Elevated interleukin-8 concentrations in amniotic fluid of mothers whose neonates subsequently develop bronchopulmonary dysplasia1

European Journal of Obstetrics & Gynecology and Reproductive Biology 78 (1998) 5–10

Elevated interleukin-8 concentrations in amniotic fluid of mothers whose neonates subsequently develop bronchopulmonary dysplasia 1 a

a

a ,b ,

a

a

Fabio Ghezzi , Ricardo Gomez , Roberto Romero *, Bo Hyun Yoon , Samuel S. Edwin , Cristina David a , James Janisse c , Moshe Mazor a a

b

Perinatology Research Branch, National Institute of Child Health and Human Development, Bethesda, Maryland, USA Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Wayne State University /Hutzel Hospital, Detroit, Michigan, USA c Center for Health Care Effectiveness Research, Wayne State University, Detroit, Michigan, USA Received 11 July 1997; received in revised form 19 September 1997; accepted 4 October 1997

Abstract Objective: To determine if an intrauterine sub-clinical inflammatory process is a risk factor for the development of bronchopulmonary dysplasia. Methods: A cohort study was conducted in patients who met the following criteria: (1) Singleton gestation; (2) preterm labor or preterm premature rupture of the membranes; (3) amniocentesis for microbiologic studies of the amniotic fluid and (4) delivery between 24 and 28 weeks of gestation. Bronchopulmonary dysplasia was defined as the need for supplemental oxygen for 28 days or longer after birth, associated with compatible chest radiographic findings. Amniotic fluid interleukin-8, was measured using a specific immunoassay. Logistic regression analysis and bootstrap procedure were used for statistical purposes. Results: Forty-seven patients met the inclusion criteria for this study. Among these patients, the prevalence of bronchopulmonary dysplasia was 23.4% (11 / 47). Amniotic fluid culture was positive in 21 out of 47 (44.7%) patients. Median (range) amniotic fluid interleukin-8 concentration was higher in patients whose neonates subsequently developed bronchopulmonary dysplasia than in those who did not (17 [9.8–583.7] ng ml 21 versus 9.6 [0.91–744] ng ml 21 , P50.057). An amniotic fluid IL-8 level greater than 11.5 ng ml 21 was far more common in mothers whose fetuses went on to develop bronchopulmonary dysplasia than in those who did not (10 / 11 [90.9%] versus 17 / 36 [47%]; P50.01). This relationship remained significant even after correcting for the effect of gestational age and birthweight (Odds ratio: 11.9; P,0.05). Conclusion: Sub-clinical intrauterine inflammation is a risk factor for the subsequent development of bronchopulmonary dysplasia. We propose that in utero aspiration of fluid with high concentration of pro-inflammatory mediators may contribute to the lung injury responsible for the development of bronchopulmonary dysplasia.  1998 Elsevier Science Ireland Ltd. Keywords: Amniotic fluid; Interleukin 8; Bronchopulmonary dysplasia; Intraamniotic infection; Cytokines

1. Introduction Bronchopulmonary dysplasia (BPD) is emerging as one of the most important sequela of prematurity [1,2]. Among infants with birthweight below 1500 g, 15–47% are *Corresponding author. Department of Obstetrics and Gynecology, Hutzel Hospital, Perinatology Research Branch, NICHD, 4707 St. Antoine Blvd., Detroit, MI 48201, USA. Tel.: 11 313 9932700; fax: 11 313 9932683. 1 Presented in part at the 17th Annual Meeting of the Society of Perinatal Obstetricians, Anaheim, California, January 20–25, 1997.

diagnosed to have BPD and a fraction will have significant respiratory handicaps during childhood [3]. Surfactant treatment and modern ventilatory support have had a major impact in the frequency and severity of respiratory distress syndrome [4]. However, the frequency of BPD in very low birthweight infants has increased steadily in the last two decades from 10% to 32% [5]. Affected infants are at risk for long term requirement of oxygen therapy and for persistence of pulmonary symptoms (i.e. wheezing, retractions, cyanosis, nasal discharge etc.) [6]. Other serious long term complications include recurrent pneumonia, right and left ventricular hypertrophy and hypertension [3]. BPD has

