Genital tract infection/inflammation is considered one cause of preterm delivery, especially early preterm delivery.1–3 Bacterial vaginosis, characterized by a decreased concentration of lactobacilli and an overgrowth of a diverse community of bacteria,4,5 has been associated with upper genital tract infection/inflammation and with preterm delivery.6–9 The higher prevalence of bacterial vaginosis in African Americans compared with non-Hispanic whites10–12 has led some to hypothesize that bacterial vaginosis might be related to racial disparities in preterm delivery risk.13 Unfortunately, randomized clinical trials using antimicrobial agents to prevent preterm delivery have yielded primarily disappointing results,14–21 and there remains a limited understanding of the complex interplay between bacterial overgrowth and host immune response in the vagina and in gestational tissues.
As part of the vaginal tract’s innate immune system, mucosal epithelial cells and inflammatory cells produce antimicrobial peptides. Antimicrobial peptides include lactoferrin, lysozyme, defensins, and catholicities,22–26 which can be produced constitutively or induced by infection. Antimicrobial peptides of the defensin class belong to two structurally distinct groups in humans, α-defensins and β-defensins.22 Of the six α-defensins identified in humans, human neutrophil peptides (HNPs) 1, 2, 3, and 4 are produced by neutrophils.24,27 Elevations in amniotic fluid HNPs 1, 2, and 3 were found in pregnant women with preterm labor and subclinical intrauterine infection.28 High HNP 2 levels detected in vaginal fluid collected at 24–29 weeks of pregnancy were linked to an increased risk of spontaneous preterm delivery at less than 32 weeks.29
Building on previous studies, this study evaluated HNPs 1, 2, and 3 in vaginal fluid sampled earlier in pregnancy, at 15–27 weeks. The goal was to assess relations among vaginal HNP 1–3 levels, bacterial vaginosis, and risk of preterm delivery and compare the findings across two race groups, non-Hispanic whites and African Americans.
MATERIALS AND METHODS
This study included participants from the Pregnancy Outcomes and Community Health (POUCH) study,30 which enrolled women in their 15th through 27th week of pregnancy from 52 clinics in five Michigan communities from 1998 to 2004. Eligibility criteria included maternal age older than 14 years, English speaking, a singleton pregnancy with no known congenital or chromosomal abnormalities, no prepregnancy diabetes, and screening at 15th through 22nd weeks of gestation for maternal serum alpha-fetoprotein (MSAFP), a biomarker of particular interest in the POUCH study. Eligible women were randomLy sampled into the study, and those with unexplained high MSAFP (more than 2 multiples of the median) were oversampled (7% of cohort). Of the 3,038 women enrolled, 19 were lost to follow-up, leaving 3,019 (99.4%) in the cohort. Study protocols were approved by institutional review boards at Michigan State University and eight hospitals in the five communities. All participants provided written consent before enrollment. Comparisons of POUCH study women with aggregate birth certificate data specific to the five study communities showed that the POUCH sample was very similar to community mothers on most factors measured, including age, parity, education levels, the proportions of women with Medicaid insurance, preterm delivery, previous stillbirth, previous preterm infant, and previous low birth weight infant. The one exception, the percentage of African Americans aged older than 30 years, was 14% in the POUCH study and 21% in the birth certificate data.30
The subcohort included all women who delivered preterm, all women with unexplained high MSAFP levels, and a random sample of women who delivered at term with normal MSAFP, with an oversampling of African Americans in this last category. In the POUCH study subcohort (N=1,371), 692 (50.5%) women reported their race/ethnicity as non-Hispanic white, 579 (42.2%) as African American, and 100 (7.3%) as another racial/ethnic group. Because of the small number of participants and the broad racial/ethnic mix, the “other” group was excluded from the following analyses. In addition, 235 (18%) non-Hispanic white and African-American women declined vaginal fluid sampling, and five had samples with insufficient volume, leaving a subcohort sample of 1,031 women for the current analyses (787 term, 244 preterm).
