Maternal serum proinflammatory cytokines in preterm labor with intact membranes: neonatal outcome and histological associations

ARTICLE

Auteur(s) : Andrzej Torbé, Ryszard Czajka, Agnieszka Kordek, Rafał Rzepka, Sebastian Kwiatkowski, Jacek Rudnicki

From the Chair and Department of Obstetrics, Gynecology and Neonatology of Pomeranian Medical University, 70-111 Szczecin, Powstańców Wielkopolskich 72, Poland

• 1. To evaluate and compare maternal serum concentrations of interleukin-1α (IL-1α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and interleukin-8 (IL-8) in pregnancies complicated by threatened preterm labor (PTL), with the concentrations in healthy controls at approximately full-term.
• 2. To determine if maternal serum concentrations of these cytokines have any value in the prediction of early-onset neonatal infection or histological chorioamnionitis.

Methods and materials

In the study group, a detailed analysis of cytokine concentrations was carried out, and the results were compared with the presence/absence of early-onset neonatal infection or histological chorioamnionitis. Fetal outcome was evaluated by a neonatologist unaware of maternal serum cytokine results, according to the clinical signs of the neonate and its microbial status. Blood cultures were taken from all 65 infants. Neonatal congenital infection was assumed if it occurred within 48 hours of delivery, based on the maternal history and the presence of three or more of the following categories of clinical signs: skin color (pallor, jaundice, cyanosis); respiratory (apnea, tachypnea >60/min, grunting, nasal flaring, intercostal or sternal retractions, need for high ventilator settings or oxygen); cardiovascular (brady/tachycardia, poor peripheral perfusion, hypotension); neurological (hypotonia, irritability, lethargy, seizures); gastrointestinal (abdominal distension, green or bloody residuals, vomiting; temperature instability), and/or positive blood culture. Chest X-rays and laboratory tests routinely performed in the management of infection included CRP levels, white blood cell count (WBC), platelet count, and the immature to total neutrophil ratio, contributed to the diagnosis. Neonatal sepsis was recognized as proven when it was microbiologically confirmed. In other cases, it was defined as suspected (clinical) sepsis [5-7].

All infected newborns received antibiotics after delivery; two died due to neonatal sepsis. For the diagnosis of histological chorioamnionitis, microscopic analysis of the placenta was performed using more than 10 polymorphonuclear leukocytes in 10, non-adjacent microscopic fields from the extraplacental membranes, chorionic plate or umbilical cord blood vessels, and examined at × 400 magnification [8].

Cytokine levels were measured using an ELISA kit (Biotrak; Amersham Pharmacia Biotech, Buckinghamshire, England). The sensitivities of the kits were less than 1 pg/ml for IL-1 beta and IL-6, and less than 2 pg/ml for IL-1 alpha and IL-8. The WBC count was determined automatically with Celldyn 1700 and Celldyn 3500 instruments (Abbott Laboratories, IL, USA); the percentage of band cells was determined by microscopic examination, and C-reactive protein (CRP) was measured quantitatively by immunoturbidometry with the Olympus AU 560 system (Olympus Diagnostica, Hamburg, Germany). The cervicovaginal fluid was cultured for aerobic and anaerobic bacteria.

Statistical analysis was performed with the Statistica 5.5 software and MedCalc 7.2 software. The Shapiro-Wilk test was used to check the distributions of the features analyzed. Differences between the groups were assayed using the Chi-squared test for discrete variables and by the Mann-Whitney U-test for continuous variables. P-values less than 0.05 were considered significant. For all cytokines studied, a receiver operating characteristic (ROC) curve analysis was used to establish the cut-off values that optimized the prediction of neonatal infection or histological chorioamnionitis. Sensitivity, specificity, positive predictive values (PPVs) and negative predictive values (NPVs) were then calculated. Also, areas under the ROC curves (AUC) were evaluated for all study tests. Additionally likelihood ratios (LR) were calculated and values greater than 5.00 were accepted as useful.

Results

Thirty (46.16%) women from the study group delivered prematurely; 35 of them (53.84%), after successful tocolytic treatment, delivered near term (> 36 weeks). All women from the control group delivered at term. Maternal serum concentrations of IL-6 and IL-1β were significantly higher in patients with PTL, when compared to healthy women. The levels of IL-1α and IL-8 were comparable between the groups (table 2).

