating BV has mixed impact on risk of preterm birth.
In 2011, 11.72% of all births in the United States occurred prior to 37 completed weeks of gestation. Prematurity was most common among non-Hispanic blacks (16.75% compared with 10.49% for non-Hispanic white). The costs to society of preterm birth are large: an estimated $26 billion dollars in 2005. Prematurity is also a major cause of infant mortality.
Infection, both overt and subclinical, is thought to account for approximately 25-40% of all preterm births. One condition, bacterial vaginosis (BV), is associated with a significant risk of preterm birth, with estimates ranging as high as an 8-fold increase. However, the effects of BV treatment on reducing rates of preterm birth have been disappointing; vaginal metronidazole therapy has been associated with an increased risk of preterm birth in some groups.
One significant limitation of previous studies is their focus on the association of BV, diagnosed by Nugent or Amsel criteria, with preterm birth, rather than on the multiple specific taxa found in conjunction with BV, which include Mobiluncus and the recently identified bacterial vaginosis-associated bacteria (BVAB) in the order Clostridiales: BVAB1, BVAB2, and BVAB3. Although it is clear that vaginal microbes associated with BV are correlated with an increased risk of preterm birth, the role of specific microbial taxa is much less clear.
Application of genetic techniques for bacterial identification has substantially eased the difficulties of screening vaginal specimens for the presence of multiple different taxa. We used quantitative polymerase chain reaction (PCR) to analyze vaginal specimens collected during the second trimester of pregnancy from women at high risk of recurrent preterm birth for the presence and relative load of bacterial taxa either associated with BV or that have been previously associated with preterm birth. These included the recently identified BVAB1, BVAB2, and BVAB3.
Materials and Methods
Study population and sample collection
Vaginal fluid for Gram stains and clinical data were collected as part of a multicenter, randomized trial of ultrasound-indicated cerclage for preterm birth prevention. Women with a singleton gestation and at least 1 previous spontaneous preterm birth of 17-33 weeks’ gestation were eligible for enrollment. Cervical length was measured at the initial sonographic cervical length evaluation, which was scheduled between 16 and 21 weeks’ gestation. At the visit, a sterile speculum examination was performed to collect vaginal fluid from the upper one-third of the vaginal sidewalls for pH and Gram stain. Serial transvaginal ultrasound was conducted throughout the study. If the cervical length shortened to less than 25 mm, the participant became eligible for randomization to cervical cerclage or a no-cerclage cohort.
The study protocol was approved by the Human Subjects Committee at the University of Alabama at Birmingham (X991227014) and similar committees at all participating sites. The use of deidentified data for this study was deemed exempt and unregulated by the Human Subjects Committee at the University of Michigan because the original consent form provided for the collection and analysis of biological specimens as it related to studying the etiology of preterm birth.
This microbiological study includes samples from participants whose cervical length shortened to less than 25 mm and were randomized to not receive cerclage and samples from participants not eligible for the intervention trial because their cervical length remained at least 25 mm. We defined preterm birth as birth less than 37 weeks’ gestation.
For this study we identified 608 women with associated Gram stain slides and ultrasound measurement. Women with an inconclusive ultrasound because of a poorly developed lower segment (n = 76) were included because their cervical length was longer than the 25 mm cutoff we used to define cervical shortening, although not measureable precisely. Of the 608 subjects, 109 were excluded for various reasons: inability to extract and test bacterial deoxyribonucleic acid (DNA) (n = 32), self-reported racial/ethnic group was other (n = 46), or missing critical data on sample collection (n = 31), leaving a total of 499 for analysis.
Quantitative PCR assay for targeted bacteria taxa
This technique enables the estimation of the number of bacterial cells present and classification of bacteria based on the sequence of a region of a gene coding for the ribosome, which is present in all cells. Resolution to the species level is not always possible (some species in the same genus may have the same genetic sequence in the region of interest); therefore, we use the term bacterial taxon throughout the manuscript.
