Maternal Plasma Interleukin-6, Interleukin-1[Beta] and C-Reactive Protein As Indicators of Tocolysis Failure and Neonatal Outcome After Preterm Delivery
By Skrablin, Snjezana; Lovric, Helena; Banovic, Vladimir; Kralik, Saska; Et al
Objective. To investigate whether maternal serum interleukin-6 (IL-6), interleukin-1/ (IL- 1/i) and high sensitive C-reactive protein (CRP) could be used as markers of tocolysis failure and adverse neonatal outcome in pregnancies with preterm labor (PL).
Methods. Forty-seven maternal blood samples taken because of PL at admission and delivery were analyzed. Control samples were taken from 20 gravidas with normal pregnancies. Differences in interleukins and CRP levels with or without chorioamnionitis, connatal infection or periventricular leukomalacia (PVL) were analyzed. Cut-off values were estimated for prediction of tocolysis failure and adverse neonatal outcome.
Results. All three parameters were significantly higher in patients delivering prematurely than in patients delivering at term. All three parameters were significantly higher with than without histologie chorioamnionitis ([>< 0.001), with than without connatal infection (p < 0.01), with than without PVL (p < 0.01 for IL-6 and IL-I//, < 0.05 for CRP), and in pregnancies with preterm premature rupture of membranes (PPROM) delivered within 48 hours compared to those more prolonged (p < 0.01). Choosing 50.9 pg/mL of IL-6 and a CRP of 19.7 as cut-offs in maternal blood admission concentrations for neonatal PVL, resulted in sensitivity of 81% and specificity of 91% and sensitivity of 91% and specificity of 81%, respectively. At respective maternal blood admission cut-off levels of 27.8 pg/mL of IL-6 and 8.9 of CRP, both parameters were effective predictors of connatal infection.
Conclusions. Maternal blood IL-6 and CRP could become useful in predicting tocolysis failure and intrauterine treat for the fetus.
Keywords: Prematurity, interleukin-6, interleukin-, C-reactive protein, connatal infection, periventricular leukomalacia
Intrauterine infection is thought to play a key role in the pathogenesis of about one third of preterm labors [1,2]. If present it is frequently followed by serious neonatal complications, periventricular leukomalacia (PVL) being the most deleterious [1- 4]. Overt infection is infrequent, but subclinical infection has been demonstrated in about 25% of patients with preterm labor and intact membranes and even more frequently with preterm premature rupture of membranes (PPROM). The diagnosis is sometimes very difficult and often hampered by the absence of an accurate diagnostic test.
It has recently become suspected that the cytokines could be promising markers, since they have been shown to be mediators of the early phase of the local inflammatory response [1,2]. They activate prostaglandin production, an important path in the onset and propagation of myometrial contractility [5-7]. In pregnancies complicated by overt intrauterine infection, elevated amniotic fluid [8,9], maternal [7,10-12] and fetal [13-15] inflammatory cytokine levels have been found. It may be speculated that silent intrauterine infection could also be diagnosed by use of maternal blood inflammatory cytokine levels [6,7].
In the present investigation we aimed to determine whether maternal serum interleukin-6 (IL-6), interleukin-1 (IL-1/0 and high sensitive C-reactive protein (CRP) estimated at diagnosis of preterm labor (PL) without clinically overt intrauterine infection could be used as markers of chorioamnionitis, connatal infection or PVL. We attempted to determine whether maternal plasma interleukin levels could aid in recognizing the risk of connatal infection and brain damage. We also attempted to estimate the cut-off interleukin levels for predicting tocolysis failure and unfavorable neonatal outcome.
Materials and methods
A prospective trial was performed at the Department of Obstetrics and Gynecology, Medical School in Zagreb, a tertiary referral center for perinatal medicine in Croatia, during the time period August 2003 to August 2004. Out of 332 preterm labors in that period we randomly selected 47 pregnant women with preterm contractions (PC) or PPROM but without any sign of overt clinical intra-amniotic infection. All patients were admitted between 27 and 33 weeks of gestation.
Preterm labor was diagnosed when either 10 or more uterine contractions per hour could be documented by uterine activity monitoring or cervical effacement followed by dilatation was identified or when rupture of fetal membranes was diagnosed by sterile speculum examination showing amniotic fluid leakage or by fern testing. Gestational age was determined from the data on last menstrual period confirmed by ultrasound data on crownrump length in the first trimester.
