Urine Proteomics for Noninvasive Monitoring of Biomarkers in Bronchopulmonary Dysplasia.

INTRODUCTION: Current techniques to diagnose and/or monitor critically ill neonates with bronchopulmonary dysplasia (BPD) require invasive sampling of body fluids, which is suboptimal in these frail neonates. We tested our hypothesis that it is feasible to use noninvasively collected urine samples for proteomics from extremely low gestational age newborns (ELGANs) at risk for BPD to confirm previously identified proteins and biomarkers associated with BPD. METHODS: We developed a robust high-throughput urine proteomics methodology that requires only 50 µL of urine. We utilized the methodology with a proof-of-concept study validating proteins previously identified in invasively collected sample types such as blood and/or tracheal aspirates on urine collected within 72 h of birth from ELGANs (gestational age [26 ± 1.2] weeks) who were admitted to a single Neonatal Intensive Care Unit (NICU), half of whom eventually developed BPD (n = 21), while the other half served as controls (n = 21). RESULTS: Our high-throughput urine proteomics approach clearly identified several BPD-associated changes in the urine proteome recapitulating expected blood proteome changes, and several urinary proteins predicted BPD risk. Interestingly, 16 of the identified urinary proteins are known targets of drugs approved by the Food and Drug Administration. CONCLUSION: In addition to validating numerous proteins, previously found in invasively collected blood, tracheal aspirate, and bronchoalveolar lavage, that have been implicated in BPD pathophysiology, urine proteomics also suggested novel potential therapeutic targets. Ease of access to urine could allow for sequential proteomic evaluations for longitudinal monitoring of disease progression and impact of therapeutic intervention in future studies.

Role of Genetic Susceptibility in the Development of Bronchopulmonary Dysplasia.

OBJECTIVE: To assess heritable contributions to bronchopulmonary dysplasia (BPD) risk in a twin cohort restricted to gestational age at birth <29 weeks. STUDY DESIGN: A total of 250 twin pairs (192 dichorionic, 58 monochorionic) born <29 weeks gestational age with known BPD status were identified. Three statistical methods applicable to twin cohorts (χ2 test, intraclass correlations [ICCs], and ACE modeling [additive genetic or A, common environmental or C, and unique environmental or E components]) were applied. Heritability was estimated as percent variability from A. Identical methods were applied to a subcohort defined by zygosity and to an independent validation cohort. RESULTS: χ2 analyses comparing whether neither, 1, or both of monochorionic (23, 19, 16) and dichorionic (88, 56, 48) twin pairs developed BPD revealed no difference. Although there was similarity in BPD outcome within both monochorionic and dichorionic twin pairs by ICC (monochorionic ICC = 0.34, 95% CI [0.08, 0.55]; dichorionic ICC = 0.39, 95% CI [0.25, 0.51]), monochorionic twins were not more likely than dichorionic twins to have the same outcome (P = .70). ACE modeling revealed no contribution of heritability to BPD risk (% A = 0.0%, 95% CI [0.0%, 43.1%]). Validation and zygosity based cohort results were similar. CONCLUSIONS: Our analysis suggests that heritability is not a major contributor to BPD risk in preterm infants <29 weeks gestational age.

Early blood gas predictors of bronchopulmonary dysplasia in extremely low gestational age newborns.

Aim. To determine among infants born before the 28th week of gestation to what extent blood gas abnormalities during the first three postnatal days provide information about the risk of bronchopulmonary dysplasia (BPD). Methods. We studied the association of extreme quartiles of blood gas measurements (hypoxemia, hyperoxemia, hypocapnea, and hypercapnea) in the first three postnatal days, with bronchopulmonary dysplasia, among 906 newborns, using multivariable models adjusting for potential confounders. We approximated NIH criteria by classifying severity of BPD on the basis of the receipt of any O2 on postnatal day 28 and at 36 weeks PMA and assisted ventilation. Results. In models that did not adjust for ventilation, hypoxemia was associated with increased risk of severe BPD and very severe BPD, while infants who had hypercapnea were at increased risk of very severe BPD only. In contrast, infants who had hypocapnea were at reduced risk of severe BPD. Including ventilation for 14 or more days eliminated the associations with hypoxemia and with hypercapnea and made the decreased risk of very severe BPD statistically significant. Conclusions. Among ELGANs, recurrent/persistent blood gas abnormalities in the first three postnatal days convey information about the risk of severe and very severe BPD.

