Preterm Resuscitation With Low Oxygen Causes Less Oxidative Stress,

Inflammation, and Chronic Lung Disease

Maximo Vento, Manuel Moro, Raquel Escrig, Luis Arruza, Gema Villar, Isabel
Izquierdo, L. Jackson Roberts, II, Alessandro Arduini, Justo Javier Escobar, Juan
Sastre and Miguel A. Asensi
Pediatrics 2009;124;e439-e449; originally published online Aug 3, 2009;
DOI: 10.1542/peds.2009-0434

The online version of this article, along with updated information and services, is
located on the World Wide Web at:
http://www.pediatrics.org/cgi/content/full/124/3/e439

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ARTICLES

Preterm Resuscitation With Low Oxygen Causes Less

Oxidative Stress, Inammation, and Chronic Lung Disease
CONTRIBUTORS: Maximo Vento, PhD, MD,a Manuel Moro,
MD,b Raquel Escrig, MD,a Luis Arruza, MD,b Gema Villar,
MD,b Isabel Izquierdo, MD,a L. Jackson Roberts, II, MD,c
Alessandro Arduini, PhD,d Justo Javier Escobar, PhD,d
Juan Sastre, PhD,d and Miguel A. Asensi, PhDd
a

Division of Neonatology, University Hospital La Fe, Valencia,

Spain; bDivision of Neonatology, University Hospital San Carlos,
Madrid, Spain; cDepartment of Pharmacology, Vanderbilt
University, Nashville, Tennessee; dDepartment of Physiology,
Faculty of Pharmacy, University of Valencia, Valencia, Spain

WHATS KNOWN ON THIS SUBJECT: In a previous study, the

feasibility of resuscitating ELGANs with an initial FIO2 of 30% and
achieving targeted SpO2 values was demonstrated.
WHAT THIS STUDY ADDS: This study conrms the feasibility of
using lower oxygen concentrations for resuscitating extremely
premature infants, but it also shows that lower FIO2 reduces
oxidative stress, expression of proinammatory cytokines, and the
incidence of BPD.

KEY WORDS
prematurity, resuscitation, oxygen, oxidative stress biomarkers,
inammatory cytokines, bronchopulmonary dysplasia
ABBREVIATIONS
ELGAN extremely low gestational age neonate
DR delivery room
BPD bronchopulmonary dysplasia
ROSreactive oxygen species
FIO2fraction of inspired oxygen
SpO2pulse oxygen saturation
GSHreduced glutathione
GSSG oxidized glutathione
TNF-tumor necrosis factor
IL-8 interleukin 8
HR heart rate
This trial has been registered at www.clinicaltrials.gov
(identier NCT00494702).
www.pediatrics.org/cgi/doi/10.1542/peds.2009-0434
doi:10.1542/peds.2009-0434
Accepted for publication Apr 14, 2009
Address correspondence to Maximo Vento, PhD, MD, Department
of Pediatrics, Neonatal Research Unit, Division of Neonatology,
University Hospital La Fe, Avenida de Campanar, 21, E46009
Valencia, Spain. E-mail: maximo.vento@uv.es
PEDIATRICS (ISSN Numbers: Print, 0031-4005; Online, 1098-4275).
Copyright 2009 by the American Academy of Pediatrics
FINANCIAL DISCLOSURE: The authors have indicated they have
no nancial relationships relevant to this article to disclose.