All patients were hospitalized and 5 mL of blood from the cubital vein was collected immediately following admission for cytokine and CRP analysis. Urinalysis and cervical smears were also carried out for all the patients for microbiologie evaluation, including aerobic and anaerobic bacteria, genital mycoplasmas and Chlamydia trachomatis, according to methods described previously [16,17]. Gravidas with signs of overt intrauterine infection at admission (fever, uterine tenderness, bloody discharge, leukocytosis and elevated band count), those with positive urinalysis and cervical smears, suspected or proved viral infection, immunodeficiency, as well as those with known immunologie or other inflammatory diseases were not included in the study. The control group consisted of 20 healthy pregnant women who delivered at term. All their children were healthy. Maternal plasma samples for IL-I, IL-6 and high sensitive CRP were taken at routine laboratory blood sampling at 27- 34 weeks of gestation, and again immediately before term delivery.
Parenteral tocolysis with ritodrine was started immediately after admission and dexamethasone was administered for fetal lung maturation at a dose of 12 mg daily for three consecutive days in all patients. All die patients delivered prematurely: 12 deliveries were completed by cesarean section because of abnormal CTG tracings, the remaining 35 delivered vaginally because of intractable contractions.
Two out of 47 developed overt clinical intrauterine infection by the time of delivery. Again, 5 mL of maternal blood from the cubital vein was collected immediately before preterm birth for IL-1/i, IL- 6 and high sensitive CRP determination. All placentas were analyzed pathohistologically for the presence of chorioamnionitis. Acute chorioamnionitis was denned as the presence of acute inflammatory changes on pathohistological examination of the placenta, membranes and umbilical cord, according to criteria previously published .
Neonatal blood was also microbiologically analyzed. Newborns underwent an ultrasound brain examination (Kretz SA 6000C) within three weeks of birth. White matter lesions were diagnosed by standard transfontanellar approach according to the following criteria: presence of cystic lesions within the periventricular white matter or persistent abnormally increased white matter echogenicity with definitive periventricular tissue loss. Neonates showing a lesion or a suspected lesion were followed up with serial scans every seven days until the lesion was stable . All ultrasound examinations were performed by an experienced neonatologist. Connatal infection was diagnosed in newborns with either bacteriological proven sepsis or early onset clinical sepsis that required antibiotic therapy irrespective of bacteriological data, and with pneumonia, as has previously been described [19,2O].
Sample preparation and IL-6, IL-I and high sensitive CRP measurements
A 5-mL sample of blood from the maternal cubital vein was collected at the time of admission (regular visit in the control group) and during the active phase of labor. These were drawn in a dry vacutainer and after 30 minutes centrifuged at 3000 rpm for 15 minutes. Supernatants were removed and stored at – 20 C until assayed.
Immunoassays: IL-1 and IL-6 were measured using an enzyme- linked immunosorbent assay kit (R&D System, UK). The assays do not cross react with any other known cytokine. The sensitivity of the test used for IL-1 was 1 pg/mL, and for IL-6 was 0.7 pg/mL. CRP was determined by particle enhanced turbidimetric immunoassay (PETIA). Latex particles coated with antibody to CRP aggregate in the presence of CRP in the sample. The increase in turbidity that accompanies aggregation is proportional to the CRP concentration. The concentration is determined by means of a mathematical function. The sensitivity of the assay is 0.5 mg/L.
The r-test was used to evaluate the differences between IL- 1/i, IL-6 and high sensitive CRP in the analyzed and control populations. A value of p < 0.05 was considered statistically significant. A paired sample r-test was needed to compare the levels of IL-6, IL-I and CRP at admission and before labor.
Receiver operating characteristic (ROC) curves were constructed to estimate the diagnostic indices (sensitivity and specificity) for IL-6, IL-I and CRP cut-off values in the prediction of tocolysis failure, preterm delivery, histologie chorioamnionitis, neonatal infection and periventricular leukomalacia. Cut-off values were approximated from the optimal sensitivity and specificity level defi\ned by ROC curves.