At enrollment, study participants met with a study nurse and provided information about demographics, current pregnancy, reproductive history, health behaviors such as smoking, and psychosocial factors. Vaginal fluid samples were collected using a fetal fibronectin specimen collection kit (Adeza International, Sunnyvale, CA). After placement of a vaginal speculum, vaginal fluid was collected from the posterior fornix with a sterile Dacron (E.I. du Pont de Nemours and Company, Wilmington, DE) swab. The swab was placed in 1 mL extraction buffer and refrigerated (4°C) for at least 24 hours. After the refrigeration period, buffer was expressed from the swab, the specimen was filtered using a 10.25-mm×4-inch serum filter (Fisherbrand Serum Filter System, Fisher Scientific, Pittsburgh, PA), divided into 0.5 mL aliquots, and stored at –80°C.
Vaginal fluid specimens were later thawed and 50 microliter aliquots were removed, refrozen, and shipped to the University of Alabama at Birmingham for assay. An enzyme-linked immunosorbent assay kit measured total HNP level, which included HNP 1, 2, and 3 (HyCult Biotechnology, Uden, the Netherlands) and was reported out from the assay as one value. This approach has been used in other studies, including one that assessed defensin levels and presence of sexually transmitted pathogens in urethral lavages.31 Thirteen women had a vaginal HNP 1–3 level below the lower limit of detection and these values were imputed as half the detection level.
At the time of vaginal fluid sampling, study nurses also obtained vaginal smears. Smears were Gram-stained and evaluated for bacterial vaginosis by a microbiologist using the Nugent scoring system.5 Scores ranged from 0 to 10, with bacterial vaginosis status categorized as negative (0–3), intermediate (4–6), and positive (7–10).
Information on three major vaginal infections, Chlamydia, Gonorrhea, and Trichomonas, was ascertained in two ways. At enrollment, just before vaginal fluid sampling, women were asked about diagnoses of these vaginal infections for two different periods, the year before the pregnancy and during pregnancy. Prenatal and labor and delivery records were abstracted, and women were classified as being culture positive or negative for any of the three infections. Culture results from the pregnancy period were available in 99% of women. In addition, the maternal interview asked women to report antibiotic use from the beginning of the pregnancy up through the time of enrollment.
Preterm delivery was defined as birth before 37 completed weeks of gestation. Gestational age was determined by the last menstrual period or by ultrasound data when the last menstrual period–derived gestational age differed from the ultrasound estimate by at least 2 weeks. Based on information abstracted from labor and delivery medical records, preterm delivery was divided into two groups: 1) Spontaneous preterm delivery included women with preterm labor defined as regular contractions that led to cervical change (2 cm or more dilatation), or spontaneous premature rupture of membranes; and 2) medically indicated preterm delivery included women who had labor induced or who had cesarean delivery before either preterm labor or premature rupture of membranes.
The overall analytic goals were, first, to assess relations between vaginal HNP 1–3 levels and maternal characteristics including bacterial vaginosis and, second, to examine associations between vaginal HNP 1–3 levels and risk of preterm delivery. All analyses incorporated sampling weights to account for the sampling schemes used to construct the cohort and subcohort, with preterms and African-American women weighted to their original proportion in the cohort, and unexplained MSAFP weighted to the original proportion in the population. The proportional sampling weights remove any bias introduced by oversampling at risk groups into the subcohort. For the comparison of mean vaginal HNP 1–3 levels between African Americans and non-Hispanic whites, HNP 1–3 levels were transformed to the natural log scale and analyzed using SAS Surveymeans and Surveyreg procedures (SAS Institute Inc., Cary, NC.32
Human neutrophil peptide 1–3 levels were dichotomized using the median value (high, median or more; low, less than median) from the distribution of HNP 1–3 levels in women who delivered at term and had normal MSAFP levels. The median was selected because effects were similar for women in the upper third and fourth quartile of HNP 1–3 levels. Race-specific analyses were performed to evaluate the bacterial vaginosis Nugent score and other maternal characteristics (age, education level, smoking, Medicaid insurance status, week of pregnancy at study enrollment, and parity) in relation to high and low HNP 1–3 levels (SAS surveyfreq, SAS Institute Inc.). The Rao-Scott χ2 test, used in complex survey designs,33 was applied to test for statistically significant associations. Maternal characteristics associated with HNP 1–3 levels at P<.10 were considered potential confounders to be included in final models.