Low urine vascular endothelial growth factor levels are associated with mechanical ventilation, bronchopulmonary dysplasia and retinopathy of prematurity.

BACKGROUND: Organ-specific vascular endothelial growth factor (VEGF) expression is decreased during the pathogenesis of bronchopulmonary dysplasia (BPD) and retinopathy of prematurity (ROP) several weeks before either disease can be diagnosed. Early measurement of organ-specific tissue VEGF levels might allow identification of infants at high risk for these diseases, but is not clinically feasible. Urine VEGF is easily measured and useful in early diagnosis of several diseases. OBJECTIVES: Our aims were to assess the correlation of urine VEGF levels measured in the first postnatal month with subsequent BPD or ROP diagnosis and to determine whether various infant characteristics influence urine VEGF levels. METHODS: 106 subjects born at <29 weeks' gestation and surviving to 36 weeks' postmenstrual age were selected from an existing database and biorepository. Urine VEGF and total protein were measured in 2-3 samples per subject. RESULTS: Urine VEGF/protein levels increased by 72% per week (p < 0.0001) during the first postnatal month. In multivariable analysis controlling for postnatal age, lower VEGF/protein was associated with higher levels of mechanical respiratory support (p = 0.006), male gender (p = 0.001) and early sepsis (p = 0.003) but not with fraction of inspired oxygen. Lower urine VEGF/protein and mechanical ventilation were each associated with BPD and ROP. In analyses adjusted for respiratory support, lower urine VEGF/protein and ROP remained associated but urine VEGF/protein and BPD did not. CONCLUSIONS: Low urine VEGF/protein levels in the first postnatal month are associated with mechanical ventilation, BPD, and ROP.

Systemic inflammation associated with mechanical ventilation among extremely preterm infants.

Little evidence is available to document that mechanical ventilation is an antecedent of systemic inflammation in preterm humans. We obtained blood on postnatal day 14 from 726 infants born before the 28th week of gestation and measured the concentrations of 25 inflammation-related proteins. We created multivariable models to assess the relationship between duration of ventilation and protein concentrations in the top quartile. Compared to newborns ventilated for fewer than 7 days (N=247), those ventilated for 14 days (N=330) were more likely to have elevated blood concentrations of pro-inflammatory cytokines (IL-1ß, TNF-α), chemokines (IL-8, MCP-1), an adhesion molecule (ICAM-1), and a matrix metalloprotease (MMP-9), and less likely to have elevated blood concentrations of two chemokines (RANTES, MIP-1ß), a matrix metalloproteinase (MMP-1), and a growth factor (VEGF). Newborns ventilated for 7-13 days (N=149) had systemic inflammation that approximated the pattern of newborns ventilated for 14 days. These relationships were not confounded by chorioamnionitis or antenatal corticosteroid exposure, and were not altered appreciably among infants with and without bacteremia. These findings suggest that 2 weeks of ventilation are more likely than shorter durations of ventilation to be accompanied by high blood concentrations of pro-inflammatory proteins indicative of systemic inflammation, and by low concentrations of proteins that might protect from inflammation-mediated organ injury.

Innate immune activation in neonatal tracheal aspirates suggests endotoxin-driven inflammation.

BACKGROUND: Tracheal aspirates (TAs) from critically ill neonates accumulate bacterial endotoxin and demonstrate mobilization of endotoxin-binding proteins, but the potential bioactivity of endotoxin in TAs is unknown. We characterized innate immune activation in TAs of mechanically ventilated neonates. METHODS: Innate immune activation in TAs of mechanically ventilated neonates was characterized using a targeted 84-gene quantitative real-time (qRT) PCR array. Protein expression of cytokines was confirmed by multiplex assay. Expression and localization of the endotoxin-inducible antimicrobial protein Calgranulin C (S100A12) was assessed by flow cytometry. Endotoxin levels were measured in TA supernatants using the Limulus amoebocyte lysate assay. RESULTS: Analyses by qRT-PCR demonstrated expression of pattern recognition receptors, Toll-like receptor-nuclear factor κB and inflammasome pathways, cytokines/chemokines and their receptors, and anti-infective proteins in TA cells. Endotoxin positivity increased with postnatal age. As compared with endotoxin-negative TAs, endotoxin-positive TAs demonstrated significantly greater tumor necrosis factor (TNF), interleukin (IL)-6, IL-10, and serpin peptidase inhibitor, clade E, member 1 (SERPINE1) mRNA, and IL-10, TNF, and IL-1ß protein. Expression of S100A12 protein was localized to TA neutrophils. CONCLUSION: Correlation of endotoxin with TA inflammatory responses suggests endotoxin bioactivity and the possibility that endotoxin antagonists could mitigate pulmonary inflammation and its sequelae in this vulnerable population.

