Preterm birth (delivery before 37 weeks' gestation) is a significant cause of perinatal morbidity and mortality.1 Identification of women at risk for preterm delivery is important, and several strategies have been suggested, including risk scoring systems, biochemical markers of inflammation, such as fetal fibronectin, and screening for various infections, including bacterial vaginosis.2 Risk scoring systems focusing on historic and epidemiologic factors associated with preterm birth have been developed to direct prevention strategies toward high-risk obstetric populations,3–9 but sensitivities and positive predictive values are low. Studies have found an association between bacterial vaginosis and spontaneous preterm delivery.10–14 Cervicovaginal fetal fibronectin using the monoclonal antibody FDC-6 was suggested as a predictor of preterm delivery in low-risk women.15–18 Another monoclonal antibody, X18A4, was identified and found to bind with high affinity to a new epitope within fetal fibronectin,19 and might be an adjunctive antibody to enhance fetal fibronectin assays.
No study has evaluated the combination of vaginal and cervical fetal fibronectin FDC-6 and X18A4, the diagnosis of bacterial vaginosis, and a population-specific risk scoring system. The purpose of this study was to determine the predictive probability of those potential markers for spontaneous preterm birth in a relatively low-risk population.
Materials and Methods
Subjects between 20 and 24 weeks' gestation were recruited from December 1995 to August 1996 from the Perinatal Centre of the Grace Maternity Hospital. The study was approved by the Human Investigation Committee of the hospital and Dalhousie University. Written informed consent was obtained from each subject by an investigator not directly involved in their care. Gestational age was based on known last menstrual period or ultrasound dating before 20 weeks' gestation. Exclusion criteria were multiple gestations, ruptured membranes, placenta previa, active bleeding, previously treated bacterial vaginosis in the pregnancy, cervical cerclages, known major fetal anomalies, and fetal death (present at the time of potential enrollment). All eligible patients attending the Perinatal Centre for prenatal care were informed of the study and offered enrollment.
Data collected at enrollment included demographics (age, race, parity, gravida, abortions), Nova Scotia perinatal risk score, preterm birth-risk score (developed specifically for the Nova Scotia population), and digital examination (cervical dilatation, effacement, and Bishop score). Vaginal swabs for bacterial vaginosis (clinical diagnosis and Gram stain), and cervical and vaginal swabs for fetal fibronectin (FDC-6 and X18A4) and a nonspecific fibronectin (CAF) were also obtained.
The Nova Scotia perinatal risk score included reproductive, obstetric, and medical histories and present pregnancy complications, to identify women at increased risk for a variety of pregnancy complications. The preterm birth-risk score was developed specifically for the Nova Scotia population by identifying major and minor risk factors for preterm delivery, based on a retrospective cohort analysis of over 25,000 pregnancies (Armson BA, Dodds L. Prediction of preterm birth in a population of Canadian women. Abstract FIGO, 1995; FC078.1:93). The scores were determined from 20–24 weeks' gestation. Major risk factors included previous preterm delivery, uterine anomaly, multiple gestation, suspected preterm labor, bleeding after 12 weeks, anemia (hemoglobin less than 100 g/L), cervical dilatation exceeding 1 cm, cervical length less than 1 cm, and abdominal surgery during the current pregnancy. Minor risk factors included smoking during pregnancy, polyhydramnios, sexually transmitted disease, and gestational diabetes. At least one major factor or two minor factors gave women positive (or high) risk scores.
Before digital cervical examination, a speculum examination was performed, and samples for fetal fibronectin were taken from the posterior fornix and cervix (separately) by placing a dry Dacron swab against the area for 10 seconds and then placing it in 750 μL of assay buffer. The solutions were centrifuged within 6 hours of collection and stored at −70C for a maximum of 6 weeks. Samples were batched and shipped to the University of Pennsylvania Medical Center where enzyme-linked immunosorbant assay was performed with three different antibodies, FDC-6, X18A4, and CAF (a general antifibronectin antibody that binds to a nonglycosylated epitope in the fibronectin central cell binding domain), as previously described.19,20 Concentrations exceeding 50 μg/mL were considered positive. Clinical information of subjects was not known to those performing the assays.
