Obstetrics & Gynecology:
Maternal Serum Cytokines in Preterm Premature Rupture of Membranes
Murtha, Amy P. MD1; Sinclair, Tammy BSN1; Hauser, Elizabeth R. PhD2; Swamy, Geeta K. MD1; Herbert, William N. P. MD3; Heine, R Phillips MD1
From the 1Department of Obstetrics and Gynecology and 2Center for Human Genetics, Duke University Medical Center, Durham, North Carolina; and 3Department of Obstetrics and Gynecology, University of Virginia, Charlottesville, Virginia.
Supported by a grant MO1-RR-30, National Center for Research Resources, Clinical Research Centers Program, National Institutes of Health.
Corresponding author: Amy P. Murtha, MD, Duke University Medical Center, Department of Obstetrics and Gynecology, Division of Maternal Fetal Medicine, Box 3967, Trent Rd, Durham, NC 27710; e-mail: email@example.com.
OBJECTIVE: To estimate whether maternal serum interleukin (IL)-6 or granulocyte colony-stimulating factor (G-CSF) obtained daily are elevated in women with preterm premature rupture of membranes who develop funisitis.
METHODS: Daily blood samples were obtained from women with preterm premature rupture of membranes and analyzed for IL-6 and G-CSF by enzyme-linked immunosorbent assay. Funisitis was determined by placental examination. Observations were stratified based on the presence or absence of funisitis and analyzed. Proportional hazards models were used to evaluate time-to-delivery on the basis of diagnostic IL-6 and G-CSF levels, determined by receiver operating characteristic curve analysis.
RESULTS: Of the 107 patients available for analysis, 54 (50%) had evidence of funisitis after delivery. Patients with funisitis were more likely to deliver at an earlier gestational age (28.5 weeks compared with 31.5 weeks, P<.001) and have Medicaid insurance (57% compared with 39%, P=.04). Serum IL-6 and G-CSF were elevated 24 to 48 hours before delivery in women with preterm premature rupture of membranes with funisitis compared with those without funisitis (IL-6, 7.5 compared with 2.8 pg/mL, P<.001; G-CSF, 121.7 compared with 56.9 pg/mL, P=.002). Using values identified by the receiver operating characteristic curve, elevated serum IL-6 in the interval 24–72 hours before delivery was significantly associated with funisitis (P<.03), even after controlling for gestational age and insurance status.
CONCLUSION: Maternal serum IL-6 and G-CSF appear to be biomarkers in the identification of women with preterm premature rupture of membranes likely to develop funisitis.
LEVEL OF EVIDENCE: II
Preterm premature rupture of membranes (PROM) affects more than 120,000 pregnancies annually in the United States and is associated with significant maternal, fetal, and neonatal risk.1 Preterm PROM occurs in approximately 2–3% of all pregnancies but is associated with 20% of all perinatal deaths. The most important complications related to preterm PROM are preterm delivery and intra-amniotic infection. Intra-amniotic infection, or chorioamnionitis, occurs in 6% of preterm deliveries without preterm PROM but occurs in at least 27% of preterm deliveries with preterm PROM; it is associated with a four-fold increase in neonatal mortality as well as increased neonatal morbidities.2,3
The cause of preterm PROM remains unclear, although both infection and inflammation seem to play important roles. Infections, including Group B Streptococcus and bacterial vaginosis, have been associated with preterm PROM.4,5 In addition to the direct proteolytic effect of bacteria, there is a pronounced inflammatory response in the setting of infection.6 This inflammatory response results in activation of neutrophils and macrophages to the site of infection and resultant production of cytokines, prostaglandins, and metalloproteases. This inflammatory response can be demonstrated in the fetal amniotic fluid as well as systemically in both mother and fetus in the setting of preterm PROM on the day of delivery.7
Patients diagnosed with preterm PROM are often managed expectantly with hospitalization and bed rest until there is clinical evidence of intra-amniotic infection or documentation of fetal lung maturity. Once preterm PROM occurs, patients are evaluated for evidence of underlying infection or labor. If neither condition is present, preterm PROM patients are admitted for inpatient observation and initiation of antibiotic and steroid therapy.