Blood protein concentrations in the first two postnatal weeks that predict bronchopulmonary dysplasia among infants born before the 28th week of gestation.

Lung inflammation contributes to the pathogenesis of bronchopulmonary dysplasia (BPD) and may be accompanied by a systematic inflammatory response. The objective of this study was to investigate the role of systemic inflammation in the development of BPD in a cohort of extremely low GA newborns (ELGANs) by examining the relationships between inflammation-associated proteins in neonatal blood samples and pulmonary outcomes. Proteins were measured in blood specimens collected on postnatal d 1-3, 5-8, and 12-15 from 932 ELGANs. Increased risk of BPD was associated with elevated blood concentrations of a variety of proinflammatory cytokines, adhesion molecules, and proteases. Reduced risk was prominently associated with increased concentrations of one chemokine, RANTES. Elevations of inflammatory proteins associated with BPD risk occurred during the first days after birth and inflammation intensified thereafter. Therefore, exposures that promote inflammation after the first postnatal days may be more critical in the pathogenesis of BPD. Fetal growth restriction, a known BPD risk factor, was not accompanied by proteins elevations and therefore does not seem to be mediated by systemic inflammation. By contrast, mechanical ventilation altered protein levels and may be associated with systemic inflammation

Does bronchopulmonary dysplasia contribute to the occurrence of cerebral palsy among infants born before 28 weeks of gestation?

OBJECTIVE: To evaluate the relationships among cerebral palsy (CP) phenotypes and bronchopulmonary dysplasia (BPD) severity and, in the process, to generate hypotheses regarding causal pathways linking BPD to CP. STUDY DESIGN: We studied 1047 infants born before the 28th week of gestation. Receipt of supplemental oxygen at 36 weeks postmenstrual age (PMA), with or without the need for mechanical ventilation (MV) at 36 weeks PMA, defined two levels of BPD. At 24 months, the children underwent neurologic examinations and CP diagnoses were made using an algorithm based on topographic localisation. RESULTS: The 536 infants with BPD were at increased risk of all three CP phenotypes. In time-oriented multivariable analyses that adjusted for potential confounders, receipt of supplemental oxygen without MV at 36 weeks PMA (BPD) was not associated with increased risk of any CP phenotype. In contrast, BPD accompanied by MV at 36 weeks PMA (BPD/MV) was associated with a nearly sixfold increased risk of quadriparesis and a fourfold increased risk of diparesis. CONCLUSIONS: Combined treatment with both MV and supplemental oxygen at 36 weeks PMA strongly predicts the more common bilateral CP phenotypes. BPD without MV at 36 weeks PMA was not significantly associated with any form of CP.

Epidemiology of bronchopulmonary dysplasia.

First described more than 40 years ago, bronchopulmonary dysplasia (BPD) remains one of the most serious and vexing challenges in the care of very preterm infants. Affecting approximately one-quarter of infants born <1500g birth weight, BPD is associated with prolonged neonatal intensive care unit hospitalization, greater risk of neonatal and post-neonatal mortality and a host of associated medical and neurodevelopmental sequelae. This seminar focuses on the epidemiology and definition of BPD as well as the current evidence pertaining to a number of potential preventive treatments for BPD: non-invasive respiratory support technologies, inhaled nitric oxide, vitamin A, and caffeine.

Chronic lung disease and developmental delay at 2 years of age in children born before 28 weeks' gestation.