abstract
OBJECTIVE: The goal was to reduce adverse pulmonary adverse outcomes, oxidative stress, and inammation in neonates of 24 to 28
weeks of gestation initially resuscitated with fractions of inspired oxygen of 30% or 90%.
METHODS: Randomized assignment to receive 30% (N 37) or 90%
(N 41) oxygen was performed. Targeted oxygen saturation values
were 75% at 5 minutes and 85% at 10 minutes. Blood oxidized glutathione (GSSG)/reduced glutathione ratio and urinary o-tyrosine,
8-oxo-dihydroxyguanosine, and isoprostane levels, isofuran elimination, and plasma interleukin 8 and tumor necrosis factor levels
were determined.
RESULTS: The low-oxygen group needed fewer days of oxygen supplementation (6 vs 22 days; P .01) and fewer days of mechanical ventilation (13 vs 27 days; P .01) and had a lower incidence of bronchopulmonary dysplasia at discharge (15.4% vs 31.7%; P .05). GSSG/
reduced glutathione 100 ratios at day 1 and 3 were signicantly
higher in the high-oxygen group (day 1: high-oxygen group: 13.36
5.25; low-oxygen group: 8.46 3.87; P .01; day 3: high-oxygen group:
8.87 4.40; low-oxygen group: 6.97 3.11; P .05). Urinary markers
of oxidative stress were increased signicantly in the high-oxygen
group, compared with the low-oxygen group, in the rst week after
birth. GSSG levels on day 3 and urinary isofuran, o-tyrosine, and
8-hydroxy-2-deoxyguanosine levels on day 7 were correlated signicantly with development of chronic lung disease.
CONCLUSIONS: Resuscitation of preterm neonates with 30% oxygen
causes less oxidative stress, inammation, need for oxygen, and risk of
bronchopulmonary dysplasia. Pediatrics 2009;124:e439e449

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e439

Rates of survival of extremely low gestational age neonates (ELGANs) (28

weeks of gestation) have increased
steadily since the late 1990s.13 However, death in the delivery room (DR)
still represents 32% of total ELGAN
deaths at discharge, and morbidity
rates are especially high at 23 to 25
weeks of gestation.4,5 Of note, the use of
positive pressure ventilation with a
high oxygen concentration has been associated with development of chronic
lung disease.6,7 Lung-stretching and hyperoxia may act synergistically on prenatally inamed fetal membranes and
may elicit a systemic inammatory response that contributes to the development of bronchopulmonary dysplasia
(BPD).8 In fact, in experimental and
clinical settings, hyperoxia has been
associated with serious injury to the
developing brain, lung, myocardium,
and kidney.9 Therefore, excessive oxygenation contributes to the formation
of reactive oxygen species (ROS).10
These highly reactive compounds react with nearby molecules (eg, protein,
lipids, DNA, and RNA), modifying their
structure and function.11 In addition, it
was shown experimentally that ROS
may act in the lung as upstream signaling molecules favoring alveolar epithelial cell apoptosis.12
To prevent adverse effects of oxygen
during resuscitation, room air instead
of 100% oxygen was used in randomized, clinical trials.13 In those studies,
room air reduced signicantly the
time of resuscitation,14,15 biomarkers
of heart (troponin C) and kidney
(N-acetyl-glucosaminidase) damage,16
and oxidative stress17; moreover, room
air reduced mortality rates13 without
increasing long-term neurologic morbidity rates for survivors.18
The antioxidant defense system develops late in gestation.19,20 Therefore, premature neonates are highly
susceptible to the negative side effects of excess oxygen.21 Consee440

VENTO et al

quently, it would be especially advisable to avoid hyperoxia during

neonatal resuscitation of preterm
neonates.22 However, few studies
monitored oxygen supplementation
during resuscitation of ELGANs.2326
Wang et al24 failed to achieve clinical
stabilization of ELGANs by using room
air. Dawson et al25 compared 2 cohorts of ELGANs, before and after establishment of room air as the initial
standard of care in the DR, and reported similar results. Harling et al23
did not nd differences in clinical recovery of premature neonates with
the use of 50% instead of 100%
oxygen during resuscitation. Conversely, in a previous trial in our
group, Escrig et al,26 using initial FIO2
of 30%, showed the feasibility of
ELGANs achieving targeted pulse oxygen saturation (SpO2) values of 85%
at 10 minutes after birth. Furthermore, neonates in the lower-oxygen
group received signicantly smaller
oxygen loads during resuscitation.26
The only medical intervention that differentiated the 2 groups in our study
was oxygen concentration during resuscitation. The remaining interventions followed established standards
of care. We hypothesized that lower oxygen levels during resuscitation would
cause less oxidative stress and inammation and would reduce the need for
oxygen supplementation and mechanical ventilation and/or the incidence of
BPD. To validate our hypothesis, we
launched a new trial enrolling ELGANs
assigned randomly to an initial FIO2 of
30% or 90%. Targeted SpO2 values were
75% at 5 minutes and 85% at 10 minutes after birth. For evaluation of oxidative stress and redox status, the
reduced glutathione (GSH)/oxidized
glutathione (GSSG) ratio was determined.14,15,17,18 In addition, we evaluated
oxidation of proteins (o-tyrosine/phenylalanine ratio), DNA (8-hydroxy-2deoxyguanosine/2-deoxyguanosine ra-

