Genomic Differences between Spontaneous versus Indicated Extreme Preterm Birth.

OBJECTIVE: Extremely preterm infants are at high risk of neonatal mortality and morbidity. Extreme preterm birth (PTB) may result from spontaneous preterm labor or preterm premature rupture of membranes or may be indicated due to preeclampsia, eclampsia, hypertension, or other causes. Our objective was to identify single nucleotide polymorphisms (SNPs) and biological pathways associated with spontaneous versus indicated extreme PTB using the neonatal genome. STUDY DESIGN: We evaluated 523 spontaneous births and 134 indicated births weighing 401 to 1,000 g at birth from the Eunice Kennedy Shriver National Institute of Child Health and Human Development Neonatal Research Network's Genomics dataset by genome-wide association study (GWAS) and pathway analysis. The TOLSURF cohort was used to replicate the results. RESULTS: In the NRN GWAS, no statistically significant results were found, although the Manhattan plot showed one almost significant peak (rs60854043 on chromosome 14 at p = 1.03E-07) along with many other modest peaks at p = 1-9E-06, for a total of 15 suggestive associations at this locus. In the NRN pathway analysis, multiple pathways were identified, with the most significant being "GO_mf:go_low_density_lipoprotein_particle_receptor_activity" at p = 1.14E-06. However, these results could not be replicated in the TOLSURF cohort. CONCLUSION: Genomic differences are seen between infants born by spontaneous versus indicated extreme PTB. Due to the limited sample size, there is a need for larger studies. KEY POINTS: · Genomic differences are seen between infants born by spontaneous versus indicated very PTB.. · Future studies with large sample sizes evaluating extreme PTB are necessary.. · Spontaneous PTB is more common than indicated extreme PTB..

The Urinary Metabolomic Fingerprint in Extremely Preterm Infants on Total Parenteral Nutrition vs. Enteral Feeds.

Total Parenteral Nutrition (TPN), which uses intravenous administration of nutrients, minerals and vitamins, is essential for sustaining premature infants until they transition to enteral feeds, but there is limited information on metabolomic differences between infants on TPN and enteral feeds. We performed untargeted global metabolomics on urine samples collected between 23-30 days of life from 314 infants born <29 weeks gestational age from the TOLSURF and PROP cohorts. Principal component analysis across all metabolites showed a separation of infants solely on TPN compared to infants who had transitioned to enteral feeds, indicating global metabolomic differences between infants based on feeding status. Among 913 metabolites that passed quality control filters, 609 varied in abundance between infants on TPN vs. enteral feeds at p < 0.05. Of these, 88% were in the direction of higher abundance in the urine of infants on enteral feeds. In a subset of infants in a longitudinal analysis, both concurrent and delayed changes in metabolite levels were observed with the initiation of enteral feeds. These infants had higher concentrations of essential amino acids, lipids, and vitamins, which are necessary for growth and development, suggesting the nutritional benefit of an enteral feeding regimen.

The hydrocortisone-responsive urinary metabolome of premature infants.

BACKGROUND: Extremely premature infants are at risk for circulatory collapse or respiratory failure that are often treated with hydrocortisone (HC); however, there is no information on the metabolic consequences of this therapy. METHODS: Longitudinal urine samples from infants <28 weeks gestation in the Trial of Late Surfactant were analyzed by untargeted UHPLC:MS/MS. Fourteen infants who received a tapering course of HC beginning at 3 mg/kg/day for ≥9 days were compared to 14 matched control infants. A secondary cross-sectional analysis by logistic regression used urines from 314 infants. RESULTS: Of 1145 urinary metabolites detected, abundance of 219, representing all the major biochemical pathways, changed at p < 0.05 in the HC-treated group with 90% decreasing; 3 cortisol derivatives increased ~2-fold with HC therapy. Only 11% of regulated metabolites remained responsive at the lowest HC dose. Regulated metabolites included two steroids and thiamin that are associated with lung inflammation in infants. HC responsiveness was confirmed in 57% of metabolites by cross-sectional analysis. CONCLUSIONS: HC treatment of premature infants influenced in a dose-dependent manner abundance of 19% of identified urinary metabolites of diverse biochemical systems, primarily reducing concentrations. These findings indicate that exposure to HC reversibly impacts the nutritional status of premature infants. IMPACT: Hydrocortisone treatment of premature infants with respiratory failure or circulatory collapse alters levels of a subset of urinary metabolites representing all major biochemical pathways. This is the first description of the scope, magnitude, timing and reversibility of metabolomic changes in infants in response to hydrocortisone, and it confirms corticosteroid regulation of three biochemicals that are associated with lung inflammatory status. The findings indicate a dose-dependency of hydrocortisone for metabolomic and anti-inflammatory effects, that prolonged therapy may lower the supply of many nutrients, and that monitoring concentrations of cortisol and inflammation markers may be a useful clinical approach during corticosteroid therapy.