INTRODUCTION: Extremely low gestational age newborns (ELGANs) are at increased risk of chronic lung disease (CLD) and of developmental delay. Some studies have suggested that CLD contributes to developmental delay. PATIENTS AND METHODS: We examined data collected prospectively on 915 infants born before the 28th week of gestation in 2002-2004 who were assessed at 24 months of age with the Bayley Scales of Infant Development-2nd Edition or the Vineland Adaptive Behavior Scales. We excluded infants who were not able to walk independently (Gross Motor Function Classification System score < 1) and, therefore, more likely to have functionally important fine motor impairments. We defined CLD as receipt of oxygen at 36 weeks' postmenstrual age and classified infants as either not receiving mechanical ventilation (MV) (CLD without MV) or receiving MV (CLD with MV). RESULTS: Forty-nine percent of ELGANs had CLD; of these, 14% were receiving MV at 36 weeks' postmenstrual age. ELGANs without CLD had the lowest risk of a Mental Developmental Index (MDI) or a Psychomotor Developmental Index (PDI) of <55, followed by ELGANs with CLD not receiving MV, and ELGANs with CLD receiving MV (9%, 12%, and 18% for the MDI and 7%, 10%, and 20% for the PDI, respectively). In time-oriented multivariate models, the risk of an MDI of <55 was associated with the following variables: gestational age of <25 weeks; single mother; late bacteremia; pneumothorax; and necrotizing enterocolitis. The risk of a PDI of <55 was associated with variables such as single mother, a complete course of antenatal corticosteroids, early and persistent pulmonary dysfunction, pulmonary deterioration during the second postnatal week, pneumothorax, and pulmonary interstitial emphysema. CLD, without or with MV, was not associated with the risk of either a low MDI or a low PDI. However, CLD with MV approached, but did not achieve, nominal statistical significance (odds ratio: 1.9 [95% confidence interval: 0.97-3.9]) for the association with a PDI of <55. CONCLUSIONS: Among children without severe gross motor delays, risk factors for CLD account for the association between CLD and developmental delay. Once those factors are considered in time-oriented risk models, CLD does not seem to increase the risk of either a low MDI or a low PDI. However, severe CLD might increase the risk of a low PDI.

Perturbation of gene expression of the chromatin remodeling pathway in premature newborns at risk for bronchopulmonary dysplasia.

BACKGROUND: One-third to one-half of all infants born before the 28th week of gestation develop bronchopulmonary dysplasia (BPD). Inflammatory regulators appear to be involved in the pathogenesis of BPD, possibly beginning in fetal life. To evaluate the feasibility of using expression profiling in umbilical cord tissue to discover molecular signatures for developmental staging and for determining risk of BPD, we conducted a cross-sectional study of infants born at less than 28 weeks of gestation (n = 54). Sections of umbilical cord were obtained at birth from 20 infants who later developed BPD and from 34 of their peers who did not develop BPD. RESULTS: Umbilical cord expression profiles at birth exhibited systematic differences in bioenergetic pathways with respect to gestational age. Infants who subsequently developed BPD had distinct signatures involving chromatin remodeling and histone acetylation pathways, which have previously been implicated in several adult onset lung diseases. These findings are consistent with prior work on inflammatory processes and bioenergetics in prematurity. CONCLUSION: This study of gene expression of the newborn umbilical cord implicates the chromatin remodeling pathways in those premature infants who subsequently develop BPD. Larger sample sizes will be required to generate prognostic markers from umbilical cord profiles.

Risk factors for persistent pulmonary hypertension of the newborn.