Despite the use of broad-spectrum antibiotics, about 30% of preterm PROM patients will develop clinical chorioamnionitis.8 Moreover, it is unclear which patients will or will not develop significant infection resulting in funisitis. Infants born with funisitis are at higher risk for neonatal sepsis as well as long-term handicap, including bronchopulmonary dysplasia and cerebral palsy.9–12 The majority of infants born with funisitis have no detectable clinical signs before delivery. Diagnosis of clinical infection is dependent on clinical signs and symptoms, including maternal fever, uterine tenderness, and fetal or maternal tachycardia, which are often not present.
Previously, we have described elevations in maternal serum IL-6 and granulocyte colony-stimulating factor (G-CSF) in term and preterm patients with chorioamnionitis on the day of delivery.7,13 Our previous investigations suggest that this rise may occur before the day of delivery and therefore would be a useful tool for the prediction of infection in the absence of clinical signs or symptoms.
This investigation was therefore designed to estimate the diagnostic accuracy of maternal serum cytokines (IL-6 and G-CSF) in preterm PROM patients for the prediction of funisitis. Specifically, we sought to determine whether maternal serum IL-6 and G-CSF rose before the onset of clinical infection or labor in preterm PROM patients diagnosed with funisitis at delivery. This was accomplished by collecting daily blood samples on participants with preterm PROM for the entire hospital stay and then evaluating placentas for evidence of funisitis.
MATERIALS AND METHODS
All patients receiving prenatal care at Duke University Medical Center were eligible for this Duke University Health System Institutional Review Board–approved prospective cohort investigation. Patients at 22–34 weeks of gestation diagnosed with PROM from July 1997 through April 2004 were offered enrollment in this study. Preterm PROM was diagnosed with either vaginal pooling of amniotic fluid or positive phenaphthazine (Nitrazine, Bristol-Myers Squibb, New York, NY) testing and positive fern testing. Participants were excluded if there was evidence of extrauterine infection, clinical chorioamnionitis, active labor with cervical dilatation greater than 3 cm, and known fetal congenital malformation incompatible with life. Participants with clinical evidence of chorioamnionitis were delivered and therefore excluded.
After informed consent was obtained, a 5-mL sample of blood was collected at enrollment. Serial daily blood samples were collected by a General Clinical Research Center nurse between 8 am and 12 pm. All samples were collected into sterile silicon-coated tubes and refrigerated at 5°C. Specimens were centrifuged at 600g for 10 minutes at 5°C with serum aliquots frozen at –80°C.
Maternal serum IL-6 and G-CSF levels were determined by standard enzyme-linked immunosorbent assay (ELISA). For IL-6 concentrations, an ultrasensitive ELISA was performed (Cytokine Core Laboratory, Baltimore, MD), with lower limits of detection of 1.2 pg/mL and interassay and intra-assay variations of less than 5%. Granulocyte colony-stimulating concentrations were performed using a standard sensitivity assay (R&D Systems, Minneapolis, MN) with lower limits of detection of 20 pg/mL, and interassay and intra-assay variation of less than 10%. Placentas were examined for evidence of histologic chorioamnionitis and funisitis by one staff pathologist who had no knowledge of the IL-6 and G-CSF levels.
All patients were monitored clinically with vital signs, including temperature and fetal heart tones, and twice-daily physical examinations for evaluation of the development of clinical evidence of infection. All patients were treated with intravenous antibiotics. Although the standard antibiotic regimen varied throughout the study period, the majority of study participants received a 7-day course of ampicillin and azithromycin. Patients between 23 and 34 weeks of gestation received betamethasone for fetal lung maturation on admission. The standard for betamethasone administration varied throughout the study period from once weekly to a single course at admission. The results of the cytokine assays were not available to the clinicians. Clinical data were collected prospectively and confirmed by chart review by the principal investigator and study coordinator. Data collected included demographic information, maternal age, race, gestational age at enrollment, gestational age at delivery, parity, tobacco, alcohol or illicit substance use, past medical history, obstetric history including history of preterm deliveries, temperature, maternal and fetal heart rate, evidence of uterine tenderness, and interval from enrollment to delivery. Funisitis was defined as the presence of neutrophils infiltrating the wall of one or more umbilical vessels as described by Redline et al.14
Descriptive statistics (mean and standard deviation for continuous variables and proportions for ordinal variables) were generated on each of the variables. Observations were stratified on the basis of the presence or absence of funisitis. Tests were performed using both parametric and nonparametric methods (t tests and Mann-Whitney U tests for continuous variables, χ2 and Fisher exact tests for ordinal variables) to allow for departures from normality in our sample. Normality was assessed using standard benchmarks for skewness and kurtosis, Q-Q plots, and by visual inspection for outliers and symmetry. Proportional hazards regression models were used to evaluate time to delivery on the basis of diagnostic IL-6 and G-CSF levels, as determined by the receiver operating characteristic (ROC) analysis described below. Logistic regression was performed to investigate the relationship of IL6 levels and G-CSF levels on the probability of funisitis after adjusting for other baseline variables that were significantly different between pregnancies with and without funisitis, including gestational age and insurance status (Table 1). All analyses were performed using SAS 9.0 (SAS Institute, Cary, NC).