The pregnancies complicated by PTL were subject to particularly careful analysis. In none of these cases were clinical signs of infection found. However, in these women laboratory indices suggesting the presence of subclinical intrauterine infection were investigated by evaluating WBC count (> 15.0 G/L) [9], the band cells percentage (> 10%) [10], CRP concentration (> 10 mg/L) [11] and by cervico-vaginal secretion culture. The frequency of occurrence is shown in table 3. In 11 positive cultures of cervico-vaginal secretion, the following microbes were found: Candida albicans (5), Bacteroides capillosus (2), Streptococcus B (2), Actinomyces naeslundii and Prevotella (1), Actinomyces naeslundii with Pepto-streptococcus (1). Although in 44.61% (n=29) of cases complicated by PTL, at least one laboratory parameter suggesting the presence of subclinical intra-amniotic infection was observed, the differences in maternal serum proinflammatory cytokines concentrations between the patients with and without positive laboratory indices of infection were not significant.

Out of 65 patients enrolled in the study group, 15 (23.08%) of their neonates developed early-onset infection. Neonatal blood cultures were positive only in five (33.3%) of the infected neonates: Streptococcus agalactiae in 3 cases and Enterococcus foecalis in 2 cases. In the remaining 10 (66.7%) neonates from the study group, suspected sepsis was diagnosed. Maternal serum concentrations of IL-1β were significantly higher in women who delivered newborns with signs of infection than in those who delivered healthy newborns (13.60 verus 1.20 pg/mL; p < 0.02). The levels of other cytokines evaluated were comparable (table 4). To determine the critical values that could predict neonatal, congenital infection, ROC curves were generated for all cytokines studied. The prognostic cut-off values for these markers are shown in table 5. ROC curve analysis revealed that the predictive performance of all cytokines studied for predicting early-onset neonatal infection was of limited clinical value (tables 5, i>6).

The placentas were examined in 48 (73.85%) patients out of the study group. Inflammatory changes were found in 20 of them. In this subgroup, maternal serum concentrations of IL-1β were significantly higher than in the subgroup without histological chorioamnionitis (28.79 versus 5.19 pg/mL; p = 0.001) (table 7). The cut-off value of IL-1β ≥ 14 pg/mL predicted inflammatory changes of the placenta with a sensitivity of 80%, specificity of 86%, positive predictive value of 80%, negative predictive value of 86% and likelihood ratio of 5.62 (table 8). ROC curve analysis also revealed that the diagnostic performance of IL-1β was superior to that of other study markers (table 9).
Table 1 Clinical characteristics of the study and control groups

Discussion

In most of the recent studies, maternal serum proinflammatory cytokine levels were mainly evaluated as regards neonatal outcome in cases of amniorrhexis. Pfeiffer et al. [14] showed that determination of IL-6 in maternal serum can contribute significantly to an earlier detection of fetal infection in patients with PROM. At a cut-off of 11 pg/mL, IL-6 reached a sensitivity of 81% and a specificity of 76% in the prediction of neonatal infection. In their study, IL-8 results showed a less significant correlation with fetal outcome. In the study of Lewis et al. [15], the presence of IL-6 in the maternal plasma, obtained prior to delivery in patients with preterm premature rupture of membranes between 24 and 35 weeks’ gestation, predicted neonatal complications. Hatzidaki et al. [5] concluded that IL-6 concentrations in maternal blood, taken during delivery, are a very sensitive, reliable, and early marker of neonatal sepsis and can provide an accurate indication of whether a neonate will develop early sepsis, thus, offering the opportunity for prompt diagnosis and aggressive therapeutic intervention. In their study, receiver-operating characteristic analysis revealed that using a cut-off concentration of 81 pg/ml for maternal serum IL-6, resulted in sensitivity of 90%, specificity 97.4%, positive predictive value 94.7%, and negative predictive value 94.9% for neonatal sepsis. Recently, Sorokin et al. [16] observed that elevated maternal serum IL-6 concentration is risk factor for preterm birth < 32 weeks and subsequent development of neonatal intraventricular hemorrhage. Conversely, Bahar et al. [1] stated that maternal serum levels of IL-6 and IL-8 were not increased in preterm labor compared to normal control women. They concluded that there is doubt regarding the usefulness of maternal serum measurement of these cytokines for the detection of early fetal infection in preterm labor.