After samples for fetal fibronectin were obtained, vaginal secretions for bacterial vaginosis were collected with premoistened cotton-tipped swabs placed in the posterior fornix. One swab was smeared in a drop of normal saline on a slide, which was examined by microscopy for clue cells. The other swab was placed in 10% potassium hydroxide to detect a fishy odor. Vaginal secretions were tested with pH paper to determine whether the pH exceeded 4.5. Clinical diagnosis of bacterial vaginosis included the presence of clue cells (representing 20% or more of epithelial cells) plus two of the following: (1) homogeneous discharge that adhered to, but was easily wiped from, the vaginal wall, (2) vaginal pH above 4.5, or (3) a fishy odor on addition of 10% potassium hydroxide.21 A dry cotton-tipped swab was used to collect secretions from the posterior fornix, and a thin smear was placed on a dry slide to air dry. This slide was used for Gram stain diagnosis of bacterial vaginosis, indicated by a score of 7–10 using the criteria of Nugent et al.22 Clinical information about subjects was not known by the lab technician reading the slides.
Digital cervical examinations were done with sterile gloves and lubricant to estimate cervical dilatation, effacement, and Bishop score. The results of digital cervical examinations were known to the attending physicians. However, results of bacterial vaginosis and fetal fibronectin tests were not available to patients' caregivers. Prenatal care was left to the discretion of the attending physicians. The standard of care at the study hospital for patients with preterm labor between 24 and 32 weeks' gestation was ritodrine as a tocolytic agent (unless contraindicated). Steroids were administered to women with preterm labor or premature rupture of membranes between 24 and 34 weeks' gestation. Antibiotics were not universally given to women not in labor with premature rupture of membranes. After delivery, patients' charts were reviewed to determine the occurrence of preterm labor, onset of spontaneous labor, date of delivery, birth weight, and suspected or confirmed neonatal or maternal infections.
The primary outcome of the study was spontaneous preterm birth (after premature rupture of membranes or spontaneous labor) before 37 weeks' gestation. Secondary outcomes included suspected and confirmed neonatal infection, and maternal infections including chorioamnionitis and endometritis. Positive results of blood, urine, or cerebrospinal fluid cultures in neonates resulting in antibiotic therapy confirmed the diagnosis of neonatal infections. Suspected neonatal infections were defined as clinical signs and need for antibiotics, but negative cultures. Clinical chorioamnionitis was defined as uterine tenderness with temperature at least 38C or foul-smelling amniotic fluid. Postpartum endometritis was defined as uterine tenderness and temperature at least 38C on at least two occasions 6 hours apart.
A sample size of 130 was estimated to give a margin of error of 25%. This sample size was based on the confidence interval around a positive predictive value (PPV) of 70%.23 Review of the Grace Maternity Hospital Atlee Database for 1992 found a spontaneous preterm delivery rate of 10.0% in patients followed primarily in the Perinatal Centre, excluding women with multiple gestation or placenta previa.
Data were analyzed using the computer program Statistix (Statistix Analytical Software, Tallahassee, FL). In the description of subjects, categoric data were expressed by percentage, continuous variables as mean and standard deviation if normally distributed, or median with quartiles if nonnormally distributed. Sensitivity, specificity, positive predictive value, negative predictive value, and likelihood ratios for each individual factor and significant combination of factors were calculated. Multiple logistic regression (using reverse elimination) was used to assess the association of significant variables with the outcome of spontaneous preterm birth with significance of P < .05.
Of 238 women approached for enrollment 142 (59.7%) gave consent. The spontaneous preterm delivery rate of the 96 women who declined enrollment was 7.4%. Of the 142 enrolled, two were lost to follow-up because they delivered outside the province, leaving 140 subjects for analysis. Table 1 summarizes the demographic characteristics of the women. Fifteen (10.7%) women delivered before 37 weeks' gestation, nine (6.4%) of whom had spontaneous preterm births. Maternal infections (chorioamnionitis or endometritis) were diagnosed in five women (3.6%), with neonatal infections suspected in seven infants (5.0%) but confirmed in none.