After institutional review board approval, 122 participants were enrolled from June 1997 through March 2004. Of the 122 participants enrolled, 15 were excluded because pathologic examination of the placenta was not available (n=8) or they delivered at an outside institution (n=7). Of the remaining 107 participants, 54 (50%) had evidence of funisitis on examination of the placenta. The average gestational age at admission was 28.0 weeks and the average gestational age at delivery was 30.0 weeks, with a mean latency period of 14.4 days. Participants were divided into those with and without funisitis for analysis. Maternal demographic and clinical characteristics are presented in Table 1. There were significantly more participants with clinical evidence of chorioamnionitis in the funisitis group than in the no-funisitis group. Of note, 59% of participants with funisitis were without clinical evidence of infection before delivery, despite having funisitis at the time of delivery. In addition, the gestational age at admission and delivery were significantly earlier in the funisitis group compared with the no-funisitis group. As expected, birthweight was also significantly lower in the funisitis group compared with the no-funisitis group. In addition, Medicaid insurance was more common in participants with funisitis compared with those without funisitis.
The results of the maternal serum IL-6 and G-CSF values are presented in Table 2. The number of patients included in each analysis was dependent on the time point analyzed. Maternal serum IL-6 and G-CSF were significantly elevated 24–48 hours before delivery in the funisitis group compared with the no-funisitis group. This difference was also seen in IL-6 when samples were obtained 48 to 72 hours before delivery. In addition, the most remote from delivery value for either IL-6 or G-CSF between 24 and 72 hours was chosen for analysis. If two samples were obtained within that interval, the value furthest from delivery was selected for comparison. Maternal serum IL-6 was significantly higher in participants that developed funisitis compared with those without funisitis (Table 2). Analysis beyond 72 hours was limited due to samples size, although when all samples obtained greater than 72 hours were combined, maternal serum IL-6 was higher in participants with funisitis.
An ROC curve (Analyze-it Software V.13, Leeds, UK) was constructed to further examine the relationship of maternal serum IL-6 and G-CSF to funisitis.15 As shown in Figure 1, IL-6 is an accurate predictor of funisitis in preterm PROM participants 24 to 72 hours before delivery (area under curve: 0.79, standard error=.05, P<.001). Cutoff values for maternal serum IL-6 and G-CSF obtained 24–72 hours before delivery were selected from ROC curves to optimize for both sensitivity and specificity to detect funisitis. Table 3 shows the diagnostic indices for IL-6 and G-CSF selected to optimize for both sensitivity (lower number) and specificity (higher number).
Interleukin-6 and G-CSF appear to be highly correlated (r=.59, P<.001) and were therefore not independently predictive of funisitis; thus, only one measure was included in the logistic and survival analysis. Survival analysis using time to delivery was performed for maternal serum IL-6 and G-CSF at 24–72 hours before delivery using the cutoff values calculated from the ROC curves above. Figure 2 shows an example of the survival curve for IL-6, where the IL-6 cutoff chosen was selected to provide a sensitive predictor of funisitis.
To control for the significant differences in gestational age at delivery and Medicaid insurance, we performed a logistic regression analysis (Table 4). Using the 1.8 pg/mL value identified by the ROC curve, an elevated maternal serum IL-6 in the interval 24–72 hours before delivery was significantly associated with funisitis (P<.03), even after controlling for gestational age at delivery and insurance status. Thus, elevations in maternal serum IL-6 values in preterm PROM patients may be a clinically useful indicator of impending infection.