Forty-seven pregnant women were admitted to our institution because of symptoms of preterm labor (PL) not accompanied by any sign of intra-amniotic or other infection. Rupture of fetal membranes (PPROM) was diagnosed in 27 patients (57%), uterine contractions (PC) in the remaining 20 gravidas (43%). By the time of delivery clinically overt intrauterine infection had occurred in only two of the patients with PPROM and they were treated with antibiotics. Pathohistological analysis of the placenta revealed acute chorioamnionitis, and the children suffered connatal streptococcus B sepsis and later developed PVL. Only three out of 27 pregnancies with PPROM and 13 out of 20 with PC could be prolonged for more than 48 hours. Histological chorioamnionitis was diagnosed in 23 (48.9%) placentas. Twenty-five infants suffered connatal infection, and 11 (23%) PVL. No child died. All children developing PVL came from PPROM pregnancies, all suffered connatal infection, and all their placentas revealed chorioamnionitis. Tocolysis failed within 48 hours in all pregnancies with proved chorioamnionitis or later neonatal PVL. All women in the control group delivered at term and all their children remained healthy. There were no differences regarding age and parity between women in the examined and control groups.
At admission, mean maternal plasma IL-6, IL-1 and high sensitive CRP levels in a group of patients admitted because of PL were significantly higher than in the control group of healthy gravidas. Also, respective cytokine and high sensitive CRP plasma levels were significantly higher in PL gravidas immediately before preterm labor, than in healthy gravidas immediately before term labor. At admission with PPROM, mean plasma IL-6 was 50.0 pg/mL, IL-1 3.5 pg/ mL and high sensitive CRP 20.4. Respective values at admission with PC were 21.9 pg/mL, 2.8 pg/mL and 6.0. The differences in IL-6, IL- I^ and high sensitive CRP plasma levels between PC and PPROM patients at both admission and delivery are highly significant. In PPROM gestations that could be prolonged for more than 48 hours IL- 6, IL1 and high sensitive CRP levels were significantly lower than in those that failed tocolysis and were delivered within 48 hours. In the PC group, levels of all three parameters were higher in patients that failed tocolysis and gave birth within 48 hours after admission, but the discriminative levels in comparison to those whose pregnancy could be prolonged was not significant. There was no difference in admission levels of all three markers between PPROM and PC group where the pregnancy could be prolonged for more than 48 hours.
Chorioamnionitis was found in 23 out of 47 placentas. Maternal plasma IL-6 and IL-1/i, as well as high sensitive CRP levels at both admission and at preterm delivery were significantly higher in the presence than in the absence of chorioamnionitis (p < 0.001). IL-6 and IL-I, but not high sensitive CRP levels, were significantly higher in preterm 'no chorioamnionitis' than in control group placentas. Connatal infection was diagnosed in 25 newborns. Admission and delivery plasma levels of all three parameters were significantly higher in mothers whose children suffered connatal infection than in those without infection and in the control group of children. In 11 cases PVL lesions were found and admission and delivery plasma levels of all three parameters were significantly higher in mothers whose children suffered PVL than in those who did not (Table I).
Significant increases in mean IL-6 and high sensitive CRP levels at delivery in comparison to admission in both the PL and control groups was observed. Significant increases in IL-1 at term delivery, in comparison to levels at 27-33 weeks of gestation in the control group of healthy gravidas was also observed. Increases in IL- I levels at delivery in comparison to admission values in the PL group were not significant (Figure 1).
Cut-off values for all three markers from ROC curve analysis were used for prediction of chorioamnionitis, connatal infection and PVL as well as tocolysis failure in the PPROM group. An IL-6 level of 27.5 pg/mL has 60% sensitivity and 100% specificity; an IL-1/ level of 2.9 pg/mL has 83% sensitivity and 64% specificity; and a high sensitive CRP level of 10.8 has 87% sensitivity and 100% specificity for prediction of tocolysis failure in PPROM patients. A maternal admission IL-6 value of 29.1 pg/mL has 91% sensitivity and 84% specificity in predicting chorioamnionitis, a value of 27.8 pg/mL has 80% sensitivity and 69% specificity in predicting connatal infection, and a value of 50.9 pg/mL has 81% sensitivity and 91% specificity in predicting PVL. Respective cut-off high sensitive CRP levels show exceptionally high specificity and sensitivity in predicting chorioamnionitis, connatal infection, and PVL (Table II).
Table I. Maternal blood IL-6, IL-I// and CRP with preform premature rupture of membranes, preterm contractions and complications (mean plasma levels at admission and range).
Preterm birth is still the leading perinatal problem in the world. In most cases, especially in pregnancies up to 34 weeks of gestation, physicians are left with a difficult decision: to deliver promptly or manage expectantly by hospitalization, bed rest and always uncertain and marginally effective ways to delay delivery by arresting contractions until there is unequivocal evidence of infection, fetal jeopardy or proof of fetal lung maturation [1,21].