Bacterial DNA was extracted from the Gram stain slides as described by Srinivasan et al. Preamplification was done using 8F-1492R universal bacterial primers based on 16S rRNA. Total bacterial and species-specific bacterial ribosomal gene copy numbers were identified using total bacteria primers and primers specific to each bacteria taxa. The targeted bacteria genera and species include Atopobium spp ; BVAB types 1, 2, and 3 in the order Clostridiales ; Escherichia coli ; Gardnerella vaginalis ; group B Streptococcus ; Lactobacillus spp ; Mobiluncus spp ; Mycoplasma spp ; and Ureaplasma spp.
We also used a primer set for lactic acid bacteria that includes lactic acid–producing bacteria of the genera Lactobacillus , Pediococcus , Leuconostoc , and Weissella . Total bacterial load was estimated using a broad-coverage universal bacterial primer for the 16S rrn gene. Primer sequences and conditions used for individual taxa were reported previously. The limit of detection was 100 copies and readings lower than the limit were considered negative. We calculated the relative proportion of 16S rrn copies from each bacterial taxon using the bacterial copy number measured by each specific bacterial assay divided by the total number of 16S rrn bacterial copies measured by the universal bacterial assay.
Statistical analysis
The prevalence of each bacterial taxon was calculated overall and stratified by cervical length. To evaluate the association between the presence of each bacterial taxon and cervix shortening to less than 25 mm, we used the ratio of the prevalence among women with shortened relative to normal cervices. In addition, we estimated the risk ratio (RR) of preterm birth relative to term birth, associated with the presence of each bacterial taxon, stratified by self-reported racial/ethnic group, and estimated the 95% confidence intervals (CIs) around the ratio.
To test for an effect of abundance, or load, of each bacterial taxon (that is, a dose response), we fit a set of logistic regression models. The models included preterm birth as the dependent variable and the participant’s self-reported racial/ethnic group (white, black, Hispanic), cervical length status (<25 mm vs ≥25 mm), and the proportion of a given bacterial taxon relative to the total bacteria load as covariates; separate models were fit for each bacterial taxon. Because of observed interactions, for Lactobacillus , we fit separate models by race and included the presence/absence of Mycoplasma and cervical length status as covariates. Statistical tests were conducted using the software program R, version 2.15.2.
Results
As expected, because of the entry criteria of the study (at least 1 prior preterm birth; see Materials and Methods ), the proportion of women with a preterm birth less than 37 weeks in this population was very high, with blacks having significantly higher rates of preterm birth than Hispanics or whites as well as significantly higher rates of cervical shortening (<25 mm). Smoking and douching habits also differed significantly by race ( Table 1 ).
| Demographic | Black (n = 207) | Hispanic (n = 155) | White (n = 137) |
|---|---|---|---|
| Birth status a | |||
| Preterm | 107 (51.7%) | 45 (29%) | 40 (29.2%) |
| Term | 100 (48.3%) | 110 (71.0%) | 97 (70.8%) |
| Age (mean ± SD) | 26.2 ± 4.7 | 27.3 ± 5.6 | 27.8 ± 5.8 |
| BMI (mean ± SD) | 31.1 ± 8.3 | 27.7 ± 5.4 | 27.5 ± 6.3 |
| Shortened cervix (<25 mm) at sampling a | 25 (14.4%) | 1 (0.7%) | 7 (6.1%) |
| Shortened cervix (<25 mm) between sampling and the end of study a | 51 (24.6%) | 12 (7.7%) | 19 (13.9%) |
| Smoking during pregnancy a | 43 (20.8%) | 2 (1.3%) | 36 (26.2%) |
| Douching before or during pregnancy a | 66 (31.9%) | 26 (16.8%) | 26 (19.0%) |
a Prevalence was significantly different by racial/ethnic group using the χ 2 test ( P < .05).
