Obstetrics & Gynecology:
Tumor Necrosis Factor–α Promoter Gene Polymorphism ‐308 and Chorioamnionitis
Simhan, Hyagriv N. MD; Krohn, Marijane A. PhD; Zeevi, Adriana PhD; Daftary, Ashi MD; Harger, Gail MS; Caritis, Steve N. MD
Departments of Obstetrics, Gynecology, and Reproductive Sciences and Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Address reprint request to: Hyagriv N. Simhan, MD, 300 Halket Street, Pittsburgh, PA 15213; E-mail: firstname.lastname@example.org.
Support provided by the National Institutes of Health (1K12 HD 01261).
Received November 26, 2002. Received in revised form February 11, 2003. Accepted February 27, 2003.
OBJECTIVE: A single-base polymorphism within the promoter of the gene for tumor necrosis factor α at position -308 relative to the transcriptional start site of the gene is correlated with differences in production of tumor necrosis factor α. There are two allelic forms: TNFA1 and TNFA2. The TNFA2 allele is associated with increased production of tumor necrosis factor α. The purpose of this study is to assess the relationship between carriage of the TNFA2 allele and clinical chorioamnionitis.
METHODS: In this retrospective cohort study, previously banked deoxyribonucleic acid from 149 women who had spontaneous labor from 37 to 42 weeks' gestation was used. Polymerase chain reaction was used for polymorphism assay. Demographic and clinical information was obtained from the medical record. Clinical chorioamnionitis was defined as at least one temperature elevation above 38C combined with at least two of the following signs: maternal or fetal tachycardia, uterine tenderness greater than expected, foul-smelling vaginal discharge, and white blood cell count more than 18,000.
RESULTS: Chorioamnionitis was present in 18 women (12.1%). Among women who did not carry TNFA2, the chorioamnionitis rate was 7.4%. Among women who carried TNFA2, the chorioamnionitis rate was 24.4%. The relative risk for chorioamnionitis with carriage of TNFA2 was 3.3 (95% confidence interval 1.3, 7.1). This increased risk was not altered after adjustment for race, type of rupture of membranes, intrauterine pressure catheter use, smoking, and prolonged rupture of membranes.
CONCLUSION: Carriage of the TNFA2 allele is associated with a more than three-fold increased risk of clinical chorioamnionitis, even when accounting for important clinical and microbiologic risk factors.
Intraamniotic infection is a major cause of maternal and neonatal morbidity. When defined by clinical criteria, intraamniotic infection has a reported frequency of 0.5–10.5%.1–3 Among neonates born at term, maternal intra-amniotic infection is powerfully associated with cerebral palsy, with an odds ratio of 9.3.4 Traditionally, the risk factors for the development of intraamniotic infection either are related to intrapartum events such as the number of vaginal examinations in labor, duration of ruptured membranes, use of internal monitors, duration of total labor, and prolonged second stage or are demographic risk factors such as nulliparity and alcohol and tobacco use.1–3 These traditional risk factors fall into two basic categories: those that increase the exposure of the host to vaginal flora and those that are related to variations in host response to that bacterial inoculum. Examples of the former include internal monitors in labor, frequent cervical examinations, or protracted labor. Factors that may represent variations in host response include alcohol and tobacco use.
Intraamniotic infection is associated with a maternal proinflammatory response, as demonstrated by elevation in proinflammatory cytokines and chemokines in amniotic fluid, maternal serum, or cervicovaginal fluid.5–9 This proinflammatory response is an important link between maternal infection and neonatal morbidity. Tumor necrosis factor α is an important proinflammatory cytokine in the cascade of host response to infection. It increases the expression of other proinflammatory mediators and induces cellular apoptosis. In large part, the production of tumor necrosis factor α is under genetic regulation.