0301-2115 / 98 / $19.00  1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0301-2115( 97 )00236-4

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F. Ghezzi et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 78 (1998) 5 – 10

also been identified as an independent risk factor for the subsequent development of cerebral palsy [7]. Finally, death has been reported to occur in 11–71% of infants with chronic lung diseases [8,9]. BPD has been traditionally attributed to the injury to immature lung tissue inflicted by the combination of barotrauma and oxygen toxicity [10–13]. However, this does not explain the development of BPD in infants with mild respiratory distress syndrome or the absence of chronic lung disease in some infants with severe hyaline membrane disease requiring prolonged and high pressure ventilation. Therefore, other unidentified factors may play a role in the genesis of chronic lung disease in the setting of prematurity. Recently, infection has been recognised as a potential etiologic factor for BPD. Nosocomial infections during the neonatal period and colonisation of the neonatal respiratory tract with Ureaplasma urealyticum have been implicated in the pathogenesis of BPD through the participation of inflammatory cytokines [14]. Little consideration has been given to the possibility that the pathogenic process leading to chronic lung disease may begin during fetal life. Strong evidence suggests a link between microbial invasion of the amniotic cavity and prematurity [15]. Moreover, subclinical microbial invasion of the human fetus in patients with preterm premature rupture of the membranes has been recently documented [16]. Fetuses with a systemic fetal inflammatory response syndrome-defined as an elevated fetal plasma interleukin (IL) 6 concentration-are at risk for neonatal complications including BPD [17]. Recent studies [18,19] have demonstrated that elevated concentrations of pro-inflammatory cytokines in the tracheo–bronchial fluid obtained from preterm neonates predict the subsequent development of BPD. The similarity in the pattern of expression of proinflammatory cytokines (ie., IL-6, IL-8) in the amniotic fluid of patients at risk for preterm delivery [15] and in the pulmonary effluent fluid [18] of infants who subsequently develop BPD suggests a possible common pathway for the initiation of preterm labor and subsequent chronic lung diseases in neonates. The purpose of this study was to examine the relationship between amniotic fluid concentration of IL-8, a chemokine capable of inducing neutrophil’s chemotaxis and activation, and the subsequent development of BPD. IL-8 was chosen because neutrophils have been identified in the tracheo–bronchial lavage fluid of newborns who develop BPD obtained shortly after birth [20]. In addition, amniotic fluid IL-8 concentrations are elevated in patients with preterm labor and microbial invasion of the amniotic cavity [21].

2. Material and methods A cohort study was conducted in patients who met the following criteria: (1) Singleton gestation; (2) preterm

labor or preterm premature rupture of the membranes; (3) delivery between 24 and 28 weeks of gestation; and (4) newborn survival for at least 28 days. Amniocentesis was performed in all patients for the assessment of the microbiologic status of the amniotic cavity. Written informed consent was obtained from patients to undergo amniocentesis and to save amniotic fluid for research purposes. Samples were collected from patients admitted to Hutzel Hospital between February 1, 1992 and January 31, 1996. The study was restricted to neonates delivering within 28 weeks of gestation because the risk of BPD above this gestational age is relatively low [8]. Preterm labor was diagnosed in the presence of a history of regular uterine contractions associated with changes in cervical dilatation or effacement. Premature rupture of the membranes was diagnosed by sterile speculum examination confirming pooling of amniotic fluid in the vagina and a positive Nitrazine test result. Collection of samples for this study was performed under the sponsorship of a protocol approved by the Institutional Review Board of Wayne State University, which has a Multiple Project Assurance, and / or of the National Institute of Child Health and Human Development. Amniotic fluid was retrieved by transabdominal amniocentesis under ultrasonographic guidance. The fluid was transported to the laboratory in a capped plastic syringe and cultured for aerobic and anaerobic bacteria as well as for Mycoplasmas. The remaining amniotic fluid were centrifuged and stored in polypropylene tubes at 2708C until assayed for IL-8. IL-8 levels in amniotic fluid was measured by a commercially available enzyme-linked immunoassay (ELISA) (R and D System. Minneapolis, MN) (Sensitivity 12.1 pg ml 21 ). Amniotic fluid samples were assayed in duplicate. The intraassay and interassay coefficient of variation were 5.6% and 14.1% respectively. Results of amniotic fluid IL-8 determinations were not used for patient management. BPD was defined according to the criteria proposed by Bancalari et al. [22]. The diagnosis required (1) the need for a ventilatory assistance during the first week of life for a minimum of three days, (2) continuing requirement for supplemental oxygen at 28 days of age and (3) typical radiographic changes. Respiratory distress syndrome and necrotizing enterocolitis were defined as previously described [23]. Proven neonatal sepsis was diagnosed in the presence of a positive culture of blood, urine or cerebrospinal fluid. Statistical analysis was performed using SPSS (SPSS Inc. Chicago, IL) and S-Plus 3.3 (MathSoft Inc., Seattle, WA) statistical softwares. Either Mann-Whitney U or Student’s t-test was used for comparison of continuous variables. Comparisons between proportion were performed with x 2 or Fisher’s exact test. Receiver operator characteristic (ROC) curves were constructed to describe the relationship between the sensitivity and the falsepositive rate for different values of amniotic fluid IL-8