Table 2 shows the prevalence of possible predictors of spontaneous preterm birth, including preterm birth-risk score, bacterial vaginosis (both clinical criteria and Gram stain using Nugent's score), and vaginal and cervical fetal fibronectin (FDC-6, X18A4, and CAF). Because of a report24 of higher incidence of fetal fibronectin-positive results up to 20 weeks' gestation, we analyzed the prevalence of possible predictors for the subgroup of subjects enrolled at a gestational age between 22 and 24 weeks (n = 91). The prevalence of predictors in that group was similar to that in the overall group. Univariate analysis showed that the variables significantly associated with primary outcome were preterm birth-risk score and vaginal fetal fibronectin FDC-6. These findings were confirmed in the subgroup of women enrolled between 22 and 24 weeks' gestation.
Multiple logistic regression was done for the potential predictors of spontaneous preterm birth, including bacterial vaginosis by Gram stain and clinical diagnoses, preterm birth-risk scores, vaginal fetal fibronectin FDC-6, vaginal fetal fibronectin X18X4, and potential confounders (including race, maternal age, and parity). The Nova Scotia perinatal risk score correlated strongly with preterm birth-risk score (P < .001) and was dropped from further analyses. Modeling was done with all variables initially, and with a second model excluding cervical fetal fibronectin FDC-6, cervical fetal fibronectin X18A4, and fibronectin CAF (because they were statistically significantly associated with other variables in the model using McNemar's test). The final model in both cases included preterm birth-risk scores (OR 16.9; 95% CI 3.0, 92.8; P = .001) and vaginal fetal fibronectin FDC-6 (OR 8.0; 95% CI 1.6, 38.3; P = .01).
Positive vaginal fetal fibronectin FDC-6 and preterm birth-risk scores were the two factors associated with the primary outcome. Combinations of those factors in predicting spontaneous preterm births were evaluated (Table 3). Positive preterm birth-risk scores and negative fetal fibronectin FDC-6 vaginal swabs (compared with both tests being negative) were significantly associated with primary outcomes (OR 11.2). Positive vaginal fetal fibronectin FDC-6 results and negative pre-term birth-risk scores (compared with both tests negative) were not significantly associated with pre-term delivery. When both factors were positive, there was a significantly increased risk (OR 34.1).
Table 4 summarizes the sensitivity, specificity, positive and negative predictive values, and the likelihood ratios of positive and negative tests for preterm birth-risk scores, vaginal fetal fibronectin FDC-6, and the combination of the two. Each variable alone had a poor positive predictive value, but when both predictors were present positive predictive values increased to 57.1% with a high likelihood ratio (19.4). When both risk factors were present, however, sensitivity decreased, specificity increased, and negative predictive value remained high.
Univariate analysis of significant predictors of spontaneous preterm birth and secondary outcomes found that preterm birth-risk scores and vaginal fetal fibronectin FDC-6 were not significantly associated with either maternal infection or suspected neonatal infection. None of the other potential predictors were associated with these outcomes.
The Nova Scotia population-specific preterm birth-risk scoring system was associated significantly with pre-term delivery. However, the risk scoring system alone does not meet the requirements of a good screening test, because the positive predictive value (21.2%) and positive likelihood ratio (3.9) are low.
This study confirmed the findings of others that suggested that vaginal fetal fibronectin FDC-6 was associated with spontaneous preterm birth in asymptomatic women.15–20 Cervical fetal fibronectin FDC-6 was not associated with preterm birth in this study. Some investigators15,16 found differences in diagnostic values between vaginal and cervical sampling, but believed that observed differences between the two sites were small and of limited clinical relevance. Unlike those earlier studies, our study found a significant difference in the association of fetal fibronectin FDC-6 and preterm delivery in the sampling site.