tio),27 and lipids (isoprostanes and

isofurans).28 Furthermore, for assessment of inammation, plasma interleukin 8 (IL-8) and tumor necrosis
factor (TNF-) levels were determined.29 We correlated oxygen levels at
resuscitation with biomarkers of oxidative stress and inammation and
clinical outcomes.

METHODS
Design
A prospective clinical trial enrolled
ELGANs (28 weeks of gestation, as
determined with fetal ultrasonography) in 2 tertiary referral centers in
Spain during a 30-month period
(September 2005 through March
2008). Eligible patients were assigned randomly to be resuscitated
with a lower (30%) or higher (90%)
fraction of inspired oxygen (FIO2). The
higher FIO2 was chosen because a recent meta-analysis showed higher
mortality rates among neonates resuscitated with pure oxygen.13 The
study was not blinded with respect to
the neonatologist in charge of the respiratory airway but it was with respect to the rest of the caregivers
in the DR. The institutional review
board of our hospital counseled
against complete blinding, citing a
lack of published experience with the
resuscitation and follow-up care of
ELGANs with lower oxygen concentrations. Interventions in the DR were
recorded scrupulously and were analyzed to evaluate possible bias. The
study protocol was approved by the
ethics and scientic research committees of both participating hospitals. Parents informed consent was
obtained for all enrolled infants.
The main clinical outcome was neonatal death (death at 28 days) plus
the incidence of BPD, dened as the
need for oxygen supplementation at
postconceptional age of 36 weeks.30
Secondary outcomes were durations

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of supplementary oxygen treatment

and mechanical ventilation and use
of surfactant. Biochemical outcomes
were biomarkers of oxidative stress
and proinammatory cytokines and
their correlation with BPD.
Women who were admitted at gestational ages of 28 weeks were asked
to sign informed consent forms and
were assigned to the high-oxygen
or low-oxygen group, according to
computer-generated random numbers in sealed envelopes. In cases of
refusal, the patients were not assigned. Moreover, if an infant was assigned randomly but was not resuscitated, then the data were not
included in the study. Twin gestations were considered as a unit for
the purpose of randomization. Targeted objectives were preductal SpO2
values of 75% at 5 minutes and 85%
at 10 minutes after birth. The sample
size was calculated to reduce the incidence of BPD from 30% to 20%
(minimum of 35 patients per group).
Moreover, patients were stratied in
groups of 27 to 28 weeks and 26
weeks of gestation, to ensure a homogeneous representation of all
gestational ages.
Patients
Eligible neonates were inborn patients with 1 of the following resuscitation criteria: (1) absence of spontaneous respiration at 1 minute;
(2) bradycardia (60 beats per
minute) for 2 minutes, hypoactivity, and/or hypotonia; or (3) ineffective ventilation, as determined
through auscultation and/or lack of
thoracic expansion. Exclusion criteria were (1) uncertain gestational
age or (2) severe congenital malformations or chromosomal abnormalities. Of a total of 106 eligible neonates, 9 in the low-oxygen group and
10 in the high-oxygen group were lost