Nowadays, we are faced with increasing evidence of connection of intrauterine infection and brain damage [9,13,22], most frequently in preterm , but also in term infants . Infection could start early in gestation, be clinically silent and very dangerous for fetal neurological outcome . Inflammatory cytokines, e.g., tumor necrosis factor (TNF), IL-6 and IL-I// have been implicated as mediators for the development of PVL, a major risk for cerebral palsy . So, the decision of how to manage pregnancy with PC or PPROM, where the possibility of hidden and silent infection is very high [13,21-24], is even more complicated. Diagnosis of silent intrauterine infection is difficult but a prerequisite for proper clinical management.
There are a number of methods for the detection of intrauterine infection, but they often take several days and show low sensitivity and specificity [1,25]. After Romero et al. showed elevated levels of certain cytokines in amniotic fluid of women with microbial invasion of the intra-amniotic cavity [26,27], many investigators tried to estimate discriminative levels of amniotic fluid IL-6, IL- I , interferons, TNF [8,16] and IL-8  in patients with preterm labor with and without concomitant intra-amniotic infection or chorioamnionitis. More recently, maternal serum inflammatory cytokine concentrations in patients with premature rupture of membranes [14,21] and preterm contractions [10,12,28], as well as in term labors  have also been analyzed.
We decided to measure inflammatory cytokines IL-1/i and IL-6 together with high sensitive CRP in a group of patients with symptoms of PL but without any clinical sign of infection. The levels were correlated with the presence or absence of chorioamnionitis and connatal infection, as well as with PVL as final outcome. We tried to determine whether all or any of the analyzed markers could aid in making the diagnosis of silent intrauterine infection and hence improve the directing of clinical decisions. Our results point strongly towards significant differences in plasma levels of IL-6, IL-1 and CRP in patients with PL in comparison to healthy gravidas and in preterm in comparison to term labor, and the correlation of elevated levels of IL-6, IL-I// and high reactive CRP with chorioamnionitis, connatal infection and final PVL is striking. The reliability of each marker is high; however, that of IL-6 appears to be the best. The levels also show interesting correlation with tocolysis failure, especially in PPROM patients; namely, there was no difference in the admission level of either marker between PPROM and PC patients if the pregnancy could be prolonged for more than 48 hours. Also, in pregnancies with similarly low mean marker levels, tocolysis success could be expected together with low risk of neonatal infectious complications, especially PVL. This finding could prove very important.
Figure 1. Differences between mean + SD maternal plasma IL-6, IL- 1/f and CRP at admission (A, open bars) and delivery (D, closed bars) in patients (N) with preterm labor and control group.
According to the results presented, as long as inflammatory markers are low, it should be possible to administer tocolysis with a reasonable expectation for pregnancy prolongation for at least a three-day corticosteroid treatment, without fear of newborn infectious complications. A maternal blood cut-off concentration at admission for IL-6 of 50.9 pg/mL, for IL- 1/ of 3.3 pg/mL and for CRP of 19.7 offers an opportunity to predict newborn PVL with great sensitivity and specificity. Similarly Hatzidaki et al.  showed a maternal blood IL-6 cut-off level of 81 pg/mL at delivery as being highly accurate in detecting early neonatal sepsis. However, our finding of elevated plasma IL-6, IL-1 and high sensitive CRP in patients with the symptoms of preterm labor is only partially consistent with some other reports [7,11,22,29]. Lencki et al. found no significant differences in maternal IL-6 and IL-I/^ levels between patients delivering prematurely with and without clinical chorioamnionitis [1O]. Also in the research of Salafia et al.  in a group of patients failing tocolysis, maternal serum cytokine levels were not associated with the presence or severity of histologie evidence of acute placenta! inflammation. Shimoya et al. could not find elevated IL-6, IL-I or IL-8 in maternal se\rum with either term or preterm histological chorioamnionitis . Bahar et al. observed no significant difference in the concentration of IL- 6, IL-8, TNF-alpha and interferon gamma measured between PL patients and healthy gravidas, as well as between women in PL with ruptured membranes and those with intact membranes . However, in the study of Lencki et al.  a certain proportion of patients with subclinical infections could have been put into a group of patients without clinical infection, and in the research of Salafia et al.  only patients who failed tocolysis were analyzed.