Although the sensitivity and specificity for vaginal fetal fibronectin FDC-6 were marginal, the positive predictive value was poor, which is consistent with other studies of low-risk populations.15–18 The positive likelihood ratio was low (3.3) suggesting that fetal fibronectin alone has limited predictive value for spontaneous preterm births in asymptomatic women. Unlike the positive likelihood ratios of the risk scoring system or vaginal fetal fibronectin FDC-6 alone, the combination of positive test results suggests a conclusive change (because the likelihood ratio is greater than 10) and indicates significant accuracy in predicting preterm delivery. One possible screening test would be to test for vaginal FFN FDC-6 in women with a positive preterm birth-risk score. Women with a positive pre-term birth-risk score and a positive vaginal fetal fibronectin FDC-6 result might need closer antenatal surveillance.
The present study did not find an association between bacterial vaginosis identified between 20 and 24 weeks' gestation and preterm birth. That might contradict the findings of earlier investigators, however, those differences might be due to the small sample sizes, gestational age of samplings, racial mix of our population, and universal access to prenatal care. Our results agree with those of Meis et al14 in which no association between bacterial vaginosis at 24 weeks' gestation and preterm delivery was found.
Researchers investigated distinct oncofetal epitopes within fibronectin other than FDC-6 and identified X18A4 as a monoclonal antibody that binds to fetal fibronectin that is distinct from FDC-6.19 Because this epitope exhibited no detectable binding to plasma fibronectin, it might provide greater detection specificity than FDC-6. In the present study, however, samples that tested positive for X18A4 were not associated with spontaneous preterm birth. Further studies with larger sample sizes will be required to confirm those findings before dismissing this epitope as not predictive of preterm delivery.
One important limitation of this study was the small sample size, possibly resulting in a lack of significance in several of the potential predictors (including bacterial vaginosis and several forms of fetal fibronectin). Significant associations were found between the preterm birth-risk score, vaginal fetal fibronectin FDC-6, and preterm birth. The percentage of women considered high risk in the present study (42.9%) was higher than that of the province (20%). Women referred to the Perinatal Centre might be at higher risk than the general obstetric population, which might limit generalizability.
Although preterm birth-risk score and vaginal fetal fibronectin FDC-6 together predicted preterm delivery, we could not conclude that by using this combination of screening tests, the rate of spontaneous preterm births will decrease. Intervention trials are needed to determine whether treatment based on results of screening tests (eg, antibiotics or antiinflammatory agents) will cost-effectively reduce spontaneous preterm births.
1. Morrison JC. Preterm birth: A puzzle worth solving. Obstet Gynecol 1990;76:5S–12S.
2. McLean M, Walters WAW, Smith R. Prediction and early diagnosis of preterm labor: A critical review. Obstet Gynecol Survey 1993;48:209–25.
3. Creasy RK, Gummer BA, Liggins GC. System for predicting spontaneous preterm birth. Obstet Gynecol 1980;55:692–5.
4. Papiernik E, Bouyer J, Dreyfus J, Collin D, Winisdorffer G, Guegen S, et al. Prevention of preterm births: A perinatal study in Haguenau, France. Pediatrics 1985;76:154–8.
5. Herron MA, Katz M, Creasy RK. Evaluation of a preterm birth prevention program: Preliminary report. Obstet Gynecol 1982;59:452–6.
6. Main DM, Richardson D, Gabbe SG, Strong S, Weller SC. Prospective evaluation of a risk scoring system for predicting preterm delivery in black inner city women. Obstet Gynecol 1987;69:61–6.
7. Holbrook RH, Laros RK, Creasy RK. Evaluation of a risk-scoring system for prediction of preterm labor. Am J Perinatol 1989;6:62–8.
8. Owen J, Goldenberg RL, Davis RO, Kirk KA, Copper RL. Evaluation of a risk scoring system as a predictor of preterm birth in an indigent population. Am J Obstet Gynecol 1990;163:873–9.