PEDIATRICS Volume 124, Number 3, September 2009

TABLE 1 Characteristics of ELGANs Resuscitated With Lower (30%) or Higher (90%) Oxygen
Concentrations at Birth and Management in DR

Gestational age, mean SD, wk

Birth weight, mean SD, g
Gender, n
Male
Female
Multiple birth, n
Small for gestational age, n
Prenatal corticosteroid therapy (full schedule), n (%)
Type of delivery, n (%)
Vaginal
Cesarean section
Cord blood pH at birth, mean SD
Apgar score, median (interquartile range)
1 min
5 min
Received supplementary oxygen during resuscitation, n (%)
Tracheal intubation in DR, n (%)
Breathing 21% oxygen at arrival at NICU, n (%)
a

Low Oxygen
(N 37)

High Oxygen
(N 41)

26.0 1.5
854.7 170.1

26.3 1.3
901.7 195.4

14
23
11
13
36 (97.2)

18
23
9
12
38 (92.7)

18 (48.6)
19 (51.4)
7.05 0.9

17 (41.4)
24 (58.6)
7.09 1.1

5 (27)
8 (59)
32 (86.5)
21 (56.7)
28 (75.6)

6 (28)
8 (59)
34 (82.9)
25 (60.9)
23 (56.0)a

Statistical comparison between groups (2 test), P .05, compared with low-oxygen group.

to randomization because parents

declined to participate. Of the remaining subjects, 4 in the low-oxygen
group and 3 in the high-oxygen group
died and did not complete the study.
Resuscitation Procedure
The resuscitation procedure was described previously.26,31 Neonates with
heart rate (HR) of 80 to 100 beats
per minute and increased work of
breathing in the rst 2 minutes of life
initially underwent ventilation with a
face mask (5 8 cm H2O), with a
T-piece resuscitator (Neopuff; Fisher
& Paykel Healthcare, Penmure, New
Zealand); if clinical responses (HR
and/or SpO2) were insufcient, then
the neonates were switched to intermittent positive pressure ventilation.
For infants with gestational ages of
27 weeks, endotracheal intubation
was performed; however, for infants
with gestational ages of 27 or 28
weeks, noninvasive ventilation was
pursued for several minutes and intubation was performed only if the
patients condition did not improve.
Initial parameters for intermittent
positive pressure ventilation were

positive inspiratory pressure of 20

cm H2O and positive end expiratory
pressure of 5 cm H2O. If no improvement was detected after 2 minutes,
then respirator parameters were
modied to achieve the targeted objectives. Immediately after birth, an
oximeter probe with sensors with
2-second averaging (Radical 7;
Masimo, Irvine, CA) was set on the
right wrist for preductal SpO2 monitoring.32 FIO2 was titrated to attain
targeted SpO2 values, and 60 to 90
seconds were allowed for responses
after each change. Abrupt FIO2
changes (10% every 30 seconds)
were avoided, to prevent pulmonary
vessel constriction.33 HR indicated
the effectiveness of resuscitation; if
bradycardia (HR of 60 beats per
minute) lasted 30 seconds, then
the FIO2 was switched to 100%. SpO2
was recorded continuously, and the
information was downloaded and analyzed once oxygen treatment was
discontinued.
Analytical Determinations
Cord blood (3 mL) was collected in
the DR before clamping, and blood

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was collected from a peripheral vein

at days 1 and 7. One milliliter of total
blood was used for GSH and GSSG assays. Two milliliters were centrifuged (2000 rpm for 20 minutes), and
the supernatant was divided into
0.5-mL aliquots and deep-frozen
(80C). One 0.5-mL aliquot was
used for TNF- and IL-8 determinations, and the rest was kept deepfrozen. Urine samples were deepfrozen (80C). GSH and GSSG levels
were determined through highperformance liquid chromatography,34 IL-8 and TNF- levels through
ow cytometry (CBA Flex Set System;
Becton-Dickinson, Franklin Lakes,
NJ), o-tyrosine, phenylalanine, 8hydroxy-2-deoxyguanosine, and 2deoxyguanosine levels through highperformance liquid chromatography/
mass spectrometry,27 and levels of isoprostane metabolites and isofurans
through gas chromatography/mass
spectrometry.28
Statistical Analyses
Baseline characteristics were determined by using SPSS 13 (SPSS Inc,
Chicago, IL). For parameters with
normal distribution (as assessed
with the Kolmogorov-Smirnov test),
1-way analysis of variance was performed and differences between
groups were calculated with Tukeys
method. The level of signicance was
.05. The Mann-Whitney U test was
used for comparison of nonpaired
samples and the Kruskal-Wallis test
for 2 nonpaired (independent)
samples for nonnormally distributed
values.35