Polytomous logistic regression models34 (SAS surveylogistic, SAS Institute Inc.) tested associations between covariates (high/low HNP 1–3 levels, bacterial vaginosis, and race) and a three-level outcome variable: term (referent group), spontaneous preterm delivery, and medically indicated preterm delivery. Results showed a statistically significant three-way interaction among race, HNP 1–3 levels, and bacterial vaginosis status. This led to the development of race-specific models to calculate unadjusted and adjusted odds ratios for the associations between HNP 1–3 levels and preterm delivery subtypes. Models with a four-level outcome, ie, same as above but spontaneous preterm delivery subdivided into less than 32 weeks and 32–36 weeks, explored the specificity of the relation between HNP 1–3 levels and timing of spontaneous preterm delivery. Beta estimates from race-specific models were used to calculate probabilities of each preterm delivery subtype for six groups defined by bacterial vaginosis (normal, intermediate, high) and HNP 1–3 (high, low) status, and results were displayed in graphs.
Maternal characteristics are presented without sampling weights to describe the subcohort and with sampling weights to compare characteristics across race groups (Table 1). Analyses of weighted data showed that, compared with non-Hispanic whites, African-American women were younger, had fewer years of education, were more likely to be insured by Medicaid, smoked less if still smoking at enrollment, and were more likely to enroll in the study before 20 weeks of gestation. In addition, preterm delivery risk was significantly greater in African-American women (14%) compared with that in non-Hispanic white women (10%). With the exception of the 13 women with undetectable levels, participants’ HNP 1–3 levels ranged from 2.0–90,340 ng/mL (weighted median 2,661 ng/mL; weighted mean 7,398 ng/mL). The weighted mean log HNP 1–3 level was significantly higher in non-Hispanic whites compared with that in African Americans.
In non-Hispanic whites two factors were associated with low HNP 1–3 levels, maternal age older than 29 years and bacterial vaginosis, although the inverse relation with bacterial vaginosis was not statistically significant (Table 2). Among African Americans, samples collected earlier in pregnancy had lower HNP 1–3 levels. The bacterial vaginosis–positive group had the largest percentage of women with high HNP 1–3 levels (57%), followed by the bacterial vaginosis–intermediate group (53%), and the bacterial vaginosis–negative group (43%).
High vaginal HNP 1–3 levels at mid pregnancy were associated with spontaneous preterm delivery (unadjusted odds ratio 2.2, 95% confidence interval 1.2–4.0) but not with medically indicated preterm delivery in African-American women (Table 3). These results remained virtually unchanged after adjusting for maternal age, week of pregnancy at sampling, bacterial vaginosis status, and culture results for Chlamydia, Gonorrhea, and Trichomonas ascertained from medical records. Inclusion of other covariates such as maternal self-report of vaginal infections in the year before the pregnancy, vaginal infections during pregnancy, and antibiotic use during pregnancy (up through the time of vaginal fluid sampling for HNP 1–3 assay and bacterial vaginosis evaluation) did not alter the results. In analogous models with non-Hispanic whites, vaginal HNP 1–3 levels were unrelated to preterm delivery. After further dividing spontaneous preterm delivery by weeks of gestation, high HNP 1–3 levels were linked to deliveries at less than 32 weeks and at 32–36 weeks in African Americans. There was some evidence that midpregnancy vaginal HNP 1–3 levels might also be increased in non-Hispanic whites who later delivered spontaneously at less than 32 weeks, but statistical power was limited.
The relation between HNP 1–3 and preterm delivery was also considered within the context of bacterial vaginosis. Because the subcohort was sampled from a prospective cohort study, weighted analyses permitted calculations of the probabilities (risks) of each preterm delivery subtype given HNP 1–3 and bacterial vaginosis status at mid pregnancy (Fig. 1). In both race groups, bacterial vaginosis status and HNP 1–3 levels were unrelated to the risk of medically indicated preterm delivery. The risk of spontaneous preterm delivery was significantly associated with high HNP 1–3 levels in African Americans, and this association persisted regardless of bacterial vaginosis status. There was no link between spontaneous preterm delivery and vaginal HNP 1–3 levels in non-Hispanic whites among any of the bacterial vaginosis groups.
We found that in African-American women, vaginal HNP 1–3 levels at the median or above were associated with bacterial vaginosis and with spontaneous preterm delivery. Once African-American women were stratified by vaginal HNP 1–3 levels, bacterial vaginosis added nothing to the prediction of spontaneous preterm delivery risk. None of the above associations were observed in non-Hispanic whites.