Blood metabolomics in infants enrolled in a dose escalation pilot trial of budesonide in surfactant.

BACKGROUND: The pathogenesis of BPD includes inflammation and oxidative stress in the immature lung. Corticosteroids improve respiratory status and outcome, but the optimal treatment regimen for benefit with low systemic effects is uncertain. METHODS: In a pilot dose escalation trial, we administered ≤5 daily doses of budesonide in surfactant to 24 intubated premature infants (Steroid And Surfactant in ELGANs (SASSIE)). Untargeted metabolomics was performed on dried blood spots using UPLC-MS/MS. Tracheal aspirate IL-8 concentration was determined as a measure of lung inflammation. RESULTS: Metabolomics data for 829 biochemicals were obtained on 121 blood samples over 96 h from 23 infants receiving 0.025, 0.05, or 0.1 mg budesonide/kg. Ninety metabolites were increased or decreased in a time- and dose-dependent manner at q ≤ 0.1 with overrepresentation in lipid and amino acid super pathways. Different dose response patterns occurred, with negative regulation associated with highest sensitivity to budesonide. Baseline levels of 22 regulated biochemicals correlated with lung inflammation (IL-8), with highest significance for sphingosine and thiamin. CONCLUSIONS: Numerous metabolic pathways are regulated in a dose-dependent manner by glucocorticoids, which apparently act via distinct mechanisms that impact dose sensitivity. The findings identify candidate blood biochemicals as biomarkers of lung inflammation and systemic responses to corticosteroids. IMPACT: Treatment of premature infants in respiratory failure with 0.1 mg/kg intra-tracheal budesonide in surfactant alters levels of ~11% of detected blood biochemicals in discrete time- and dose-dependent patterns. A subset of glucocorticoid-regulated biochemicals is associated with lung inflammatory status as assessed by lung fluid cytokine concentration. Lower doses of budesonide in surfactant than currently used may provide adequate anti-inflammatory responses in the lung with fewer systemic effects, improving the benefit:risk ratio.

Composition and origin of lung fluid proteome in premature infants and relationship to respiratory outcome.

BACKGROUND: Infants born at extremely low gestational age are at high risk for bronchopulmonary dysplasia and continuing lung disease. There are no early clinical biomarkers for pulmonary outcome and limited therapeutic interventions. OBJECTIVES: We performed global proteomics of premature infant tracheal aspirate (TA) and plasma to determine the composition and source of lung fluid proteins and to identify potential biomarkers of respiratory outcome. METHODS: TA samples were collected from intubated infants in the TOLSURF cohort before and after nitric oxide treatment, and plasma was collected from NO CLD infants. Protein abundance was assayed by HPLC/tandem mass spectrometry and Protein Prospector software. mRNA abundance in mid-gestation fetal lung was assessed by RNA sequencing. Pulmonary morbidity was defined as a need for ventilatory support at term and during the first year. RESULTS: Abundant TA proteins included albumin, hemoglobin, and actin-related proteins. 96 of 137 detected plasma proteins were present in TA (r = 0.69, p<0.00001). Based on lung RNAseq data, ~88% of detected TA proteins in injured infant lung are derived at least in part from lung epithelium with overrepresentation in categories of cell membrane/secretion and stress/inflammation. Comparing 37 infants at study enrollment (7-14 days) who did or did not develop persistent pulmonary morbidity, candidate biomarkers of both lung (eg., annexin A5) and plasma (eg., vitamin D-binding protein) origin were identified. Notably, levels of free hemoglobin were 2.9-fold (p = 0.03) higher in infants with pulmonary morbidity. In time course studies, hemoglobin decreased markedly in most infants after enrollment coincident with initiation of inhaled nitric oxide treatment. CONCLUSIONS: We conclude that both lung epithelium and plasma contribute to the lung fluid proteome in premature infants with lung injury. Early postnatal elevation of free hemoglobin and heme, which are both pro-oxidants, may contribute to persistent lung disease by depleting nitric oxide and increasing oxidative/nitrative stress.