Our study revealed that, only the IL-1β concentrations were significantly higher in the serum of mothers whose babies developed early-onset infection than in mothers whose newborns were born healthy. However, its predictive values, as well as the values of the other cytokines studied, were poor.

Only a few previous studies have evaluated the relationship between maternal serum cytokines to histological chorioamnionitis. Salafia et al. [4] evaluated concentrations of IL-1β and IL-6 in maternal serum from 32 consecutive patients at 20-36 weeks were experiencing progressive labor and tocolytic failure, and observed that serum levels of these cytokines were not associated with the presence or severity of histological evidence of acute placental inflammation. Nowak et al. [17] observed that maternal serum levels of IL-6 and IL-8 during both term and preterm labor were elevated in comparison to levels found in pregnant women who were not in labor. However, the elevated levels of these cytokines were not connected to any histological chorioamnionitis. Wenstrom et al. [18] considered that women with subclinical inflammation might be identified by analysis of the amniotic fluid following amniocentesis and, unfortunately, cannot be identified by maternal serum Il-6 levels before the procedure. These studies point out that the maternal compartment differs from the fetal compartment and the inflammatory responses in the fetal compartment are not necessarily reflected in maternal serum [1]. On the other hand, Greig et al. [19] stated that women in preterm labor and who delivered preterm with evidence of histological chorioamnionitis had significantly higher serum concentrations of interleukin-6 than did those in preterm labor who delivered in the absence of chorioamnionitis. In their study, the cut-off value of IL-6 > 6 pg/mL predicted placental inflammatory changes, with a sensitivity of 100%, specificity of 67%, PPV 88% and NPV of 100%. Previously, the same group of researchers [20] reported that maternal serum interleukin-6 concentrations are elevated in patients with preterm, premature rupture of membranes with histological chorioamnionitis. They suggested that evaluation of this cytokine in maternal serum may enable physicians to diagnose infection before the onset of clinical symptoms. Recently, Murtha et al. [21] reported that maternal serum IL-6 appears to be a biomarker for the identification of women with preterm, premature rupture of membranes likely to develop funisitis. Maeda et al. [22] also evaluated whether maternal serum IL-6 and IL-8 levels are useful for the diagnosis of histological chorioamnionitis. They examined blood samples and placentas from 29 women who delivered preterm between 22 and 34 weeks of gestation and found that only IL-6 determinations are useful for the prediction of placental changes. Oleszczuk et al. [23] found elevated IL-6 levels in maternal serum from pregnant women with signs of preterm labor and with laboratory markers of infection. They concluded that this cytokine may be the marker of preterm labor caused by infection. Shimoya et al. [24], who examined serum from 22 mothers with chorioamnionitis and 81 mothers without chorioamnionitis at term delivery, reported elevated concentrations of IL-8, but no increase in the concentration of IL-1α, IL-1β or IL-6. They concluded that measurement of maternal IL-8 is useful for rapid, prenatal screening for histological chorioamnionitis at term.

In our study, only maternal serum levels of IL-1β were significantly higher in patients with histological chorionamnionitis than in those without the condition. The cut-off value of this cytokine, ≥ 14 pg/mL, seemed to be a good predictor for the prognosis of inflammatory changes in the placenta, with a sensitivity of 80%, specificity of 86%, PPV of 80%, NPV of 86% and LR 5.60. The AUC was also the greatest for IL-1β, which confirms this marker as the best predictor of histological chorioamnionitis. We identified IL-1β as being a more sensitive marker of histological chorioamnionitis than other cytokines studied, including IL-6. This discrepancy, between ours and others’ findings, could be explained by the fact that our samples were taken early, immediately after establishing the diagnosis of new-onset, preterm labor, none of the patients having clinical signs of chorioamnionitis. It is known that the increase in serum IL-1β concentrations occurs earlier, before that of IL-6 in response to infection [25], and that IL-1 β stimulates the production of other cytokine mediators [26-29].

In conclusion, our observations suggest that the evaluation of maternal serum proinflammatory cytokines, obtained shortly after establishing the diagnosis of new-onset, preterm labor, is of limited value. Only IL-1β seems to be moderately valuable in the prediction of histological chorioamnionitis. However, this observation needs further investigation.

Acknowledgements

References

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