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To evaluate the hypothesis that elevated levels of inflammation-related proteins in early postnatal blood predict impaired mental and motor development among extremely preterm infants.
We measured concentrations of 25 inflammation-related proteins in blood collected on postnatal days 1, 7, and 14 from 939 infants born before 28 weeks gestation. An elevated level was defined as a concentration in the highest quartile for gestational age and day of blood collection. We identified impaired development at 24 months of age using the Bayley Scales of Infant Development. The primary outcomes were scores on the Mental or Motor Scale below 55 (more than 3 standard deviations below the mean).
For 17 of the 25 inflammation-related proteins, one or more statistically significant association (p < 0.01) was found between an elevated blood level of the protein and a developmental impairment. Elevations on multiple days were more often associated with developmental impairment than elevations present for only one day. The highest number of elevations was found in day-14 blood.
In extremely preterm infants, elevated levels of inflammation-related proteins in blood collected on postnatal days 7 and 14, especially when sustained, are associated with impaired mental and motor development at age two years.
Preterm newborns are at increased risk for long-term impairments, especially cognitive impairment.(1) Factors contributing to the underlying brain damage probably include infection and inflammation.(2) Experimental inflammation-induced brain damage is mediated, in part, by inflammatory cytokines and other inflammation-related proteins.(3) Elevated levels of such proteins are present in preterm infants who had brain damage identified with neuroimaging,(4) but we are aware of only one study describing associations between such proteins and clinical dysfunctions at age 2 years. In that study of 67 infants born before 32 weeks gestation, levels of proinflammatory and modulatory cytokines in blood obtained during the first 72 postnatal hours were associated with motor, but not cognitive, impairment.(5) We evaluated the hypothesis that elevated levels of inflammation-related proteins in blood collected on day 1, 7, and 14 after extremely preterm birth predict impaired mental and motor development at 24 months of age.
The Extremely Low Gestational Age Newborn (ELGAN) Study was designed to identify characteristics and exposures that increase the risk of structural and functional neurological disorders in children born before 28 weeks gestation.(6) During the years 2002-2004, we invited the participation of women who delivered before 28 weeks gestation at any of 14 participating institutions. The study was approved by the Institutional Review Boards at each site.
The gestational age estimates were based on a hierarchy of the quality of available information. Most desirable were estimates based on the dates of embryo retrieval or intrauterine insemination or fetal ultrasound before the 14th week (62%). When these were not available, we used (in order of preference) fetal ultrasound at 14 or more weeks (29%), last menstrual period without fetal ultrasound (7%), and gestational age recorded in the log of the neonatal intensive care unit (1%).
Drops of blood were collected on filter paper on the first postnatal day (range: 1-3 days), the 7th postnatal day (range: 5-8 days), and the 14th postnatal day (range: 12-15 days), Dried blood spots were stored at −70°C in sealed bags with desiccant until processed.
For protein elution, 12mm punched biopsies of the frozen blood spots were submerged in 300 μL phosphate buffered saline containing 0.1% Triton X100 and 0.03% Tween-20 (Fisher, Hampton, NH) ,vortexed for 30 sec, and incubated on a shaker for 1h at 4°C. The buffer and biopsy were then transferred over the filter of a SpinX tube, centrifuged at 2000 x g followed by collection of the filtered eluted blood. An additional wash of the punch was performed in 100 μL for a final elution volume of 400 μL.
Proteins were measured in duplicate using the Meso Scale Discovery (MSD) multiplex platform and Sector Imager 2400 (MSD, Gaithersburg, MD). Multiplex assays, measuring up to 10 proteins simultaneously, were optimized to allow detection of each biomarker within the linearity range of the eluted samples. The total protein concentration in each eluted sample was determined by BCA assay and the measurements of each analyte normalized to mg total protein. More details about the procedures used to measure inflammation-related proteins are presented elsewhere.(7)
Developmental assessments at 24-months corrected age included the Bayley Scales of Infant Development-Second Edition (BSID-II),(8) and an assessment of gross motor function using the Gross Motor Function Classification System(9). Certified examiners administered and scored the BSID-II. All examiners were experienced users of the BSID-II and, specifically for the ELGAN Study, attended a one-day workshop where published guidelines for test administration and videotaped examinations were reviewed. Examiners were aware of the child’s enrollment in the ELGAN Study and corrected age, but not the child’s medical history.
When a child’s visual or neurological impairments precluded assessment with the BSID-II, or more than 2 items were omitted or judged to be ‘unscorable,’ the child was classified as not testable on that scale. The Adaptive Behavioral Composite of the Vineland Adaptive Behavior Scales, obtained for 26 of 33 children who were considered not testable with the BSID-II Mental Scale (i.e., Mental Development Index), was used to approximate the Mental Scale score (10). Among infants not testable with the BSID-II Motor Scale (i.e., Psychomotor Development Index), 32 were assessed with the Vineland Adaptive Behavior Scales, and the Vineland Adaptive Behavior Scales Motor Skills Domain score was used to approximate the Motor Scale score.