OBJECTIVE: Persistent pulmonary hypertension of the newborn, a clinical syndrome that results from the failure of the normal fetal-to-neonatal circulatory transition, is associated with substantial infant mortality and morbidity. We performed a case-control study to determine possible antenatal and perinatal predictors of persistent pulmonary hypertension of the newborn. METHODS: Between 1998 and 2003, the Slone Epidemiology Center enrolled 377 mothers of infants with persistent pulmonary hypertension of the newborn and 836 mothers of matched control subjects. Within 6 months of delivery, study nurses interviewed participants regarding demographic, medical, and obstetric characteristics. RESULTS: Factors that were independently associated with an elevated risk for persistent pulmonary hypertension of the newborn were infant male gender and black or Asian maternal race compared with white race. High prepregnancy BMI (>27 vs <20) was also associated with persistent pulmonary hypertension of the newborn, as were diabetes and asthma. Compared with infants who were delivered vaginally, the risk for persistent pulmonary hypertension of the newborn was higher for those who were born by cesarean section. Compared with infants who were born within 37 to 41 gestational weeks, the risk was higher for those who were born between 34 and 37 completed weeks and for those born beyond 41 weeks. Compared with infants within the 10th and 90th percentiles of birth weight for gestational age distribution, the risk was higher for infants above the 90th percentile. CONCLUSIONS: Our findings suggest an increased risk for persistent pulmonary hypertension of the newborn associated with cesarean delivery; late preterm or postterm birth; being large for gestational age; and maternal black or Asian race, overweight, diabetes, and asthma. It remains unclear whether some of these factors are direct causes of persistent pulmonary hypertension of the newborn or simply share common causes with it; however, clinicians should be alert to the increased need for monitoring and intervention among pregnancies with these risk factors.

Progress in discovery and evaluation of treatments to prevent bronchopulmonary dysplasia.

BACKGROUND: Recent improvements in the survival of extremely preterm infants have been accompanied by evolution in the pathogenesis and histopathology of bronchopulmonary dysplasia (BPD). Although oxygen and barotrauma-induced injury remain important contributing factors, pulmonary developmental arrest appears to play an equally important causal role in prolonged respiratory illness, especially among the most immature surviving preterm newborns. To date, clinical trials have failed to demonstrate a substantial benefit of a single treatment or preventive strategy for BPD. OBJECTIVES: To evaluate the current evidence in favor of treatments that might prevent BPD. METHODS: Review of clinical studies of preventive treatment strategies for BPD. RESULTS: High frequency oscillatory ventilation, permissive hypercapnea, and inhaled nitric oxide might offer benefit to infants at risk of BPD. These and other potential preventive therapies for BPD, such as superoxide dismutase, inositol, and alpha(1)-proteinase inhibitor, deserve further study. CONCLUSIONS: Although some current treatments offer promise, no preventive therapy for BPD has proven safe and effective, except for intramuscular vitamin A. Additional studies of respiratory technologies, management strategies, and protective molecules are needed.

Selective serotonin-reuptake inhibitors and risk of persistent pulmonary hypertension of the newborn.

BACKGROUND: Persistent pulmonary hypertension of the newborn (PPHN) is associated with substantial infant mortality and morbidity. A previous cohort study suggested a possible association between maternal use of the selective serotonin-reuptake inhibitor (SSRI) fluoxetine late in the third trimester of pregnancy and the risk of PPHN in the infant. We performed a case-control study to assess whether PPHN is associated with exposure to SSRIs during late pregnancy. METHODS: Between 1998 and 2003, we enrolled 377 women whose infants had PPHN and 836 matched control women and their infants. Maternal interviews were conducted by nurses, who were blinded to the study hypothesis, regarding medication use in pregnancy and potential confounders, including demographic variables and health history. RESULTS: Fourteen infants with PPHN had been exposed to an SSRI after the completion of the 20th week of gestation, as compared with six control infants (adjusted odds ratio, 6.1; 95 percent confidence interval, 2.2 to 16.8). In contrast, neither the use of SSRIs before the 20th week of gestation nor the use of non-SSRI antidepressant drugs at any time during pregnancy was associated with an increased risk of PPHN. CONCLUSIONS: These data support an association between the maternal use of SSRIs in late pregnancy and PPHN in the offspring; further study of this association is warranted. These findings should be taken into account in decisions as to whether to continue the use of SSRIs during pregnancy.

Strategies for preventing bronchopulmonary dysplasia.