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Table 1 Clinical characteristics of patients according to the development of BPD in their neonates

Maternal age (year) Primiparous Primigravid Gestational age at admission Amniocentesis to delivery interval (hear) Gestational age at delivery Positive Amniotic fluid culture for microorganisms Histologic chorioamnionitis Proportion of patients with PROM

BPD (n511)

No BPD (n536)

P

24.566.3 6 2 25.361.1 24 (1–96) 25.461.1 4 8 2

22.365.4 13 8 25.361.9 40.5 (1–1120) 26.361.1 17 17 12

NS a NS b NS c NS a NS c ,0.05 a NS b NS b NS b

BPD, Bronchopulmonary dysplasia; NS, not significant; PROM, premature rupture of the membranes. Data are presented as mean6standard deviation of the mean, median and range or numbers. a Student t-test, b Mann Whitney U test, c Fisher exact test.

concentrations. To assess the reliability of the threshold derived from the ROC curve of our population, a bootstrap procedure was used. The mean, median, standard deviation and 95% confidence interval of the distribution based on 1 000 bootstrap samples was calculated. Diagnostic indices (sensitivity and specificity) as well as predictive values were calculated for amniotic fluid IL-8 concentrations in the identification of newborns who would develop BPD. Logistic regression was used to investigate the relationship between the occurrence of BPD and explanatory variables such as gestational age, birthweight, cervical dilatation at admission and amniotic fluid IL-8 concentration.

3. Results Forty-seven patients with preterm labor or preterm premature rupture of the membranes met the inclusion criteria for this study. The prevalence of BPD was 23.4% (11 of 47). Microbial invasion of the amniotic cavity was detected in 44.7% (21 of 47) of cases. Ureaplasma Urealyticum and Mycoplasma hominis were the microorganisms most frequently isolated from the amniotic fluid (nine of twenty-one [42.8%]). Mixed flora was isolated from the amniotic cavity in five cases (23.8%). Other microorganisms isolated were: Fusobacterium nucleatum

(n52), Streptococcus viridans (n52), Gardnerella vaginalis (n51), Candida albicans (n51), Staphylococcus aureus (n51). The proportion of patients who received steroids was similar between women whose infants developed BPD and those whose newborn did not develop BPD (4 / 11 [36.4%] versus 15 / 36 [41.6%]). Tables 1 and 2 describe the clinical characteristics of mothers and neonates according to the development of BPD. Median (range) amniotic fluid IL-8 concentrations were higher in patients whose neonates developed BPD than in those whose neonates did not develop BPD (17 [9.8–583.7] ng ml 21 versus 9.6 [0.91–744] ng ml 21 , P50.057). ROC curve analysis was conducted to examine the diagnostic performance of amniotic fluid IL-8 in the identification of BPD (area under the curve50.69, SE50.07, P,0.01). An amniotic fluid IL-8 concentration of 10.7 ng ml 21 or greater was used for the calculation of diagnostic indices and predictive values. This was based on the results of the bootstraping procedure which indicated a mean (6standard deviation) of 10.7 (61.6) ng ml 21 and a median (95% confidence interval) of 9.8 (9.8–15.0) ng ml 21 . Sensitivity, specificity and positive and negative predictive values were 90.9%, 52.7%, 37%, 95%, respectively. Similar diagnostic indices and predictive values were obtained using the cutoff of 11.5 ng ml 21 which was derived without the bootstraping procedure by conventional analysis of the