Polymorphisms have been described for many human cytokine genes.10–12 These represent normal allelic variation, frequently within the regulatory region of cytokine genes. Specific polymorphisms are associated with increased susceptibility to certain infectious diseases and increased severity of autoimmune disease.13 A polymorphism in the promoter region of tumor necrosis factor α (at position -308) is associated with high production of tumor necrosis factor α. The substitution of adenosine for guanine at this position is responsible for the increase in promoter activity. The individual with guanine at both positions (homozygous G/G) displays normal production of tumor necrosis factor α. This is known as the TNFA1 allelic variant. The high production allelic variant (TNFA2) may be homozygous (A/A) or heterozygous (G/A). There is a known relationship between preterm premature rupture of membranes (PROM) and intrauterine infection and inflammation.14 Because the genetic predisposition toward increased tumor necrosis factor–α production is related to preterm PROM,15 we sought to ascertain its relationship to another infectious complication of pregnancy: intraamniotic infection in labor. We hypothesize that women who carry the TNFA2 allele have a more robust inflammatory response and therefore are more likely to have clinical intraamniotic infection at term.
MATERIALS AND METHODS
This retrospective cohort was comprised of 149 women who had spontaneous labor with a singleton gestation at 37–42 weeks' gestation at Magee-Womens Hospital between June 1997 and June 2001. Deoxyribonucleic acid from maternal blood specimens was previously extracted and banked during the Prenatal Exposures and Preeclampsia Prevention Project longitudinal cohort study. The current project was approved by our institutional review board, and the consenting subjects in the Prenatal Exposures and Preeclampsia Prevention Study cohort all agreed to have their blood samples used for subsequent genetic studies related to adverse obstetric outcome. In this analysis, we excluded women with cervical cerclage, vaginal bleeding, preeclampsia, diabetes mellitus, and collagen vascular disease. Demographic and clinical information were obtained from the medical record and a computerized perinatal database maintained at our institution. To insure accuracy of the database, validation of several variables was performed through correlation with medical record charts, showing 96–100% accuracy for each variable.
Tumor necrosis factor–α promoter polymorphism status was determined using commercially available polymerase chain reaction kits (One Lambda, Canoga Park, CA). The amplified fragments were detected by gel electrophoresis and ethidium bromide staining. Patterns of positive and negative amplification yielded the relevant phenotype for an investigator who was blinded to all clinical and demographic data, including the outcome of interest.
Clinical chorioamnionitis was defined as at least one temperature elevation above 38C combined with at least two of the following signs: maternal or fetal tachycardia, uterine tenderness greater than expected, foul-smelling vaginal discharge, and white blood cell count more than 18,000.
Continuous variables were compared using the Mann–Whitney U test. Categoric variables were compared using the two-tailed Fisher exact test. For these analyses, α < .05 was considered statistically significant. The univariate and multivariable (adjusted) odds ratios for the development of intraamniotic infection by TNFA2 status were determined using logistic regression. Variables considered as possible confounders or effect modifiers included bacterial vaginosis, Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, group B β-hemolytic streptococcus status, race, age, marital status, gravidity, smoking, urinary tract infection in pregnancy, administration of antibiotics in labor, prelabor rupture of membranes, prolonged second stage of labor of more than 4 hours, use of intrauterine pressure catheter in labor, epidural use, and prolonged predelivery rupture of membranes of more than 12 hours. Confounders were included in the logistic model if they demonstrated a significant association with either intra-amniotic infection or TNFA2 status, with P < .10 considered significant. Candidate confounders that demonstrated a significant association with either intraamniotic infection of TNFA2 status were placed into a two-variable logistic regression model along with TNFA2 status. All statistical analyses were performed using Stata 7.0 for Windows (Stata Corp., College Station, TX).