PURPOSE OF REVIEW: Neonatologists and pulmonary biologists have long sought preventive treatments for bronchopulmonary dysplasia (BPD). The purpose of this review is to highlight recent reports of a number of potential treatments intended to prevent BPD and to discuss the controversies surrounding preventive strategies. RECENT FINDINGS: The evolution of BPD from a disorder of pulmonary injury affecting moderately preterm infants, to one characterized by a developmental pulmonary arrest among survivors of extreme prematurity has important implications for BPD prevention. Recent recognition that the pathogenesis of BPD might have prenatal origins raises new challenges and opportunities for studies of BPD prevention; however, most current preventive strategies for BPD focus on respiratory management. Neither past nor current clinical trials have shown a conclusive benefit of a single preventive treatment strategy. Promising but still largely unproven preventive respiratory treatments include: high frequency oscillatory ventilation, permissive hypercapnea, and inhaled nitric oxide. Observational and recent laboratory data support the need for randomized clinical trials of continuous positive airway pressure versus mechanical ventilation. Additionally, clinical trials are needed to address the deficit in our knowledge of the potential benefits and risks of postnatal low dose corticosteroid treatment. Further study of superoxide dismutase, inositol, and alpha-1 proteinase inhibitor also are warranted on the basis of recent clinical trials or meta-analyses. SUMMARY: Only Vitamin A has proven a safe and effective preventive treatment for BPD. Additional studies of respiratory technologies, management strategies, and protective molecules are needed. Directed cytokine and genetic therapies are on the horizon.

Bronchopulmonary dysplasia is not associated with ultrasound-defined cerebral white matter damage in preterm newborns.

Bronchopulmonary dysplasia (BPD) and cerebral white matter damage (WMD) are neonatal disorders that occur most commonly in those who are born much before term. In a large multicenter database, we sought to determine whether the two disorders occur together more frequently than expected and whether BPD and other neonatal respiratory characteristics are more common among infants who develop ultrasound-defined WMD than among those who do not. In a sample of 904 infants who were born before the 30th week of gestation and survived until 36 wk postmenstrual age, we did not find a co-occurrence of BPD and WMD above what would be expected by chance. Confounding does not seem to account for this lack of association between WMD and BPD. In conclusion, our findings do not support the hypothesis that BPD contributes to the occurrence of sonographically defined WMD.

Urine bombesin-like peptide elevation precedes clinical evidence of bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of very low birth weight infants, associated with oxygen therapy, barotrauma, and/or infections. Improved medical care has led to a paradoxically increased incidence of BPD due to greater infant survival. Early prediction of BPD has proven challenging. Increased pulmonary neuroendocrine cells containing bombesin-like peptide immunoreactivity occur in infants with BPD. We hypothesized that elevated urine bombesin-like peptide levels precede BPD. One hundred thirty-two infants, 28-weeks gestation or less, were studied. Urine bombesin-like peptide levels, determined by radioimmunoassay, were normalized for creatinine. BPD was defined as oxygen dependence at 36 weeks postmenstrual age. A first urine bombesin-like peptide level greater than 20,000 pg/mg creatinine (12,500 fmol/mg) between postnatal days 1-4 occurred among 54% of the infants who later developed BPD (p < or = 0.001), versus 10% among non-BPD infants (specificity 90%). Multivariable logistic regression analyses revealed that elevated urine bombesin-like peptide levels are associated with BPD (odds ratio 9.9, 95% confidence interval: 3.4, 29) (p < or = 0.001) after adjusting for all confounding factors. Thus, elevated bombesin-like peptide levels in these infants at 1-4 days after birth are associated with a 10-fold increased risk of developing BPD. Utilizing urine bombesin-like peptide for screening might permit early therapeutic interventions to reduce disease progression and could provide a target for new preventive therapies.

Risk of cranial ultrasound abnormalities in very-low-birth-weight infants conceived with assisted reproductive techniques.

OBJECTIVE: To evaluate risks of cranial ultrasound abnormalities among very-low-birth-weight (VLBW) infants conceived with fertility therapy (ovulation induction only or with assisted reproductive techniques [ART]) and of multiple gestation pregnancies. STUDY DESIGN: The incidences of cranial ultrasound abnormalities in 1473 VLBW infants conceived with and without fertility therapy and born of multiple versus singleton pregnancies were compared, using logistic regression models. RESULTS: Infants conceived with ART were less likely to have intraventricular hemorrhage (IVH). Twins and triplets had risks of cranial ultrasound abnormalities similar to those of singletons. Twins and triplets conceived with ART were at lower risk of IVH. CONCLUSION: VLBW infants conceived with ART do not appear to be at increased risk of cranial ultrasound abnormalities. Likewise, twins and triplets were not at increased risk of these abnormalities.