Table 2 Neonatal characteristic according to the development of bronchopulmonary dysplasia

Birthweight Proven neonatal sepsis Respiratory distress syndrome Intraventricular haemorrhage Necrotizing enterocolitis Length of hospitalization (days)

BPD (n511)

No BPD (n536)

P

794 (610–1040) 2 10 3 1 83.3614.1

940 (570–1440) 4 28 5 1 49.7632.7

NS a NS c NS d NS c NS c ,0.005 a

BPD, Bronchopulmonary dysplasia; NS, not significant. Data are presented as median and range, mean6standard deviation of the mean or numbers. a Student t-test, b Mann Whitney U test, c Fisher exact test, d x 2 test.

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Table 3 Clinical characteristics of patients whose infant developed bronchopulmonary dysplasia Case no. 1 2 3 4 5 6 7 8 9 10 11

Gestational age at admission (week) 24 24 24.0 24.7 25 25.0 25.6 26.1 25.7 27 27

Interval amniocentesis to delivery (hours)

Gestational age at delivery (week)

Birth weight (g)

7

24.0

790

24 28.0 46 82 96.0 11.5 5 87 1 24

24.1 24.2 25.0 25.5 25.6 25.7 26.1 26.2 27 27.1

610 780 794 830 760 715 1020 1040 980 1040

Amniotic fluid culture

Amniotic fluid IL 8 (ng ml 21 )

Histologic chorioamnionitis

Neonatal complications

Ureaplasma urealyticum, Mycoplasma hominis Gardnerella vaginalis Ureaplasma urealyticum negative negative negative negative negative negative Streptococcus viridans negative

583.7

positive

RDS, Anemia

11.5 135.0 17.4 67.6 17.0 12.8 9.8 28.8 14.6 12.1

negative positive positive positive negative negative positive positive positive positive

RDS Pneumonia, Anemia RDS, IVH RDS, IVH, Sepsis RDS, Retinopathy RDS, NEC RDS, Anemia RDS, Anemia RDS, IVH, Sepsis RDS, Pneumonia

RDS, Respiratory distress syndrome: IVH, Intraventricular hemorrhage; NEC, Necrotizing enterocolitis.

ROC curve. An amniotic fluid IL-8 greater than 10.7 ng ml 21 was far more common in mothers whose fetuses went on to develop BPD than in those who did not (90.9% (10 / 11) versus 47.2% (17 / 36), P50.01). The clinical and neonatal characteristics of infants who developed BPD are reported in Table 3. Logistic regression analysis indicated that an elevated amniotic fluid IL-8 ($10.7 ng ml 21 ) was significantly associated with the subsequent development of BPD after adjusting for gestational age at delivery and birthweight (IL-8 Odds ratio: 11.9, 95% confidence intervals: 1.3–107.5, P50.02). To further investigate the relationship between amniotic fluid IL-8 concentrations and the occurrence of BPD, 44 women in whom the cervical dilation at admission was available were identified. After adjusting for cervical dilatation and gestational age at admission, IL-8 remained a significant predictor of BPD (Odds ratio: 12.6, 95% confidence intervals: 1.1–147, P50.04).