RESULTS
General Characteristics of the
Population and Management in the
DR and NICU
Table 1 shows no differences in the
general characteristics of the population or management in the DR. The
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VENTO et al

FIGURE 1
A, FIO2 received by ELGANs in the low-oxygen group (initial FIO2 of 30%) and in the high-oxygen group
(initial FIO2 of 90%) in the rst 30 minutes after birth. Students t test comparisons (minute to minute)
found signicant differences between groups at minutes 2, 3, 4, and 6 after birth (aP .01). B, HR,
expressed in beats per minute (bpm), in the low-oxygen (Lox) and high-oxygen (Hox) groups in the rst
30 minutes after birth.

number of neonates breathing room

air on arrival in the NICU was signicantly higher in the low-oxygen group
(P .05).
FIO2, HR, and SpO2 in the DR
Reliable SpO2 readings were achieved
at 76 13 seconds. Figure 1 depicts
both FIO2 and HR at 60-second intervals
from the rst reliable reading to 30
minutes after birth. The initial FIO2 in

the low-oxygen group was increased in

a stepwise manner to 55% at 5 minutes; conversely, the initial FIO2 in the
high-oxygen group was reduced in a
stepwise manner, reaching 55% at 5
minutes. Signicant differences in FIO2
values used in the rst 4 minutes of life
were found (P .01), whereas no differences were found thereafter. No differences in HR between groups were
found.

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day 1 (high-oxygen group: 13.36 5.25;
low-oxygen group: 8.46 3.87; P .01)
and day 3 (high-oxygen group: 8.87
4.40; low-oxygen group: 6.97 3.11;
P .05), however, the high-oxygen
group had signicantly greater GSSG/
GSH ratios than did the low-oxygen
group.
Urinary Isoprostane Metabolites and
Isofurans

FIGURE 2
SpO2 values in the rst 30 minutes after birth for the low-oxygen (Lox) group (initially resuscitated with
FIO2 of 30%) and the high-oxygen (Hox) group (initially resuscitated with FIO2 of 90%). No signicant
differences between groups were found.

SpO2 in the Low-Oxygen and

High-Oxygen Groups
Figure 2 shows no signicant differences in SpO2 values between the lowoxygen group and the high-oxygen
group at any time point in the rst 30
minutes after birth. Both groups
reached targeted SpO2 values at 5 and
10 minutes.
Clinical Outcomes
Table 2 shows outcomes for relevant
items related to management and
conditions. Patients in the lowoxygen group received signicantly
fewer days of oxygen supplementation (P .01), mechanical ventilation (P .01), and continuous posi-

tive airway pressure treatment (P

.05) and developed less BPD (P
.05), compared with infants in the
high-oxygen group. No differences
regarding rates of neonatal death,
nosocomial infections, patent ductus
arteriosus, grade III/IV intraventricular/periventricular hemorrhage, or
retinopathy of prematurity were
found (Table 2).
Analytical Results
Blood GSSG/GSH Ratio
Figure 3 shows no signicant differences in GSSG/GSH ratios at birth (umbilical cord) between the high-oxygen
group and the low-oxygen group. On