9. Mercer BM, Goldenberg RL, Das A, Moawad AH, Iams JD, Meis PJ, et al. The preterm prediction study: A clinical risk assessment system. National Institute of Child Health and Human Development Maternal-Fetal Medicine Units Network. Am J Obstet Gynecol 1996;174:1885–95.
10. Minkoff H, Grunebaum AN, Schwarz RH, Feldman J, Cummings M, Crombleholme W, et al. Risk factors for prematurity and premature rupture of membranes: A prospective study of vaginal flora in pregnancy. Am J Obstet Gynecol 1984;150:965–72.
11. Gravett MG, Nelson HP, DeRouen T, Critchlow C, Eschenbach DA, Holmes KK. Independent associations of bacterial vaginosis and chlamydia trachomatis infection with adverse pregnancy outcome. JAMA 1986;256:1899–903.
12. Hillier SL, Nugent RP, Eschenbach DA, Krohn MA, Gibbs RS, Martin DH, et al. Association between bacterial vaginosis and preterm delivery of a low-birth-weight infant. The vaginal infections and prematurity study group. N Engl J Med 1995;333:1737–42.
13. Riduan JM, Hillier SL, Utomo B, Wiknjosastro G, Linnan M, Kandun N. Bacterial vaginosis and prematurity in Indonesia: Association in early and late pregnancy. Am J Obstet Gynecol 1993;169:175–8.
14. Meis PJ, Goldenberg RL, Mercer B, Moawad A, Das A, McNellis D, et al. The preterm prediction study: Significance of vaginal infections. National Institute of Child Health and Human Development Maternal-Fetal Medicine Units Network. Am J Obstet Gynecol 1995;173:1231–5.
15. Lockwood CR, Wein R, Lapinski R, Casal D, Berkowitz G, Alvarez M, et al. The presence of cervical and vaginal fetal fibronectin predicts preterm delivery in an inner-city obstetric population. Am J Obstet Gynecol 1993;169:798–804.
16. Hellemans P, Gerris J, Verdonk P. Fetal fibronectin detection for prediction of preterm birth in low-risk women. Br J Obstet Gynaecol 1995;102:207–12.
17. Greenhagen JB, Van Wagoner J, Dudley D, Hunter C, Mitchell M, Logsdon V, et al. Value of fetal fibronectin as a predictor of preterm delivery for a low-risk population. Am J Obstet Gynecol 1996;75:1054–6.
18. Goldenberg RL, Mercer BM, Meis PJ, Copper RL, Das A, McNellis D. The preterm prediction study: Fetal fibronectin testing and spontaneous preterm birth. National Institute of Child Health and Human Development Maternal-Fetal Medicine Units Network. Obstet Gynecol 1996;87:643–8.
19. Feinberg RF, Kliman HJ, Bedian V, Monzon-Bordonaba F, Menzin AW, Wang CL. Monoclonal antibody X18A4 identifies an oncofetal fibronectin epitope distinct from the FDC-6 binding site. Am J Obstet Gynecol 1995;172:1526–36.
20. Feinberg RF, Wang CL. Monoclonal antibody FDC-6 exhibits binding to human plasma fibronectin: A caveat for cervicovaginal oncofetal fibronectin testing? Am J Obstet Gynecol 1994;171:1302–8.
21. Amsel R, Totten PA, Spiegel CA, Chen KCS, Eschenbach D, Holmes KK. Nonspecific vaginitis: Diagnostic criteria and microbial and epidemiologic associations. Am J Med 1983;74:14–22.
22. Nugent RP, Krohn MA, Hillier SL. Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation. J Clin Microbiol 1991;29:297–301.
23. Arkin CF, Wachtel MS. How many patients are necessary to assess test performance? JAMA 1990;263:275–8.
24. Lockwood CJ, Senyei AE, Dische MR, Casal D, Shah KD, Thung SN, et al. Fetal fibronectin in cervical and vaginal secretions as a predictor of preterm delivery. N Engl J Med 1991;325:669–74.