Objective
We determined whether an environmental exposure to bacterial vaginosis (BV) modified genetic susceptibilities for spontaneous preterm delivery within genes that regulate the inflammatory response.
Study Design
Maternal DNA samples and vaginal smears for Gram staining were collected from 743 women (68 preterm births). We used a 1536–single nucleotide polymorphism (SNP) custom chip to study associations between genotype distributions and preterm birth.
Results
For 8 SNPs in 3 genes (protein kinase C alpha, fms-like tyrosine kinase 1, and interleukin 6), the odds ratios for preterm birth ranged from 1.9–4.0 among women with susceptible genotypes who were BV positive. The odds ratios for preterm birth were 2.0-5.0 times greater among women who were BV positive than among women who were BV negative. The significance of these differences was demonstrated by logistic regression analyses for genotype/BV interaction.
Conclusion
These results demonstrate that the risk of preterm delivery that is associated with tag SNPs in genes that regulate the inflammatory response is modified by an environmental exposure such as bacterial vaginosis.
Preterm birth complicates >12% of pregnancies in the United States, and efforts to prevent spontaneous preterm delivery have been disappointing. Subclinical genital tract infection is a risk factor for preterm delivery, and proinflammatory mediators produced by the mother and fetus in response to genital tract infection appear to play a fundamental role in the pathogenesis of preterm delivery. Previous investigators have associated bacterial vaginosis (BV) with preterm delivery, and treatment of asymptomatic BV during the second trimester reduces the rate of recurrent preterm birth. However, treatment of asymptomatic BV does not prevent preterm birth in the general obstetric population, and recurrent preterm birth comprises only a subset of all spontaneous preterm deliveries. Therefore, more widespread interventions that will target inflammatory pathways to prevent preterm birth are needed.
Given the association between inflammation and preterm delivery, a number of investigators have studied genetic variation at the level of single nucleotide polymorphisms (SNPs) within genes that regulate the inflammatory response to determine whether this association is more consistent in women with genetic predisposition. We previously demonstrated that an interaction between a single genetic susceptibility (carriage of the rare allele of a SNP at the –308 position in the promoter of the tumor necrosis factor-alpha gene) and an environmental factor (BV) was associated with an increased risk for spontaneous preterm delivery. More recently, we used a 1536-SNP custom chip panel to demonstrate that tag SNPs in maternal genes that regulate the inflammatory response were associated with an increased risk of spontaneous preterm delivery. Tag SNPs are representative SNPs in regions of the genome with high linkage disequilibrium that enable investigators to identify genetic variation without genotyping every SNP in a chromosomal region. We decided to study tag SNPs to identify regions within genes that are associated with spontaneous preterm delivery so that we can attempt to identify functional SNPs in these regions in subsequent studies. In the present study, we hypothesized that gene-environment interactions are critical in the pathogenesis of preterm birth. Specifically, associations between the risk of preterm birth and allelic variation in genes that control inflammatory pathways are modified by the presence of asymptomatic BV. To test our hypothesis, we analyzed tag SNPs from candidate genes in women who were screened for asymptomatic BV during pregnancy to identify an increased risk of spontaneous preterm delivery at <37 weeks’ gestation.
Materials and Methods
We enrolled 2 groups of women into a large cohort for 2 purposes: (1) women with periodontal disease were enrolled into a randomized clinical trial to study the effect of periodontal treatment on rates of preterm birth, and (2) women without periodontal disease were enrolled into an observational group to complete secondary genotyping studies. The study was approved by the Institutional Review Board at the University of Pennsylvania (protocol # 801404). Subjects were enrolled at their first prenatal visit at 3 prenatal care clinics in the metropolitan Philadelphia area. Patients with singleton pregnancies between 6 and 20 weeks’ gestation (using standard pregnancy dating criteria) were eligible for enrollment. Subjects were screened for periodontal disease by trained research nurses, and women with periodontal disease were assigned randomly to treatment and placebo arms to determine whether treatment of periodontal disease reduced the incidence of spontaneous preterm birth. Women without periodontal disease at enrollment were included in the larger cohort as an observational group. Because treatment of periodontal disease and the presence of periodontal disease were not associated with rates of preterm delivery, subjects with and without periodontal disease were included in the overall cohort for genotyping studies.