If the interval from sample collection to delivery was short once the IL-6 level reached a threshold, then the utility of this test may not be clinically relevant. We therefore evaluated the interval between the sample time and delivery for participants who achieve an IL-6 level greater than 8 pg/mL (Table 5). The interval from sample collection to delivery and the IL-6 level from participants who achieve an IL-6 level greater than 8 pg/mL is presented. In addition, the interval of sample collection to delivery and IL-6 level from the sample immediately preceding the sample with an IL-6 level of 8 pg/mL is also presented. If the participant never reached an IL-6 level greater than 8 pg/mL, they were not included in this analysis. There are fewer participants without funisitis who achieved an IL-6 level greater than 8 pg/mL than those with funisitis. The median latency from sample collection to delivery in participants with funisitis was nearly 4 days. In addition, the median IL-6 level in participants with funisitis in this subgroup analysis remains significantly higher than those without funisitis. Even after adjusting for gestational age at delivery and insurance, the odds ratio for funisitis is 7.8 (95% 95% confidence interval 2.9–21.7) for those with IL-6 levels more than 8 pg/mL relative to IL-6 levels that do not achieve 8 pg/mL.
The results of this investigation suggest that maternal serum IL-6 predicts individuals destined to develop funisitis up to 72 hours before delivery. We previously demonstrated that maternal serum IL-6 and G-CSF are elevated on the day of delivery in participants with both chorioamnionitis and funisitis.13,16,17 Others have also shown that maternal serum IL-6 is significantly elevated at the time of delivery in preterm PROM participants when labor or clinical evidence of infection are present. In addition, Hatzidaki et al18 have demonstrated that elevated maternal serum IL-6 at delivery is associated with worse neonatal outcomes. The intent of our investigation was to determine how early (or remote from delivery) this rise in maternal serum IL-6 occurs. A unique feature of this investigation was the collection of daily blood samples, which made it possible to evaluate the predictive value of specific time points before delivery that could not otherwise be predicted before delivery.
Several investigations have demonstrated a rise in amniotic fluid IL-6 in the presence of labor as well as intra-amniotic infection.19–21 In an investigation performed in Sweden, amniotic fluid IL-6 levels were significantly higher in preterm PROM participants with intra-amniotic infection diagnosed by polymerase chain reaction compared with those without infection.21 Even in term patients it is clear that a rise in amniotic fluid IL-6 and G-CSF are associated with intra-amniotic infection as well as active labor.16,19,21–26 Although it is clear that amniotic fluid IL-6 can be an important predictor of infection, the clinical usefulness of this test is limited by the need for repeated invasive procedures in the setting of preterm PROM, which carry their own innate risks.27
Several investigations have attempted to determine whether maternal serum IL-6 is elevated in patients delivering preterm. Bahar and colleagues28 measured maternal serum IL-6, IL-8, tumor necrosis factor, and interferon-γ in term and preterm labor. In this investigation they were unable to identify a rise in any of the cytokines evaluated. In this investigation samples were collected at the time of admission, the interval from sample collection to delivery was varied, and it was unclear whether there was clinical evidence of labor or infection at the time of sample collection. In addition, this investigation included both participants with intact membranes and preterm PROM, making subgroup analysis difficult due to small sample size. It is likely that the results of this investigation are in contrast to our findings because of the timing of sample collection and the small number of participants with preterm PROM.
The identification of a systemic inflammatory response to infection up to 72 hours before delivery may be critical in the management of preterm PROM patients. The ability to predict impending infection in participants with preterm PROM would allow the clinician an opportunity to offer changes in management strategies that could potentially effect the risk of neonatal complications. Further investigations would be required to evaluate various treatment strategies in the setting of rising maternal serum IL-6 or G-CSF. Such changes in management may have significant implications for the outcome of the preterm neonate. If the intervention chosen is delivery in the setting of rising IL-6, then it is important that the cutoff value optimize for specificity to limit the number of false positives. Alternatively, if reinitiation of antibiotics was chosen as the intervention of choice, then a more sensitive cutoff level would be appropriate. Cutoff values for maternal serum IL-6 and G-CSF were therefore chosen from ROC curves to optimize for both sensitivity and specificity.
This investigation is an important step in understanding the role of systemic inflammation in the development of funisitis. The fact that maternal serum IL-6 and G-CSF rise as much as 72 hours before the onset of labor or clinical infection in preterm PROM may become an important tool in the care of preterm PROM patients. Additional investigations are needed to confirm our findings and to determine alternate management strategies in the setting of elevated maternal serum IL-6 in preterm PROM patients.
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