The overall frequency of clinical intraamniotic infection was 18 of 149 (12.1%). The demographic and clinical characteristics of the cohort, stratified by intraamniotic infection status, are described in Table 1. Of the 149 women in the cohort, 41 (27%) carried the TNFA2 allele, associated with increased tumor necrosis factor–α production. All of these women were heterozygotes (G/A). There were no homozygous TNFA2 carriers (A/A). The frequency of intraamniotic infection among women who carried TNFA2 was ten of 41 (24.4%). The frequency of intraamniotic infection among women who did not carry TNFA2 was eight of 108 (7.4%). The crude odds ratio describing the strength of the association of TNFA2 carriage with intraamniotic infection is 4.0 (95% confidence interval [CI] 1.5, 11.1). Variables that demonstrate a relationship with TNFA2 status or intraamniotic infection are race, type of membrane rupture, intrauterine pressure catheter use, smoking, and prolonged membrane rupture. The point estimate of the odds ratio describing the strength of the association of TNFA2 carriage with intraamniotic infection from the five logistic regression models that include TNFA2 status and each confounder is 3.9–4.2 and is significant (P < .01) for each model. The relative risk of developing intraamniotic infection among women carrying TNFA2 versus that of women carrying TNFA1 is 3.3 (95% CI 1.3, 7.1). Even when accounting for important demographic and clinical covariates, the risk of intraamniotic infection is more than three times greater among women with the genetic predisposition for increased tumor necrosis factor–α production.
Tumor necrosis factor α is a major pyrogenic acute phase reactant in the inflammatory cascade that is ubiquitous in the maternal and fetal compartments in the setting of chorioamnionitis.6 Thus, the role of tumor necrosis factor α in clinical intraamniotic infection has intuitive appeal. Our data demonstrate that women who are genetically predisposed to increased tumor necrosis factor–α production are more likely to develop clinical infection during labor. We hypothesize that these women respond to a given infectious stimulus with a more robust inflammatory response. In the case of tumor necrosis factor α, a more vigorous response is perhaps more likely to induce fever in the host as well as increase the production and release of other chemotactic elements of the inflammatory cascade.
The tumor necrosis factor–α polymorphism -308 is a significant risk factor for severe and fatal disease in patients with meningococcal infection.13 Similarly, Gambian children with malaria who were homozygous for the TNFA2 allele were at highest risk for severe neurological sequelae. The frequency of the tumor necrosis factor–α high production allele is also associated with rheumatoid arthritis and systemic lupus erythematosus.16 There is a recent report of the relationship of the tumor necrosis factor–α polymorphism at the promoter region -308 and preterm birth. Roberts and colleagues performed a case–control study of black women with delivery before 37 weeks after idiopathic preterm labor or preterm PROM compared with black women who delivered after 37 weeks and had no history of preterm delivery. These investigators evaluated the carrier frequency of the TNFA2 allelic variant. There was not a significant association between preterm birth after idiopathic preterm labor and TNFA2. There was, however, a significant association between TNFA2 and preterm birth after preterm PROM, which is known to have a powerful etiological contribution from inflammation.14,15
We found that intrauterine pressure catheter use was associated with intraamniotic infection. Importantly, this relationship is not necessarily causative. Intrauterine pressure catheter use may be a surrogate marker of labor disorder or some other root factor. Interestingly, we found that neither prolonged rupture of membranes nor prolonged second stage of labor were associated with intraamniotic infection. Other investigators have, however, noted that labor abnormalities are associated with intraamniotic infection.1–3 Although group B streptococcus carriage and bacterial vaginosis have been associated with upper genital tract infections in pregnant and non-pregnant women such as posthysterectomy cuff cellulitis, postpartum endometritis, and pelvic inflammatory disease, we did not demonstrate an association between these microbiologic factors and clinical intraamniotic infection.17–21
Intrauterine infection in labor is an important source of morbidity for women and their newborns. Among the most devastating clinical consequences of infection in labor is cerebral palsy. Inflammation in the fetus or newborn is a major contributor to neonatal neurological injury. Nelson and associates performed a case–control study of 31 children with spastic cerebral palsy and 65 control children, matched for race. The diagnosis of cerebral palsy was made at age three or later in all of the cases. Using the dried heelstick blood spot obtained at birth, the investigators measured a variety of cytokines, chemokines, and coagulation products in case and control children. The concentration of these products in the heelstick spot reflects the concentration at time of birth. Children with cerebral palsy had significantly higher heelstick concentrations of interleukins 1, 6, and 8; tumor necrosis factor α; and RANTES (regulated on activation, normal T cell expressed and secreted) than children without cerebral palsy.22 Most of the children in this study were born at term. Understanding the predisposition toward intraamniotic infection has important implications for both maternal and neonatal morbidity.
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