The BISD-II manual defines a significant delay as a Mental or Motor Scale below 70, i.e., 2 standard deviations below the mean for the standardization sample. However, in very preterm infants, the predictive ability of a Mental Scale below 55 is higher than that of a score below 70(11). In addition, neonatal illness is more strongly associated with a score below 55 than with a score below 70.(12) Thus, we classified the BSID-II outcomes into 3 categories: Mental or Motor Scale less than 55 (more than 3 standard deviations below the mean), 55-69 (between 2 and 3 standard deviations below the mean), or greater than 69 (within 2 standard deviations of the mean or higher). The primary outcomes were scores on the Mental or Motor Scale below 55 (more than 3 standard deviations below the mean).
For analyses involving the BSID-II Mental Scale, we excluded infants with significantly impaired gross motor function, defined as an inability to walk independently (a Gross Motor Function Classification System level ≥ 1), because their failure on certain Mental Scale test items might have been attributable to impaired motor function, not impaired mental development.
For all analyses, we classified a protein biomarker concentration as elevated if it was in the highest gestational age- and postnatal day-specific quartile. To describe associations between gestational age- and postnatal day-specific elevations of protein concentrations and Mental or Motor Scale less than 55 or 55-69, we used multinominal logistic regression to estimate odds ratios. The referent group was infants with Mental or Motor Scale of 70 or greater. All models adjusted for gestational age. The aim of our etiological study was not to develop a prediction model for developmental impairment; hence we did not include all candidate predictors. To balance the risks of type 1 and type 2 errors, we chose to describe the precision of odds ratio estimates with 99% confidence intervals.
Mothers (n=1249) of 1506 infants gave their informed consent. For this analysis, we limited the sample to the 939 children from whom blood was obtained for analyses of protein levels and whose development was assessed at 24 months of age. The overall follow up rate was 85% (1018/1200) and blood protein data were available for 92% (n=939) of the infants who were evaluated at 24 months. Thus of 1200 survivors, 182 were not included in this analysis because they did not return for follow up and 79 were not included because blood protein data were not available. (Figure; available at www.jpeds.com)
Eleven percent of the cohort scored below 55 on the Mental Scale (i.e., Mental Development Index), and 11% had scores of 55-69. On the Motor Scale (i.e., Psychomotor Development Index), 16% scored below 55, and 15% scored 55-69. Among children with a Mental Scale less than 55, 43% had a Motor Scale less than 55; among those with a Motor Scale less than 55, 55% had a Mental Scale less than 55. Low scores were especially likely among infants with gestational age 23-24 weeks or birth weight of 750 grams or less, and those born to mothers with preeclampsia. (Table I; available at www.jpeds.com).
Infants included in this study (i.e., those from whom we collected blood samples and who returned for developmental evaluations) were less likely than surviving infants who were not included in this study to have a mother with less than 12 years of formal education, received public insurance, and had cervical insufficiency (Table II; available at www.jpeds.com). Children in our sample were slightly more likely than others to have experienced late onset sepsis, necrotizing enterocolitis, chronic lung disease, ventricular enlargement or a parenchymal echolucent lesion on cranial ultrasound.
Associations between elevated protein levels and a Mental Scale below 55 were most evident in specimens collected on day 14 (13 proteins including cytokines and their receptors, chemokines, adhesion molecules, acute phase proteins, and growth factors), less in specimens collected on day 7 (4 proteins), and not evident in day-1 blood (Table III). A Mental Scale between 55 and 69 was not predicted by any protein elevation on any day.
No protein elevations in day-1 blood, one elevation in day-7 blood, and eight elevations in day-14 blood were associated with Motor Scale below 55. A Motor Scale between 55 and 69 was predicted by an elevated level of one protein in day-1 blood, no proteins in day-7 blood, and 4 proteins in day-14 blood (Table IV).
For 12 proteins, an elevated concentration on two days a week apart predicted a Mental Scale below 55, whereas such a low Mental Scale was predicted by only four proteins elevated on a single day only (Table V). Three proteins elevated on multiple days were associated with a Mental Scale 55-69, but no single-day elevation was associated with a score in that range (Table VI; available at www.jpeds.com).
For eight proteins, an elevated concentration on two days predicted a Motor Scale below 55, but only two single-day elevations were predictive of a score this low. Four of the five proteins whose elevated concentrations were associated with a Motor Scale 55-69 were elevated on multiple days (Table VI).
The two days of elevated protein concentrations in Table 3 might have been any combination of two or three of the three days blood was sampled (Table VII). Because most of the elevated concentrations that predicted low developmental indices occurred on day 14 (Table IV), we compared the strength of associations with elevated concentrations on day 14 and elevated concentrations on days 7 and 14.
For 11 proteins, elevated concentrations were predictive of a Mental Scale less than 55, regardless of whether the elevations were present only in day-14 blood or were present on both day 7 and day 14. However, for five other proteins, elevated concentrations predicted a Mental Scale below 55 only if identified on both days 7 and 14. A similar pattern was found for a Motor Scale below 55, which was associated with 6 protein elevations regardless of whether they were identified on day 14 only or days 7 and 14, and two elevations only if present on both days 7 and 14. Two protein elevations (for RANTES and IL-6R) were associated with a decreased risk of motor impairment.