Figure 4A shows no differences in

urinary isoprostane metabolite levels between the groups at either day
1 or day 7. Figure 4B shows signicantly greater elimination of isofurans in the high-oxygen group on
day 1 (P .01) but no signicant differences on day 7.
Urinary o-Tyrosine/Phenylalanine
Ratio
Figure 5A shows increased urinary
elimination of o-tyrosine in the highoxygen group, compared with the lowoxygen group, on day 1 (P .05) and
day 7 (P .05).
8-Hydroxy-2-deoxyguanosine/
2-Deoxyguanosine Ratio in Urine
Figure 5B depicts signicantly increased urinary elimination of oxidized guanosine in the high-oxygen
group on both day 1 (P .01) and day
7 (P .01).
Plasma TNF- Levels
Figure 6A shows serial determinations
of plasma TNF- levels in weeks 1, 2, 3,

TABLE 2 Variables Recorded During Hospitalization and Long-Term Outcomes of ELGANs Resuscitated With Lower (30%) or Higher (90%) Oxygen
Concentrations
Group

Low-oxygen (N 37)
High-oxygen (N 41)

Duration of Oxygen Duration of Duration of CPAP Surfactant

Treatment, Median Mechanical Therapy, Median Treatment,
(IQR), d
Ventilation
(IQR), d
n (%)
Median
(IQR), d
6 (167)
22 (7132)a

13 (657)
27 (1098)a

4 (138)
12 (645)b

13 (36)
13 (33)

BPD, n
(%)

ROP, n Nosocomial
(%) Septicemia,
n (%)

6 (15.4) 4 (10.8)
13 (31.7)b 6 (14.6)

10 (27.0)
9 (23.5)

PDA, n
(%)

IPVH (Grade Neonatal Death

III/IV), n (%) (28 d), n (%)

19 (51.3)
27 (65.8)

7 (18.9)
5 (12.2)

4 (10.8)
3 (7.3)

IQR indicates interquartile range; CPAP, continuous positive airway pressure; ROP, retinopathy of prematurity (stage 2, stage 3, or plus disease)52; PDA, patent ductus arteriosus; IPVH,
intraventricular/periventricular hemorrhage.53 BPD was dened as described.54
a P .01, versus low-oxygen group.
b P .05, versus low-oxygen group.

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Plasma IL-8 Levels

Figure 6B shows IL-8 levels in the rst 5
weeks after birth. IL-8 levels were signicantly higher in the high-oxygen
group in the rst 3 weeks after birth
(P .01) but not thereafter.
Association of Oxidative Stress
Markers With BPD
FIGURE 3

GSSG/GSH ratio 100 in cord blood on days 1 and day 3 in ELGANs resuscitated with initial FIO2 of 90%
(high-oxygen [Hox] group) or 30% (low-oxygen [Lox] group). Mann-Whitney comparisons between
groups were as follows: aP .01, versus low-oxygen group; bP .05, versus low-oxygen group.

and 5 after birth. TNF- levels were

signicantly lower in the low-oxygen
group than in the high-oxygen group at
the end of the rst week of life (high-

oxygen group: 3.47 1.12 pg/mL; lowoxygen group: 2.64 0.94 pg/mL; P
.05), but no differences were found
thereafter.

Figure 7A shows blood GSSG and urinary isofuran levels for neonates who
later developed BPD (n 19) or did not
develop BPD (n 59). Blood GSSG levels on day 3 and urinary isofuran levels
at the end of the rst week were correlated signicantly (P .01) with the
development of BPD. Moreover, the otyrosine/phenylalanine and 8-hydroxy2-deoxyguanosine/2-deoxyguanosine
ratios also were correlated signicantly with later development of BPD
(Fig 7B).

DISCUSSION

FIGURE 4
Urinary levels of isoprostane metabolites (A) and urinary isofurans (B), determined through gas
chromatography/mass spectrometry, in ELGANs resuscitated with an initial FIO2 of 90% (high-oxygen
[Hox] group) or 30% (low-oxygen [Lox] group) at days 1 and 7 after birth. Mann-Whitney comparisons
between groups were as follow: aP .01, versus low-oxygen group.

