Objective
The purpose of this study was to estimate the effect of bacterial vaginosis on midtrimester cervical length in women at increased risk for recurrent spontaneous preterm birth.
Study Design
We conducted a secondary analysis of prerandomization data from a multicenter trial of ultrasound-indicated cerclage. Women with previous spontaneous preterm birth at <34 weeks’ gestation underwent initial cervical length assessment and vaginal fluid collection at 16-21 weeks 6 days gestation. Gram stains were scored with Nugent criteria. With serial scans, the shortest cervical length was observed.
Results
Records for 949 women had complete data. In unadjusted regression models, Nugent score ( P = .003) and vaginal fluid pH ( P = .008) were related inversely to cervical length. Women with bacterial vaginosis based on Nugent score ≥7 ( P = .04) or pH ≥5 ( P = .016) had significnatly lower cervical length than unaffected women; however, all of these effects were null after covariate adjustment.
Conclusion
Nugent score, pH level, and bacterial vaginosis are associated inversely with cervical length; however, these relationships become null after adjustment for relevant covariates.
Preterm birth (PTB) is the leading cause of perinatal morbidity and death ; most PTBs occur spontaneously and not for maternal-fetal indications. The incidence of PTB continues to rise largely because of our poor understanding of the pathophysiologic evidence and the paucity of effective interventions. There are multiple plausible causes for spontaneous PTB, which include asymptomatic upper genital tract infection. A Cochrane review estimated that one-third of all PTB is associated with largely asymptomatic intrauterine infection that results directly from genital tract colonization. Numerous microorganisms are associated with asymptomatic infection; the most common associated condition is bacterial vaginosis (BV).
The diagnosis of BV can be made with clinical Amsel criteria ; however, vaginal pH testing is a valuable screening tool because of its efficiency and cost-effectiveness. Vaginal-fluid Gram stain with quantitative assessment of the microbial flora has high sensitivity and specificity and is accepted widely as an alternate diagnostic technique; with the use of these 2 methods, BV can be diagnosed by a Nugent score of ≥7 or vaginal pH of ≥5.
Recent studies have confirmed an association between BV and spontaneous PTB, with adjusted odds ratios between 1.4 and 6.9. Multiple putative mechanisms by which BV leads to PTB exist; however, the exact pathway remains speculative. A possible mechanism for the link between asymptomatic genital infection and PTB is the bacterial stimulation of prostaglandin release or bacterial endotoxin that is introduced into the amniotic fluid and leads to cytokine release and spontaneous labor. In women at high risk for spontaneous PTB, the detection and treatment of asymptomatic BV appeared to reduce the rate of preterm premature rupture of membranes and low birthweight markedly, although the rate of subsequent PTB was not statistically different. We hypothesized that the pathophysiologic effect of BV could operate through changes in the cervical matrix and result in shortened midtrimester cervical length, which is recognized widely as a harbinger of spontaneous PTB.
The aim of this study was to determine whether BV, which is diagnosed by either Nugent score or vaginal pH, would predict midtrimester cervical length in women who are at increased risk for recurrent PTB.
Methods
Recruitment and protocol
This is a planned, secondary analysis of prerandomization data from the National Institute of Child Health and Human Development–sponsored randomized trial of cerclage for PTB prevention. Sixteen US clinical centers enrolled patients between January 2003 and November 2007. Healthy multiparous women who entered prenatal care were screened to identify those women with at least 1 previous spontaneous PTB between 17 weeks 0 days and 33 weeks 6 days’ gestation.
Exclusion criteria were fetal anomaly, planned history-indicated cerclage for a clinical diagnosis of cervical insufficiency and clinically significant maternal-fetal complications that would increase the risk of an indicated PTB. Qualifying women were invited to consent for the ultrasound screening phase of the study. Further details of the study protocol are described elsewhere.
Consenting women underwent serial transvaginal ultrasound examinations to measure cervical length, the first of which was scheduled in the gestational age window 16 weeks 0 days to 21 weeks 6 days gestation. Follow-up scans were scheduled every 2 weeks, unless the cervical length was observed to be 25-29 mm, after which the scan frequency was increased to weekly. Women with a cervical length that remained at least 25 mm by the final sonographic evaluation, which was scheduled to be no later than 22 weeks 6 days gestation, were ineligible for random assignment and resumed their obstetric care. If, on any evaluation, the cervical length was <25 mm, the woman became eligible for random assignment. Cervical length was obtained by the standard technique as described by Iams et al. The shortest observed cervical length from the serial evaluations was chosen for this analysis.