Although no protein elevations present only on day 14 were associated with a Mental Scale 55-69, two protein elevations on day 7 and day 14 were associated with an increased risk, and two other protein elevations were associated with decreased risk (Table VIII; available at www.jpeds.com). Two protein elevations on both days 7 and 14 predicted a Motor Scale score of 55-69, and an elevated concentration of another protein on days 7 and 14 was associated with decreased risk, and two protein elevations predicted a Motor Scale of 55-69 regardless of whether they were identified on day 14 or days 7 and 14 (Table VIII).
Extremely preterm infants who had elevated blood levels of inflammation-related proteins in the first 2 postnatal weeks were at increased risk for severely limited development two years later. This observation supports the concept that systemic inflammation near the time of birth contributes to developmental impairments after extremely preterm birth. It is also possible that the protein elevations reflect other putative risk factors for brain damage, such as intracranial hemorrhage, lung injury, necrotizing enterocolits, sepsis, and blood gas abnormalities. Protein elevations in day-14 blood were most predictive, suggesting a role for postnatal inflammation.
Five proteins conveyed prognostic information only if they were elevated on both day 7 and 14, and protein elevations on multiple days were more often associated with developmental impairment than elevations present for only one day, suggesting that persistent or recurrent inflammation heightens the risk. (13) The increased risk associated with persistent or recurrent inflammation might represent sensitization, i.e., enhanced vulnerability of the developing brain to inflammation-related damage after a prior exposure to an inflammatory stimulus.(14) Postnatal inflammation followed by feedback loops is another possible explanation for finding that persistent postnatal inflammation predicts low MDI.(13)
Alterations in gene expression after endotoxin infusion into adult humans are extinguished within 24 hours.(15) Thus even if protein degradation occurs over a longer period in preterm neonates, protein elevations in blood collected on days 7 and 14 are probably are not a consequence of inflammation initiated before delivery that resolves soon after delivery. We offer three possible explanations for the observation that of the 13 proteins whose elevated concentrations predicted a Mental Scale below 55, nine were identified only on day 14. First, vulnerability of the neonatal brain to inflammation-induced damage might increase with advancing postnatal age, due to a postnatal decrease in the levels of protectors provided by the placenta or mother.(16) Second, high levels of inflammatory proteins in day-7 and day-14 blood might result from prenatal brain damage or damage in the first postnatal days. Blood concentrations of inflammation-related proteins can increase following stroke, indicating that a non-infectious stimulus leading to brain damage can result in a systemic inflammatory response.(17) Third, systemic inflammation in the second postnatal week might contribute to, or result from, damage to lung(18) or gut(19), either of which might contribute to the risk of developmental impairment.(12, 20)
Perinatal and early neonatal infections have been associated with elevated cytokines (21, 22) and with cognitive and motor limitations.(23) (24) Our finding that infants who had elevated concentrations of inflammation-related proteins during the first postnatal month are at increased risk of cognitive limitation raises the possibility that the systemic inflammatory response we identified is the intermediary between perinatal inflammatory stimuli and brain damage.(3, 25) In a prior study of inflammation-related proteins and developmental outcome, higher cord blood levels of tumor necrosis factor-α were associated with the risk of a BSID Motor Scale more than one standard deviation below the mean, but no association was found with low scores on the BSID Mental Scale.(5) However, that study included only 67 infants and defined developmental delay as a developmental index below 85, rather than the more severe impairment (index below 55) we considered.
Our study has several limitations. First, a child’s BSID Mental Scale below 55 at 18-24 months is only modestly predictive of an intelligence quotient below 70 at 8 years of age (positive predictive value = 0.52).(11) Second, we measured only a few of the proteins that are differentially expressed with fetal systemic inflammation.(26) Similarly, we did not have information about many of proteins that might protect the brain of preterm infants, although we did identify several proteins (RANTES, IL-6, and VEGF) for which elevated concentrations were associated with a lower risk of impairment.
Our findings have implications for preventing developmental impairments in extremely preterm infants. Protein biomarkers in neonatal blood might serve as indicators of processes that underlie associations between clinical triggers for inflammation, such as necrotizing enterocolitis(27) and sepsis,(20) and subsequent developmental impairments. These biomarkers might also be useful for monitoring interventions that target inflammation-related brain damage. Our observations provide a potential explanation for associations, observed by others,(28, 29) between genetic polymorphisms in genes for cytokines and the risk of brain disorders in infants.
In conclusion, among extremely preterm infants, indicators of systemic inflammation during the second postnatal week are associated with increased risks of developmental impairment at age two years.
Supported by the National Institute of Neurological Disorders and Stroke (NS 40069). This study was completed as a cooperative agreement with the National Institute of Neurological Disorders and Stroke. The sponsor of the study participated in the study design, but not the data collection or data analysis.
The authors declare no conflicts of interest.
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