The overall prevalence of selected bacterial taxa varied by racial/ethnic group, with significant differences for Atopobium (χ 2 test, P = .001), BVAB1 ( P = .00004), BVAB2 ( P = .001), BVAB3 ( P = .008), Gardnerella ( P = .004), group B Streptococcus ( P = .004), and Mycoplasma ( P = .00008) ( Figure 1 ). The prevalence of BVAB1 and Mycoplasma were positively and significantly associated with a shortened cervix and Ureaplasma negatively associated with a shortened cervix, but the prevalence of other taxa tested did not vary by cervical length ( Table 2 ).
| Bacteria taxa | Prevalence | Cervix a | PR and 95% CI | ||
|---|---|---|---|---|---|
| All women | Shortened | Not shortened | PR | (95% CI) | |
| Atopobium | 73.2% | 73.9% | 73.0% | 1.17 | (1.00–1.37) |
| BVAB1 | 39.3% | 41.7% | 38.5% | 1.52 b | (1.11–2.08) b |
| BVAB2 | 22.8% | 29.6% | 20.8% | 1.21 | (0.68–2.17) |
| BVAB3 | 19.4% | 21.7% | 18.8% | 1.44 | (0.80–2.60) |
| Escherichia coli | 98.8% | 100.0% | 98.4% | 1.00 | (0.96–1.04) |
| Gardnerella | 35.9% | 44.3% | 33.3% | 1.29 | (0.87–1.91) |
| Group B Streptococcus | 76.3% | 91.2% | 71.5% | 1.11 | (0.95–1.30) |
| Lactic acid bacteria | 99.0% | 99.1% | 99.0% | 1.00 | (0.96–1.04) |
| Lactobacillus | 99.8% | 100.0% | 99.7% | 0.99 | (0.95–1.03) |
| Mobiluncus | 68.5% | 73.9% | 66.9% | 1.14 | (0.93–1.39) |
| Mycoplasma | 31.4% | 60.6% | 22.8% | 2.02 b | (1.47–2.78) b |
| Ureaplasma | 46.5% | 39.8% | 48.5% | 0.59 b | (0.34–1.03) b |
a Whether cervix subsequently shortened to <25 mm, as measured using vaginal ultrasound
Race/ethnicity strongly modified the crude associations between an individual bacterial taxon during the second trimester and risk of subsequent preterm birth ( Table 3 ). Several taxa were overall positively associated with preterm birth, but the risk ratios differed considerably among racial/ethnic groups, sometimes even shifting from hazardous (risk ratio greater than 1) to protective (risk ratio less than 1).
| Variable | All, preterm (n = 233), Term (n = 312) | Black, Preterm (n = 107), Term (n = 100) | Hispanic, Preterm (n = 45), Term (n = 110) | White, Preterm (n = 40), Term (n = 97) |
|---|---|---|---|---|
| Atopobium | 1.44 (1.1–1.87) a | 1.02 (0.72–1.43) | 1.77 (0.97–3.2) | 1.24 (0.65–2.37) |
| BVAB1 | 1.01 (0.83–1.24) | 0.99 (0.76–1.29) | 1.02 (0.6–1.76) | 0.64 (0.34–1.19) |
| BVAB2 | 1.2 (0.97–1.48) | 1.16 (0.88–1.52) | 1.06 (0.54–2.07) | 0.97 (0.51–1.87) |
| BVAB3 | 0.55 (0.39–0.78) a | 0.55 (0.36–0.83) a | 0.31 (0.08–1.2) | 0.38 (0.13–1.14) |
| Escherichia coli | 1.5 (0.46–4.87) | 2.62 (0.45–15.22) | b | c |
| Gardnerella | 1.08 (0.88–1.31) | 1.04 (0.8–1.35) | 1.24 (0.75–2.04) | 0.78 (0.41–1.49) |
| Group B Streptococcus | 1.08 (0.85–1.37) | 0.87 (0.64–1.19) | 2.38 (1.14–4.98) a | 0.74 (0.42–1.31) |
| Lactic acid bacteria c | — | — | — | — |
| Lactobacillus d | — | — | — | — |
| Mobiluncus | 1.36 (1.07–1.73) a | 1.42 (0.99–2.04) | 1.91 (1.00–3.65) a | 0.85 (0.5–1.45) |
| Mycoplasma | 1.83 (1.52–2.22) a | 1.97 (1.50–2.57) a | 2.81 (1.76–4.5) a | 0.88 (0.46–1.66) |
| Ureaplasma | 0.84 (0.68–1.02) | 1 (0.77–1.31) | 0.79 (0.47–1.31) | 0.53 (0.29–0.95) a |
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