Objective
We sought to evaluate gestational age, cervical length, amniotic fluid interleukin (IL)-6, and selected proteomic biomarkers as independent predictors of adverse outcome in preterm premature rupture of membranes (PPROM).
Study Design
This was a prospective cohort study of 65 consecutive women with PPROM (20.0-34.6 weeks). Gestational age, cervical length, amniotic fluid IL-6, and proteomic biomarkers (calgranulins A and C, and neutrophil defensins 1 and 2) were evaluated at diagnosis. The predictive value for intraamniotic infection and neonatal composite morbidity was calculated by logistic regression.
Results
Proteomic biomarkers were independent predictors of intraamniotic infection (odds ratio, 22.1; P = .011) and neonatal composite morbidity (odds ratio, 17.6; P = .02). With the exception of a trend between gestational age and neonatal morbidity ( P = .054), none of the other parameters were independent predictors of outcome measures.
Conclusion
Selected proteomic biomarkers were the only independent predictors of adverse outcomes in PPROM. Contrary to what is reported in preterm labor with intact membranes, gestational age, cervical length, and IL-6 were not.
Preterm premature rupture of membranes (PPROM) is responsible for approximately one-third of preterm births and a substantial proportion of neonatal mortality and serious morbidity. Neonatal outcome depends on the occurrence of complications in relation with prematurity or infection. Predicting the risk of neonatal adverse outcomes is a critical need and constitutes the basis for improving pregnancy management and parental counseling. Subclinical intraamniotic inflammation (IAI), which is present in a substantial proportion of patients with either preterm labor (PTL) or PPROM, is proposed as the strongest predictor of adverse outcome in these patients, even in the absence of demonstrable positive cultures.
Identification of the risk of adverse outcome in patients with PPROM remains challenging. The role of clinical features with an independent prognostic value among pregnancies with PTL and intact membranes, such as gestational age and short cervical length, has so far not been investigated in women with PPROM. Concerning biochemical markers, the performance of amniotic fluid interleukin (IL)-6 to predict IAI and adverse perinatal outcome is poorer in pregnancies presenting with PPROM with respect to that described for women with PTL and intact membranes. Recently, new biomarkers of inflammation in amniotic fluid, integrated in a mass restricted score, such as calgranulins A and C, and neutrophil defensins have shown promising results as predictors of intraamniotic infection and neonatal sepsis in patients at risk. In cohorts containing only women with PTL and intact membranes, these inflammatory proteins predict IAI with a similar accuracy to IL-6, and their combination with IL-6 seems to further increase the predictive value of each biomarker alone. However, the value of calgranulins and neutrophil defensins in cases with only PPROM remains to be investigated.
The purpose of this study was to evaluate the independent and combined value of clinical parameters, including gestational age and cervical length at diagnosis, and amniotic fluid IL-6 and proteomic biomarkers (calgranulins A and C, and neutrophil defensins 1 and 2) to predict adverse outcomes in pregnancies complicated with PPROM.
Materials and Methods
A prospective cohort study was performed from September 2005 through June 2008 at the Hospital Clínic of Barcelona, Catalonia, Spain. The reference population of this study were pregnant women with PPROM at <34 completed weeks. Gestational age was established according to the first-trimester ultrasound scan. Multiple pregnancies, women with clinical signs of chorioamnionitis at admission, or those cases where amniocentesis was not possible (ie, anhydramnios), were not eligible for this study. Consecutive women who fulfilled the inclusion criteria were considered for analysis. Written informed consent was obtained to donate amniotic fluid for research purposes. The institutional review board of Hospital Clinic approved the collection and use of these samples and information for research purposes.
Transvaginal cervical length was measured on admission and transabdominal amniocentesis was performed within the first 48 hours. Cultures of genital mycoplasma, aerobic and anaerobic bacteria, as well as amniotic fluid glucose concentration, white blood cell counts, and Gram stains were performed immediately after collection and their results were available for clinical management. The remaining amniotic fluid was centrifuged at 4000 rpm for 10 minutes at 4°C and stored at −70°C until assayed for IL-6 and selected proteomic biomarkers, the results of which were not used for clinical decisions. After delivery, placentas were collected and placed in 10% neutral buffered formalin for standard histological examination. All maternal and neonatal medical records were reviewed and perinatal outcome was recorded.
A complete course of antenatal steroids, betamethasone 12 mg intramuscular injection with 2 doses given 24 hours apart, was administered from 24.0-34.0 weeks. Tocolysis was considered in all cases of clinical symptoms of PTL in the absence of clinical chorioamnionitis, abruptio placentae, and fetal compromise. Prophylactic parenteral broad-spectrum antibiotics were given at admission for a period of 5 days. The antibiotic regimen of choice in our institution was parenteral ampicillin 1 g every 6 hours and gentamycin 80 mg every 8 hours. In line with other clinical management guidelines, labor was induced if PPROM occurred ≥34.0 weeks. If PPROM occurred <31.0 weeks, women were expectantly managed. At or >31.0 weeks of gestation, labor was induced if pulmonary maturity was documented.
Women with subclinical intraamniotic infection were treated with parenteral antibiotics during 10 days according to the antibiogram of the microorganism isolated. Maternal and fetal statuses were closely monitored for signs of chorioamnionitis, labor, and/or fetal compromise. Induction of labor was based on virulence of the microorganism, gestational age, maternal or fetal clinical signs of chorioamnionitis, or documented lung maturity.