4. Discussion A growing body of experimental and clinical evidence indicates that several markers of lung inflammation (i.e. neutrophils [18], elastase [18], IL-6 [19], IL-8 [18], IL-1b [24], fibronectin [25], leukotriene B 4 [18], complement component C 5 -derived anaphylatoxin [26]) are increased in lung secretions / lavage of infants who subsequently develop BPD. This observation has been interpreted as indicating that the pathologic mechanism responsible for the development of chronic lung disease involves a prolonged inflammatory process [18]. The precise etiology of the inflammatory process remains to be determined. Although barotrauma and oxygen toxicity have been considered to contribute to lung damage [10–13], a role for infection has been recently suggested [14,27]. Newborns who develop nosocomial infections are prone to develop BPD even in the absence of severe respiratory distress

syndrome [14]. In addition, Ureaplasma urealyticum has been isolated more frequently from the tracheo–bronchial secretions of neonates with bronchopulmonary dysplasia [27]. In a recent report, infants exposed to histologic chorioamnionitis had a higher rate of BPD than those not exposed [19]. Our study indicates that fetuses exposed to amniotic fluid with a high concentrations of IL-8 have a significantly increased risk of developing BPD even after adjustment for gestational age at admission and at birth as well as for birthweight which is a more accurate parameter than gestational age. An elevation of amniotic fluid IL-8 has been associated with microbial invasion of the amniotic cavity and histologic chorioamnionitis [15]. Therefore, intrauterine infection may be involved in the genesis of the lung injury which leads to BPD. However, in our study the frequency of positive amniotic fluid cultures for microorganisms was not significantly different between patients whose fetuses develop BPD and those whose fetuses did not. A possible explanation for this is the limitations of standard microbiologic techniques to detect infections with a small inoculum size, caused by fastidious microorganisms or which are localized to the extra-amniotic space. Recent evidence gathered with the use of molecular microbiologic techniques indicates that even patients with negative amniotic fluid culture in whom microbial footprints are detected in amniotic fluid by PCR to conserved microbial DNA have elevated amniotic fluid cytokines and are at risk for impending preterm delivery [28]. In addition, our study did not include cultures for Clamydia trachomatis [29] and viruses (i.e. cytomegalovirus) [30], organisms which have been implicated in the pathogenesis of BPD. A role for an inflammatory process which is not caused by infection in the etiology of chronic lung disease cannot be excluded at this time. The conclusions of the present study are consistent with a number of observations reported by other investigators. Watterberg et al. [19] reported that infants who developed

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BPD had a higher median IL-1b in the tracheo–bronchial lavage fluid collected within 24 hours of birth than those who did not develop BPD. Yoon et al. [31] reported that neonates born to mothers with elevated amniotic fluid concentrations of pro-inflammatory cytokines (i.e. IL-8, IL-6 and IL-1b) have an increased risk for developing BPD. These findings support a role for an intrauterine insult capable of eliciting an inflammatory response before birth in the genesis of BPD. An important clinical observation is that an amniotic fluid IL-8 concentration below 10.7 ng / ml had a negative predictive value of 95% for the subsequent development of BPD in infants born at or below 28 weeks of gestation. A similar negative predictive value has been reported in a study restricted to infants born prior to 33 weeks of gestation [31]. Therefore, low concentrations of inflammatory cytokines in amniotic fluid identify infants at lower risk for chronic sequelae of prematurity such as BPD and periventricular leukomalacia [32]. How would an intraamniotic inflammatory response predispose to the subsequent development of BPD? Color and spectral Doppler studies of fluid movement in the perinasal area have demonstrated influx of amniotic fluid during fetal breathing. Inspirated amniotic fluid with a high concentration of pro-inflammatory cytokines and / or chemokines (with or without demonstrable microorganisms) may reach the lower tracheo–bronchial tree of the fetus and elicit an antenatal inflammatory response. Indeed, the presence of neutrophils and macrophages has been demonstrated in the tracheo–bronchial aspirate obtained shortly after birth in infants who subsequently developed BPD [18]. We propose that intrauterine exposure to inflammatory mediators may play a substantial role in predisposing preterm neonates to chronic lung disease. Our study does not exclude a role for post-natal barotrauma or oxygen toxicity in the genesis of chronic lung disease.

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Acknowledgements We thank Drs. Joel Agar and Elizabeth J. Atkinson Ph.D for invaluable assistance and advice in the analysis of our observations.

[20]

[21]

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