Maternal DNA was isolated from buccal swab samples that were collected at enrollment in 1122 subjects. Two different web-based reference databases that link the genome to biologic systems, BioCarta ( www.biocarta.com/genes/index.asp ) and KEGG (Kyoto Encyclopedia of Genes and Genomes, www.genome.jp/kegg/pathway.html ), were searched to identify genes in pathways that regulate the inflammatory response. We selected tag SNPs that were based on Illumina SNP scores and design scores (Illumina, Inc, San Diego, CA). We designed an Illumina Golden Gate 1536-SNP custom chip panel (Illumina, Inc, San Diego, CA) that was used for our genotyping studies. Associations between genotype distributions and spontaneous preterm delivery (<37 weeks’ gestation) were examined by Fisher’s exact tests and reported elsewhere. Only tag SNPs in which the allele frequency of the rare allele was at least 5% were analyzed. Agreement of genotype frequencies with Hardy-Weinberg equilibrium expectations was evaluated by a C 2 goodness-of-fit test. For the purpose of the current exploratory study, we focused on tag SNPs in pathways in which at least 3 SNPs were associated strongly with spontaneous preterm delivery ( P < .01; Table 1 ).
| Database | Pathway | Total SNPs studied, n | SNPs associated with preterm birth, n |
|---|---|---|---|
| BioCarta | Cytokine network | 169 | 9 |
| BioCarta | Dendritic cells that regulate T-helper 1/T-helper 2 development | 60 | 3 |
| KEGG | Cytokine-cytokine receptor interaction | 436 | 8 |
| KEGG | Janus kinases and signal transducers and activators of transcription signaling | 197 | 4 |
| KEGG | Leukocyte transendothelial migration | 411 | 12 |
a www.biocarta.com/genes/index.asp ;
Cervicovaginal fluid samples were collected at 26-28 weeks’ gestation from women in the cohort, and histologic slides were prepared for Gram staining for asymptomatic BV. Results were interpreted by experienced microbiologists according to the criteria of Nugent et al; BV was defined as a Gram-stain score ≥7.
Demographics and outcomes data were double entered into our research database by trained research staff from the clinical research computing unit. Only 3 women who were enrolled in the cohort delivered between 20 and 26 weeks’ gestation; however, >300 women did not undergo collection of cervicovaginal fluid samples between 26 and 28 weeks gestation. Women with medically or obstetrically indicated preterm deliveries also were excluded from our analyses, leaving 743 women from whom cervicovaginal fluid samples were collected at 26-28 weeks’ gestation.
Comparisons of demographics and outcomes data (Student t tests, χ 2 tests) were performed between women with asymptomatic BV and women who screened negative for BV. Frequency tables were constructed to compare rates of spontaneous preterm delivery based on the 3 possible genotypes for each SNP (AA, AB, and BB) among women in 3 groups: (1) overall cohort, (2) BV positive, and (3) BV negative. For the frequency tables, probability values were derived with the Fisher’s exact tests that treated the genotype as a 3-level categoric variable, with the assumption of an additive effect of the SNP alleles. Logistic regression analysis was used to test for significant interactions between genotypes and BV status to determine whether the rate of spontaneous preterm delivery was different among women with susceptible genotypes who were BV positive than among women with susceptible genotypes who were BV negative. For each SNP, the regression model evaluated the interaction with BV, then we added race (African American vs other), periodontal disease status, current diagnosis of sexually transmitted disease, and history of spontaneous preterm delivery. Next, a backwards elimination strategy was used to assess the impact of confounding. Factors that precipitated a change in any estimated odds ratio of ≥15% were considered confounders and were retained in subsequent modeling. Odds ratios and CIs for spontaneous preterm delivery were derived from our logistic regression model, in which we assumed a dose-response for the association between preterm delivery and the genotype (0, 1, or 2 copies of the minor allele). To determine the significance of the modifying effect of BV on the relationship between allele and preterm birth, we incorporated the genotype/BV interaction term into our logistic regression model.
Results
In our cohort of 743 subjects, 306 women (41.2%) screened positive for BV at 26-28 weeks’ gestation; 437 women screened negative. Demographics and clinical outcomes were compared between the BV-positive and BV-negative groups, and there were no differences in rates of spontaneous preterm delivery, gestational age at delivery, and birthweight between the 2 groups ( Table 2 ). Sixty-eight women experienced spontaneous preterm delivery at <37 weeks’ gestation; 675 women delivered at term (overall spontaneous preterm delivery rate, 9.2%).
| Measurement | Bacterial vaginosis[+] (n = 306) | Bacterial vaginosis[–] (n = 437) | P value a |
|---|---|---|---|
| Maternal age, y b | 23.3 ± 5.1 | 23.6 ± 5.4 | .44 |
| African American, n (%) | 266 (87.2) | 365 (83.7) | .19 |
| Nulliparous, n (%) | 130 (42.5) | 191 (43.7) | .74 |
| History of spontaneous preterm delivery, n (%) | 29 (9.5) | 28 (6.4) | .12 |
| Sexually transmitted diseases during the current pregnancy, n (%) c | 84 (27.5) | 83 (19.0) | < .01 |
| Periodontal disease, n (%) d | 214 (69.9) | 272 (62.2) | .03 |
| Spontaneous preterm delivery for the current pregnancy, n (%) | 32 (10.5) | 36 (8.2) | .37 |
| Gestational age at delivery, wk b | 39.0 ± 2.0 | 39.1 ± 1.8 | .25 |
| Birthweight, g b | 3149 ± 538 | 3202 ± 552 | .20 |
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