Dose-escalation trial of budesonide in surfactant for prevention of bronchopulmonary dysplasia in extremely low gestational age high-risk newborns (SASSIE).

BACKGROUND: Initial trials of lung-targeted budesonide (0.25 mg/kg) in surfactant to prevent bronchopulmonary dysplasia (BPD) in premature infants have shown benefit; however, the optimal safe dose is unknown. METHODS: Dose-escalation study of budesonide (0.025, 0.05, 0.10 mg/kg) in calfactatant in extremely low gestational age neonates (ELGANs) requiring intubation at 3-14 days. Tracheal aspirate (TA) cytokines, blood budesonide concentrations, and untargeted blood metabolomics were measured. Outcomes were compared with matched infants receiving surfactant in the Trial Of Late SURFactant (TOLSURF). RESULTS: Twenty-four infants with mean gestational age 25.0 weeks and 743 g birth weight requiring mechanical ventilation were enrolled at mean age 6 days. Budesonide was detected in the blood of all infants with a half-life of 3.4 h. Of 11 infants with elevated TA cytokine levels at baseline, treatment was associated with sustained decrease (mean 65%) at all three dosing levels. There were time- and dose-dependent decreases in blood cortisol concentrations and changes in total blood metabolites. Respiratory outcomes did not differ from the historic controls. CONCLUSIONS: Budesonide/surfactant had no clinical respiratory benefit at any dosing levels for intubated ELGANs. One-tenth the dose used in previous trials had minimal systemic metabolic effects and appeared effective for lung-targeted anti-inflammatory action.

Correction: Genetic variation in CRHR1 is associated with short-term respiratory response to corticosteroids in preterm infants at risk for bronchopulmonary dysplasia.

In the original version of this article, the Supplementary Information file contained incorrect reference numbers. 'Supplemental Table S1' has now been replaced with the corrected version, in which the correct reference numbers are cited. The authors would like to apologise for this error.

Development and validation of an assay for quantifying budesonide in dried blood spots collected from extremely low gestational age neonates.

Budesonide is a potential therapeutic option for the prevention of bronchopulmonary dysplasia in mechanically ventilated premature neonates. The dose and concentrations of budesonide that drive effective prophylaxis are unknown, due in part to the difficulty in obtaining serial blood samples from this fragile population. Of primary concern is the limited total blood volume available for collection for the purposes of a pharmacokinetic study. Dried blood spots (DBS), which require the collection of <200 µL whole blood to fill an entire card, are an attractive low-blood volume alternative to traditional venipuncture sampling. We describe a simple and sensitive method for determining budesonide concentrations in DBS using an ultra-high-performance liquid chromatography - tandem mass spectrometry assay. Budesonide was liberated from a single 6 mm punch using a basified methyl tert-butyl ether extraction procedure. The assay was determined to be accurate and precise in the dynamic range of 1 to 50 ng/mL. The validated assay was then successfully applied to DBS collected as part of a multi-center, dose-escalation study of budesonide administered in surfactant via intra-tracheal instillation to premature neonates between 23 and 28 weeks gestational age. These findings show that DBS are a useful technique for collecting pharmacokinetic samples in premature neonates and other pediatric populations.

Surfactant status and respiratory outcome in premature infants receiving late surfactant treatment.