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VENTO et al

The fetal to neonatal transition is characterized by initiation of breathing and

changes in the cardiopulmonary
circulation, which increases tissue oxygenation abruptly.33 For adequate
postnatal adaptation, both effective
ventilation and establishment of functional residual capacity are essential.
Frequently, ELGANs are incapable of establishing effective respiration and
therefore require positive pressure
ventilation and oxygen treatment in
the DR.7 Of note is that high oxygen and
lung-stretching lead to increased ROS
production and expression of proinammatory cytokines.8 In addition,
prenatal fetal lung colonization with
vaginal ora and/or commensal organisms in ELGANs predisposes them
to lung inammation.36 We hypothesized that use of a high oxygen concentration in the DR would constitute a superimposed factor contributing to
lung inammation and architectural
remodeling and leading to chronic
lung disease. To validate this hypothesis, we launched a clinical trial to com-

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FIGURE 5

Urinary o-tyrosine/phenylalanine (o-Tyr/Phenyl) 1000 ratio (A) and urinary 8-hydroxy-2deoxyguanosine/2-deoxyguanosine 100 ratio (8oxodG/2dG 100) (B), both determined through
high-performance liquid chromatography/tandem mass spectrometry, on days 1 and 7 after birth in
ELGANs resuscitated with an initial FIO2 of 90% (high-oxygen [Hox] group) or 30% (low-oxygen [Lox]
group). Mann-Whitney comparisons between groups were as follows: aP .05, bP .01, versus
low-oxygen group.

pare the clinical and biochemical consequences of using 2 different oxygen

loads during resuscitation. Furthermore, to attenuate the inuence of
lung-stretch damage, we used gentle
ventilation for both groups.31 To date,
only 2 randomized studies have compared different FIO2 values for the resuscitation of ELGANs.24,26 Those studies conrmed that room air was
insufcient for ELGANs to achieve targeted SpO2 values in the DR, whereas
use of FIO2 of 30% was effective, thus
PEDIATRICS Volume 124, Number 3, September 2009

avoiding the need for rescue therapy

with pure oxygen. Our study conrms
the suitability of an initial FIO2 of 30%
and titration of oxygen supplementation according to SpO2 values. Oxygenation of patients with impaired lung
function is an important and lifesaving measure. Although oxygenation relieves the immediate lifethreatening consequences transiently,
it also may exacerbate lung injury.37
Hyperoxia leads to increased production of oxygen free radicals, which di-

rectly cause damage and induce impaired remodeling of the lung

structure through activation of proinammatory cytokines.38,39 Oxygen free
radical-induced TNF receptor activation of pulmonary cells upregulates
inammatory effector gene expression.39 In addition, oxygenation may
weaken or even suppress the adenosine A2A receptor-mediated antiinammatory mechanism and thereby exacerbate lung injury.40 In our study, the
GSSG/GSH ratio, a reliable marker of
intracellular oxidative stress and redox status,41,42 was increased signicantly in the group receiving higher oxygen levels, which conrmed previous
ndings that indicated that high oxygen exposure on reoxygenation increases oxidative stress and prolongs
prooxidant status.14,15,17,18 Moreover,
correlation between oxidative stress
and later development of BPD was
established in this study. Increased otyrosine/phenylalanine and 8-hydroxy2-deoxyguanosine/2-deoxyguanosine
ratios both indicated that neonates receiving high oxygen levels generated
more ROS, especially hydroxyl radicals,
which have been associated with the
pathophysiologic processes of hypoxicischemic encephalopathy.43 Interestingly, in a piglet model of asphyxia, the
rates of urinary elimination of otyrosine/phenylalanine and 8-hydroxy2-deoxyguanosine/2-deoxyguanosine
were correlated signicantly with
the amount of oxygen administered
during resuscitation.27 Increased levels of markers of protein and DNA
oxidation also were correlated with
later development of BPD. We also
measured prostaglandin F2-like compounds, which are considered the
most-reliable markers of lipid peroxidation. Under hyperoxic conditions, these compounds incorporate
an oxygen molecule into their inner
structure, leading to the formation of
isofurans. Isofurans are excellent
markers of hyperoxia-derived lipid

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was a positive correlation with isofuran levels.