The one other cohort study on vaginal defensin levels, bacterial vaginosis, and pregnancy outcome reported that elevated defensins were linked to intermediate bacterial vaginosis but not to positive bacterial vaginosis and to preterm delivery at less than 32 weeks of gestation but not to all preterm delivery at less than 37 weeks.29,35 The sample in this study was described as 44% Black and 56% non-Black, similar to our sample, but unlike our results, they found no evidence of effect modification by race. They measured HNP 2, and the interval from vaginal fluid sampling (24th through 29th week) to early delivery (less than 32 weeks) was short. We measured HNP 1–3, and vaginal samples were collected earlier in pregnancy (15th through 27th week; approximately 90% before 25 weeks), indicating that higher vaginal defensin levels may not be just a marker of impending labor.
The functional or pathologic significance of high defensin levels in vaginal fluid during pregnancy is unclear. The vagina is routinely exposed to a variety of microbes that may elicit neutrophil activation and defensin production, which could be protective or contribute to the risk of preterm delivery. Expression of defensins in the vaginal tract could be constitutive, inducible by infectious/inflammatory stimuli, or both.31,36 Human neutrophil peptide 1–3 levels seem to be induced in urethral lavages of men infected with Neisseria gonorrhoeae and Chlamydia,31 and defensins have been shown to be inducible by proinflammatory cytokines.37 In the study by Simhan et al,38 vaginal fluid defensins were positively associated with levels of interleukin-8.
To explain the positive link between vaginal HNP 1–3 levels and bacterial vaginosis in African Americans but not in non-Hispanic whites, we considered confounding due to other vaginal infections such as Trichomonas vaginalis,38,39 but adjustment for sexually transmitted diseases (culture results in pregnancy) did not change our findings. Effect modification by race group might be due to differences in host innate immune response to bacterial vaginosis. Alternatively, because bacterial vaginosis is broadly defined, higher HNP 1–3 levels might signify the presence of specific bacterial vaginosis–related bacteria that are more prevalent in African-American bacterial vaginosis cases than in non-Hispanic white bacterial vaginosis cases, such as Mobiluncus species.40
When we divided preterm delivery by gestational weeks, statistical power was limited, but results suggested that high vaginal HNP 1–3 levels were associated with spontaneous preterm delivery at less than 32 weeks in both non-Hispanic whites and African Americans. Among African Americans, the association persisted for the 33–36 week spontaneous preterm delivery as well. These findings could be interpreted as evidence that the pathway to spontaneous preterm delivery marked by elevated HNP 1–3 is of similar importance in the two race groups for the earliest deliveries and continues to be important for the later spontaneous preterm deliveries in African Americans. Our HNP 1–3 level cutoff for “high,” at the median or above, was based on the distribution of HNP 1–3 levels in both non-Hispanic white and African-American women. When we tried using race-specific medians, only 5% of non-Hispanic white and 4% of African-American women were assigned to a different category, and our results and conclusions were unchanged.
A major strength of our study was the composition of the cohort, which consisted of women sampled from 52 clinics and from a wide range of socioeconomic backgrounds. In addition, vaginal HNP 1–3 levels were measured at mid pregnancy, providing evidence for the early appearance of an inflammatory process and a maternal immune response linked to preterm delivery. The use of less invasive methods (vaginal fluid sampling) for detecting a maternal inflammatory response offers greater clinical applications. We also separated medically indicated preterm delivery from spontaneous preterm delivery, thereby demonstrating specificity and biologic coherence in our findings. A limitation of this study was the single, simultaneous measurement of bacterial vaginosis status and HNP 1–3 levels, precluding establishment of a temporal relation between bacterial vaginosis and elevated HNP 1–3 levels.
The observed effect modification by race group in the relations between bacterial vaginosis status, HNP 1–3 levels, and preterm delivery is a new finding and requires additional studies to explore underlying explanations for these results. It may be informative to compare the vaginal microbial ecology across different race groups in relation to vaginal HNP 1–3 levels. In addition, elevated HNP 1–3 levels may be a marker for particular high-risk vaginal milieus that are not distinguished by the current bacterial vaginosis Nugent scoring system.
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