BACKGROUND: Many premature infants with respiratory failure are deficient in surfactant, but the relationship to occurrence of bronchopulmonary dysplasia (BPD) is uncertain. METHODS: Tracheal aspirates were collected from 209 treated and control infants enrolled at 7-14 days in the Trial of Late Surfactant. The content of phospholipid, surfactant protein B, and total protein were determined in large aggregate (active) surfactant. RESULTS: At 24 h, surfactant treatment transiently increased surfactant protein B content (70%, p < 0.01), but did not affect recovered airway surfactant or total protein/phospholipid. The level of recovered surfactant during dosing was directly associated with content of surfactant protein B (r = 0.50, p < 0.00001) and inversely related to total protein (r = 0.39, p < 0.0001). For all infants, occurrence of BPD was associated with lower levels of recovered large aggregate surfactant, higher protein content, and lower SP-B levels. Tracheal aspirates with lower amounts of recovered surfactant had an increased proportion of small vesicle (inactive) surfactant. CONCLUSIONS: We conclude that many intubated premature infants are deficient in active surfactant, in part due to increased intra-alveolar metabolism, low SP-B content, and protein inhibition, and that the severity of this deficit is predictive of BPD. Late surfactant treatment at the frequency used did not provide a sustained increase in airway surfactant.

Genetic variation in CRHR1 is associated with short-term respiratory response to corticosteroids in preterm infants at risk for bronchopulmonary dysplasia.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is an orphan disease and advances in prevention and treatment are lacking. The clinical efficacy of systemic corticosteroid therapy to reduce the severity of lung disease and BPD is highly variable. Our objective was to assess whether candidate SNPs in corticosteroid metabolism and response genes are associated with short-term phenotypic response to systemic corticosteroids in infants at high risk for BPD. METHODS: Pharmacogenetic analysis of data from a large randomized controlled trial (TOLSURF) in infants treated with dexamethasone or hydrocortisone using multivariate linear regression. The primary outcome was a change in respiratory severity score (RSS, mean airway pressure x FiO2) at day 7 of corticosteroid treatment. RESULTS: rs7225082 in the intron of CRHR1 is significantly associated with the magnitude of decrease in RSS 7 days after starting treatment with systemic corticosteroid (meta-analysis P = 2.8 × 10-4). Each T allele at rs7225082 is associated with a smaller absolute change in RSS at day 7, i.e., less response to systemic corticosteroids. CONCLUSIONS: Genetic variability is associated with corticosteroid responsiveness with regard to respiratory status in preterm infants. Identification of genetic markers of corticosteroid responsiveness may allow for therapeutic individualization, with the goal of optimizing the risk-to-benefit ratio for an individual child.

Exome sequencing identifies gene variants and networks associated with extreme respiratory outcomes following preterm birth.

BACKGROUND: Previous studies have identified genetic variants associated with bronchopulmonary dysplasia (BPD) in extremely preterm infants. However, findings with genome-wide significance have been rare, and not replicated. We hypothesized that whole exome sequencing (WES) of premature subjects with extremely divergent phenotypic outcomes could facilitate the identification of genetic variants or gene networks contributing disease risk. RESULTS: The Prematurity and Respiratory Outcomes Program (PROP) recruited a cohort of > 765 extremely preterm infants for the identification of markers of respiratory morbidity. We completed WES on 146 PROP subjects (85 affected, 61 unaffected) representing extreme phenotypes of early respiratory morbidity. We tested for association between disease status and individual common variants, screened for rare variants exclusive to either affected or unaffected subjects, and tested the combined association of variants across gene loci. Pathway analysis was performed and disease-related expression patterns were assessed. Marginal association with BPD was observed for numerous common and rare variants. We identified 345 genes with variants unique to BPD-affected preterm subjects, and 292 genes with variants unique to our unaffected preterm subjects. Of these unique variants, 28 (19 in the affected cohort and 9 in unaffected cohort) replicate a prior WES study of BPD-associated variants. Pathway analysis of sets of variants, informed by disease-related gene expression, implicated protein kinase A, MAPK and Neuregulin/epidermal growth factor receptor signaling. CONCLUSIONS: We identified novel genes and associated pathways that may play an important role in susceptibility/resilience for the development of lung disease in preterm infants.

Ancestry and genetic associations with bronchopulmonary dysplasia in preterm infants.