The decidua is thought to be the primary source of inflammatory cytokines in human reproductive tissue as it contains the largest pool of immunocompetent cells in this area [8,3O]. So, the finding of elevated IL-6 and IL-I not only in amniotic fluid but also in maternal serum with intrauterine infection could be expected. Moreover, the increase of CRP  and elevation in IL-6 level were suspected previously to precede overt clinical infection [U]. Although small in number of patients, our group represents a uniquely homogenous group of gravidas with PL but without clinical signs of intrauterine infection. Further studies are obviously required to clarify the differences in the literature reports. According to our results, and some previous results [15,31], at least a portion of patients in the group that failed tocolysis could have suffered subclinical infection that aided in the failure of tocolytic therapy. And, tocolysis failure could be predicted by a high inflammatory cytokine level.
Table II. Sensitivity and specificity of maternal plasma IL-6, IL- and CRP at admission in the prediction of tocolysis failure, chorioamnionitis, connatal infection and periventricular leukomalacia.
During pregnancy there is an alteration in maternal immunity within the uterus where innate, proinflammatory immune responses are tightly regulated to prevent immunological rejection of the fetal allograft. Disruption of the delicate balance of cytokines by bacteria or other factors increases the production of proinflammatory cytokines at the maternal-fetal interface and activates die parturition mechanism prematurely . Hence, a parallel increase in IL-1 , IL-6 and CRP not only with inflammation could be expected. IL-I is released from gestational tissues in response to bacterial products or other stimuli. Together with TNF- alpha it stimulates amnion and decidua cells to produce prostaglandin E2. Simultaneously, decidual cells respond to IL-1 with increased IL-6 production. Further, IL-6 stimulates hepatic C- reactive protein production and at the same time aggravates prostaglandin E2 production. So, IL-1/S, IL-6 and CRP all play a role in the host response to infectioninduced , but also immunologically-induced preterm labor [34-36]. According to our results, if significantly elevated they may discriminate infection- induced PL and PL precipitated by other factors. If beyond critical levels, IL-6 and CRP are highly sensitive and specific for prenatal detection of chorioamnionitis, connatal infection, and what is most important, for estimating the risk of PVL when no other clinical sign of intrauterine infection exists.
These data show a direct connection between preterm labor with high cytokine levels and placental and connatal infection with final PVL. Not all preterm labors are associated with infection, but maternal IL-6, IL-I// and CRP levels are still significantly higher than in normal term labors. Maternal type 1 cytokine bias as the cause for preterm labor in certain proportion of patients  delivering prematurely could be an explanation for the occurrence of such high inflammatory cytokine levels. With our results the difference in IL-6, IL-I and CRP levels can be used to discriminate preterm labor cases complicated by infection and those precipitated by numerous other causes [35,36], thus enabling us to undertake optimal therapeutic modalities. Caution must be taken, however, because activation of the cytokine network may cause white matter damage even when bacteriological data are negative and placental inflammation does not exist [1,9,20,23].
Maternal plasma cytokine levels increase both in term and in preterm labors but the levels are significantly greater in preterm labor. The increase in preterm births is significantly higher than in term deliveries. Especially high levels can be registered in patients with PPROM . Although some authors suggest and report different molecular pathways for preterm labor and PPROM , the connection of high cytokine levels and proven neonatal infection points to a connection of the majority of PPROM cases with intrauterine inflammation.
In conclusion, as previously shown for amniotic fluid concentrations [8,9,37,38] and umbilical blood levels [13-15], our results demonstrate that maternal IL-6, IL-\/i and high sensitive CRP levels provide information about the risk of fetal infection and PVL with preterm labor. The process responsible for at least some cases of PVL and cerebral palsy obviously begins during intrauterine life without clinical signs of overt infection, implying that effective strategies for the prevention of cerebral palsy associated with PVL must begin in utero.
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SNJEZANA SKRABLIN1, HELENA LOVRIC1, VLADIMIR BANOVIC1, SASKA KRALIK2, ALEKSANDAR DIJAKOVIC1, & DRZISLAV KALAFATIC1
1Department of Perinatal Medicine, Medical School, Zagreb, Croatia and 2Department of Biochemistry, Medical School, Zagreb, Croatia
(Received 10 November 2006; revised 18 December 2006; accepted 18 December 2006)
Correspondence: Snjezana Skrablin, MD, PhD, University of Zagreb Medical School, Department of Perinatal Medicine, Petrova 13, Zagreb H)OOO, Croatia. Tel: +385 1 4810866. Fax: +385 1 4633512. E-mail: ivan.kuvacicwzg.htnet.hr
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