Before the initial sonographic cervical length evaluation, a sterile speculum examination was performed to collect vaginal fluid for pH and Gram stain. With a dry, cotton-tipped applicator or Dacron swab, a vaginal secretion specimen from the upper one-third of the vaginal sidewalls was obtained. The swab was touched to the reagent block on the indicator strip (ColorpHast; Capitol Scientific Inc, Austin, TX). The pH was read after the color stabilized, but before the paper dried completely. The vaginal pH was recorded. If the color fell between 2 values on the chart, the pH was rounded to the higher value. The pH was not made available to managing physicians. With a second sterile cotton or Dacron swab, vaginal fluid was obtained from the upper one-third of the vaginal sidewalls. The vaginal fluid was spread on a glass slide, allowed to air dry, and transported to the laboratory for a Nugent score determination. Only a thin layer of vaginal secretion was used. The swab was rolled gently across the entire glass slide, from end to end, as evenly as possible; “blobbing” was avoided. After the slide was allowed to air dry, the slide was labeled on the frosted part of the slide with the center number, the patient’s study identification, and the date of collection; no fixative was used.
After trial completion, the slides underwent Gram staining in batch and were analyzed. Five oil-immersion fields were examined by a single trained investigator (M.M.P.) for the presence of Lactobacillus, Gardnerella vaginalis, Bacteroides , and Mobiluncus morphotypes with the depicted microscopic scoring scale ( Table 1 ). The Gram stains were then scored according to Nugent criteria ( Table 2 ). A Nugent score of 0-10 was assigned to each slide.
| Score | Morphotype |
|---|---|
| 0 | 0 |
| <1 | 1+ |
| 1-4 | 2+ |
| 5-30 | 3+ |
| >30 | 4+ |
| Morphotype | Quantity | Points |
|---|---|---|
| Lactobacilli | 4+ | 0 |
| 3+ | 1 | |
| 2+ | 2 | |
| 1+ | 3 | |
| 0 | 4 | |
| Gardnerella vaginalis/Bacteroides | 0 | 0 |
| 1+ | 1 | |
| 2+ | 2 | |
| 3+ | 3 | |
| 4+ | 4 | |
| Mobiluncus | 0 | 0 |
| 1+/2+ | 1 | |
| 3+/4+ | 2 |
Statistical analysis
Linear regression was used to model the effects of both Nugent score and pH on the shortest observed cervical length ( Figure ) . Similar analyses examined the effect of the diagnosis of BV that was based on both criteria. Multivariable linear regression models were used to evaluate these effects in the presence of covariates that are predictive of PTB. Table 3 shows the predictors that were included in the model for coefficients and probability values. SAS software (version 9.2; SAS Institute, Cary, NC) was used for all statistical analyses. A level of .05 was selected to indicate statistical significance.
| Covariate adjusted models | Coefficient (95% CI) | P value |
|---|---|---|
| Nugent score | –0.10 (–0.28 to 0.07) | .25 |
| Race | ||
| Black | 0.77 (–0.62 to 2.17) | .28 |
| Hispanic | 2.95 (1.43–4.47) | < .01 |
| Asian/other | 2.82 (1.13–4.50) | < .01 |
| White | Referent | — |
| Age | 0.08 (–0.02 to 0.17) | .11 |
| Gestational age of earliest previous preterm birth | 0.16 (0.05–0.26) | < .01 |
| Gestational age at 1st sonogram | 1.20 (0.81–1.58) | < .01 |
| Cervical length at 1st sonogram | 0.45 (0.41–0.49) | < .01 |
| Cigarette use | 0.93 (–0.53 to 2.39) | .21 |
| pH | –0.73 (–1.48 to 0.03) | .06 |
| Race | ||
| Black | 0.76 (–0.62 to 2.14) | .28 |
| Hispanic | 2.86 (1.34–4.38) | < .01 |
| Asian/other | 2.80 (1.12–4.48) | < .01 |
| White | Referent | — |
| Age | 0.07 (–0.02 to 0.17) | .12 |
| Gestational age of earliest previous preterm birth | 0.16 (0.05–0.26) | < .01 |
| Gestational age at 1st sonogram | 1.17 (0.79–1.56) | < .01 |
| Cervical length at 1st sonogram | 0.45 (0.41–0.49) | < .01 |
| Cigarette use | 0.95 (–0.50 to 2.40) | .20 |
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