Preterm birth was defined as delivery following spontaneous labor. PPROM was defined by the presence of clinical amniotic fluid leakage after sterile speculum inspection confirmed by alkaline pH in the absence of vaginal infection.
Anhydramnios was considered if the largest vertical pocket was <10 mm. Clinical chorioamnionitis was defined following the criteria of Gibbs et al as a temperature elevation >37.8°C and ≥2 of the following criteria: uterine tenderness, malodorous vaginal discharge, fetal tachycardia (>160 beats/min), maternal tachycardia (>100 beats/min), and maternal leukocytosis >15,000/mm 3 . Intraamniotic infection was considered in patients with a positive amniotic fluid culture. Histological chorioamnionitis was defined as an infiltration of the chorion and/or amnion by polymorph nuclear leucocytes and funisitis as the presence of neutrophil infiltration into the umbilical vessels walls or into Wharton jelly. Neonatal composite morbidity included the presence of any of the following criteria: intraventricular hemorrhage, respiratory distress syndrome, congenital sepsis, and periventricular leukomalacia by ultrasound suspicion. Puerperal endometritis was considered in patients with a temperature higher or equal to 38°C on 2 occasions 4 hours apart (excluding the day of delivery), associated with uterine tenderness, foul-smelling lochia, and no other apparent source of infection.
The presence of subclinical intraamniotic infection and the occurrence of neonatal composite morbidity were considered perinatal adverse outcomes.
To compare maternal and neonatal results we defined “presence of proteomic biomarkers” on detection of at least 1 inflammatory protein in amniotic fluid (calgranulin A or C, or neutrophil defensin 1 or 2), and “absence of proteomic biomarkers” when none of these proteins were detected.
Laboratory methods
IL-6 levels were measured by enzyme-linked immunoassay (Biosource International, Inc., Camarillo, CA). The minimum detectable level of IL-6 was 0.2 ng/mL. The proteomic profile was based on the identification of calgranulin A and C, and neutrophil defensin 1 and 2 using sodium dodecyl sulfate polyacrylamide gel electrophoresis methodology and confirmed by Western blot. Details about the methodology used for proteomic biomarker determinations were previously reported and are available in a supplemental Appendix .
Statistical analysis
Data were collected in a Microsoft Access database (Microsoft, Redmond, WA) made for this purpose. Statistical analyses were performed with the SPSS 14.0 statistical software (SPSS, Chicago, IL). Receiver operating characteristic curve analysis was employed to display the relationship between sensitivity and false positive rate (1-specificity) and to select the best cutoff value for IL-6 to diagnose intraamniotic infection. Two-by-two contingency tables were constructed and χ 2 test or Fisher’s exact test analysis of independence was used to identify significant differences among test performances. Continuous data were compared with the Student t test.
Univariate and logistic regression were performed to investigate the relationship between gestational age at membrane rupture, cervical length, proteomic profile, and amniotic fluid IL-6 and the occurrence of intraamniotic infection and neonatal composite morbidity. Kaplan-Meier probability plots were generated based on the interval between gestational age at membrane rupture and at delivery in groups with high or low levels of IL-6 according to the cutoff proposed and in groups with presence or absence of selected proteomic biomarkers in amniotic fluid. A P value of < .05 was used to indicate significance.
Results
From September 2005 through June 2008, 65 consecutive women met the inclusion criteria and signed informed consent forms, from 136 women presenting with PPROM between 20.0-34.6 weeks. Table 1 displays screening enrollment and indication for delivery. Gestational age and cervical length when PPROM was diagnosed and gestational age at delivery of the entire study population were (median and range) 29.2 (19–34.5) weeks, 27.7 (5–45) mm, and 30.7 (20.2–34.5) weeks, respectively ( Table 2 ). The overall rate of intraamniotic infection was 27.7% (18/65). The microorganisms isolated from the amniotic fluid included Ureaplasma urealyticum (n = 8), Enterococcus faecalis (n = 3), Streptococcus agalactiae (n = 1), Candida albicans (n = 1), Fusobacterium species (n = 1), Streptococcus pneumoniae (n = 1), Gardnerella vaginalis (n = 1), Listeria monocytogenes (n = 1), and Peptostreptococcus spp (n = 1).
| Characteristic | n |
|---|---|
| Maternal age, y, X (SD) | 31.8 (5.5) |
| Nulliparity, n (%) | 34 (52.3) |
| Previous preterm delivery, n (%) | 5 (7.7) |
| GA at PPROM, wk, X (SD) | 29.2 (4.1) |
| GA PPROM <28 wk, n (%) | 20 (30.8) |
| GA PPROM <31 wk, n (%) | 38 (58.5) |
| Bishop index, X (SD) | 2.4 (2.5) |
| Cervical length, mm, X (SD) | 27.7 (9.8) |
| GA delivery, wk, X (SD) | 30.7 (3.3) |
| GA delivery <24 wk, n (%) | 3 (4.5) |
| GA delivery <28 wk, n (%) | 12 (18.5) |
| GA delivery <31 wk, n (%) | 27 (41.5) |
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