Bronchopulmonary dysplasia in premature infants is a common and often severe lung disease with long-term sequelae. A genetic component is suspected but not fully defined. We performed an ancestry and genome-wide association study to identify variants, genes, and pathways associated with survival without bronchopulmonary dysplasia in 387 high-risk infants treated with inhaled nitric oxide in the Trial of Late Surfactant study. Global African genetic ancestry was associated with increased survival without bronchopulmonary dysplasia among infants of maternal self-reported Hispanic white race/ethnicity [odds ratio (OR) = 4.5, P = 0.01]. Admixture mapping found suggestive outcome associations with local African ancestry at chromosome bands 18q21 and 10q22 among infants of maternal self-reported African-American race/ethnicity. For all infants, the top individual variant identified was within the intron of NBL1, which is expressed in midtrimester lung and is an antagonist of bone morphogenetic proteins ( rs372271081 , OR = 0.17, P = 7.4 × 10-7). The protective allele of this variant was significantly associated with lower nitric oxide metabolites in the urine of non-Hispanic white infants ( P = 0.006), supporting a role in the racial differential response to nitric oxide. Interrogating genes upregulated in bronchopulmonary dysplasia lungs indicated association with variants in CCL18, a cytokine associated with fibrosis and interstitial lung disease, and pathway analyses implicated variation in genes involved in immune/inflammatory processes in response to infection and mechanical ventilation. Our results suggest that genetic variation related to lung development, drug metabolism, and immune response contribute to individual and racial/ethnic differences in respiratory outcomes following inhaled nitric oxide treatment of high-risk premature infants.

Maternal Black Race and Persistent Wheezing Illness in Former Extremely Low Gestational Age Newborns: Secondary Analysis of a Randomized Trial.

OBJECTIVE: To evaluate the relationship between maternal self-reported race/ethnicity and persistent wheezing illness in former high-risk, extremely low gestational age newborns, and to quantify the contribution of socioeconomic, environmental, and biological factors on this relationship. STUDY DESIGN: We assessed persistent wheezing illness determined at 18-24 months corrected (for prematurity) age in survivors of a randomized trial. Parents/caregivers were surveyed for wheeze and inhaled asthma medication use quarterly to 12 months, and at 18 and 24 months. We used multivariable analysis to evaluate the relationship of maternal race to persistent wheezing illness, and identified mediators for this relationship via formal mediation analysis. RESULTS: Of 420 infants (25.2 ± 1.2 weeks of gestation and 714 ± 166 g at birth, 57% male, 34% maternal black race), 189 (45%) had persistent wheezing illness. After adjustment for gestational age, birth weight, and sex, infants of black mothers had increased odds of persistent wheeze compared with infants of nonblack mothers (OR = 2.9, 95% CI 1.9, 4.5). Only bronchopulmonary dysplasia, breast milk diet, and public insurance status were identified as mediators. In this model, the direct effect of race accounted for 69% of the relationship between maternal race and persistent wheeze, whereas breast milk diet, public insurance status, and bronchopulmonary dysplasia accounted for 8%, 12%, and 10%, respectively. CONCLUSIONS: Among former high-risk extremely low gestational age newborns, infants of black mothers have increased odds of developing persistent wheeze. A substantial proportion of this effect is directly accounted for by race, which may reflect unmeasured environmental influences, and acquired and innate biological differences. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01022580.

Race Effects of Inhaled Nitric Oxide in Preterm Infants: An Individual Participant Data Meta-Analysis.

OBJECTIVE: To assess whether inhaled nitric oxide (iNO) improves survival without bronchopulmonary dysplasia (BPD) for preterm African American infants. STUDY DESIGN: An individual participant data meta-analysis was conducted, including 3 randomized, placebo-controlled trials that enrolled infants born at <34 weeks of gestation receiving respiratory support, had at least 15% (or a minimum of 10 infants in each trial arm) of African American race, and used a starting iNO of >5 parts per million with the intention to treat for 7 days minimum. The primary outcome was a composite of death or BPD. Secondary outcomes included death before discharge, postnatal steroid use, gross pulmonary air leak, pulmonary hemorrhage, measures of respiratory support, and duration of hospital stay. RESULTS: Compared with other races, African American infants had a significant reduction in the composite outcome of death or BPD with iNO treatment: 49% treated vs 63% controls (relative risk, 0.77; 95% CI, 0.65-0.91; P = .003; interaction P = .016). There were no differences between racial groups for death. There was also a significant difference between races (interaction P = .023) of iNO treatment for BPD in survivors, with the greatest effect in African American infants (P = .005). There was no difference between racial groups in the use of postnatal steroids, pulmonary air leak, pulmonary hemorrhage, or other measures of respiratory support. CONCLUSION: iNO therapy should be considered for preterm African American infants at high risk for BPD. iNO to prevent BPD in African Americans may represent an example of a racially customized therapy for infants.

The Randomized, Controlled Trial of Late Surfactant: Effects on Respiratory Outcomes at 1-Year Corrected Age.

OBJECTIVE: To determine the effects of late surfactant on respiratory outcomes determined at 1-year corrected age in the Trial of Late Surfactant (TOLSURF), which randomized newborns of extremely low gestational age (≤28 weeks' gestational age) ventilated at 7-14 days to late surfactant and inhaled nitric oxide vs inhaled nitric oxide-alone (control). STUDY DESIGN: Caregivers were surveyed in a double-blinded manner at 3, 6, 9, and 12 months' corrected age to collect information on respiratory resource use (infant medication use, home support, and hospitalization). Infants were classified for composite outcomes of pulmonary morbidity (no PM, determined in infants with no reported respiratory resource use) and persistent PM (determined in infants with any resource use in ≥3 surveys). RESULTS: Infants (n = 450, late surfactant n = 217, control n = 233) were 25.3 ± 1.2 weeks' gestation and 713 ± 164 g at birth. In the late surfactant group, fewer infants received home respiratory support than in the control group (35.8% vs 52.9%, relative benefit [RB] 1.28 [95% CI 1.07-1.55]). There was no benefit of late surfactant for No PM vs PM (RB 1.27; 95% CI 0.89-1.81) or no persistent PM vs persistent PM (RB 1.01; 95% CI 0.87-1.17). After adjustment for imbalances in baseline characteristics, relative benefit of late surfactant treatment increased: RB 1.40 (95% CI 0.89-1.80) for no PM and RB 1.24 (95% CI 1.08-1.42) for no persistent PM. CONCLUSION: Treatment of newborns of extremely low gestational age with late surfactant in combination with inhaled nitric oxide decreased use of home respiratory support and may decrease persistent pulmonary morbidity. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01022580.

Early Cumulative Supplemental Oxygen Predicts Bronchopulmonary Dysplasia in High Risk Extremely Low Gestational Age Newborns.

OBJECTIVE: To assess the prognostic accuracy of early cumulative supplemental oxygen (CSO) exposure for prediction of bronchopulmonary dysplasia (BPD) or death, and to evaluate the independent association of CSO with BPD or death. STUDY DESIGN: We performed a secondary analysis of the Trial of Late Surfactant, which enrolled 511 infants born at ≤28 weeks gestational age who were mechanically ventilated at 7-14 days of life. Our primary outcome was BPD or death at 36 weeks postmenstrual age, as determined by a physiological oxygen/flow challenge. Average daily supplemental oxygen (fraction of inspired oxygen - 0.21) was calculated. CSO was calculated as the sum of the average daily supplemental oxygen over time periods of interest up to 28 days of age. Area under the receiver operating curve (AUROC) values were generated to evaluate the accuracy of CSO for prediction of BPD or death. The independent relationship between CSO and BPD or death was assessed in multivariate modeling, while adjusting for mean airway pressure. RESULTS: In the study infants, mean gestational age at birth was 25.2 ± 1.2 weeks and mean birth weight was 700 ± 165 g. The AUROC value for CSO at 14 days was significantly better than that at earlier time points for outcome prediction (OR, 0.70; 95% CI, 0.65-0.74); it did not increase with the addition of later data. In multivariate modeling, a CSO increase of 1 at 14 days increased the odds of BPD or death (OR, 1.7; 95% CI, 1.3-2.2; P < .0001), which corresponds to a 7% higher daily supplemental oxygen value. CONCLUSION: In high-risk extremely low gestational age newborns, the predictive accuracy of CSO plateaus at 14 days. CSO is independently associated with BPD or death. This index may identify infants who could benefit from early intervention to prevent BPD.