FIGURE 6

TNF- (A) and IL-8 (B) levels in plasma, as determined through ow cytometry, during weeks 1, 2, 3, and
5 after birth in ELGANs initially resuscitated with FIO2 of 90% (high-oxygen [Hox] group) or 30%
(low-oxygen [Lox] group). Mann-Whitney comparisons between groups were as follows: aP .05, bP
.01, versus low-oxygen group.

peroxidation, whereas isoprostane

metabolites reect oxidative stress
under normoxic conditions.44,45 Interestingly, although isoprostane metabolite levels were no different in
the 2 groups, isofuran levels were
signicantly higher in the highoxygen group, evidencing hyperoxiaderived oxidative stress during
reoxygenation. It is noteworthy that a
signicant number of patients with
increased levels of isofurans later
developed BPD. Previous studies correlated oxidative stress and development of BPD.46,47 In addition, Ahola et
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VENTO et al

al48 found that, among ELGANs needing ventilatory assistance, those who
later developed BPD had signicantly
higher plasma free 8-epi-prostaglandin
F2 levels on days 3 and 7, compared
with survivors without BPD. Reuter et al49
did not nd such correlation between
BPD and urinary elimination of 8-isoprostaglandin F2 in preterm neonates. Apparently not all isoprostane
metabolites have similar predictive
value regarding the development of
BPD, and we did not nd a correlation
between isoprostane metabolite
elimination and BPD, although there

The present study has several limitations that need to be addressed.

First, this was not a blinded study.
Our group previously studied the potentially harmful effects of pure oxygen resuscitation in term infants and
might have been inuenced in subsequent care by the initial patient assignment. We recorded and analyzed
scrupulously all data and decisions
made in the DR and NICU, and caregivers who were able to observe the
oxygen blender did not make decisions that might have put the patients at risk of hypoxia or hyperoxia
to inuence the randomization assignment. Moreover, caregivers in
the DR did not care for the neonates
during their hospitalizations. Another limitation is the number of patients studied, which might have precluded obtaining more-denitive
data regarding the correlation between initial management in the DR
and subsequent development of BPD.
Although the sample size was powered for 10% reduction of BPD, the
number of patients in the lower gestational age range (26 weeks of
gestation), who more frequently develop BPD, should be increased in future studies. Despite these limitations, our study showed that an
increased oxygen load in the rst few
minutes of life caused oxidative
stress and inammation, which was
correlated with later development of
BPD. Only a few minutes of oxygen excess have been shown to cause longterm oxidative stress in term infants
with mature antioxidant defense systems,14 and even serious conditions
such as cancer.50 It was proposed recently that oxygen serves as a morphogen to promote development by inuencing the production of metabolic
oxidants such as ROS. Therefore, an excess of oxygen for even a short period

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ARTICLES

could trigger structural changes in the

immature lung.51
We conclude that ELGAN resuscitation
should be initiated with lower FIO2
(30%) and titrated according to SpO2
values, to avoid oxidative damage and
inammation. However, there is a need
for randomized, blinded studies that
are powered adequately for evaluation
of the correlation between oxygen levels during resuscitation and development of BPD.

ACKNOWLEDGMENTS
This study was funded by a grant from
the Spanish Ministry of Health (Instituto Carlos III) (grant FIS PI05/1505) to
Dr Vento, a research fellowship from
the Research Foundation Hospital La
Fe to Dr Escrig, a research grant from
Mutua Madrilena to Dr Moro, Consolider grant CSD-2007-00020 to Mr Arduini and Dr Sastre, and National Institutes of Health Grant GM42056 to Dr
Roberts.
FIGURE 7
A, Blood GSSG levels at day 3 after birth and urinary isofuran (Iso F) levels at day 7 after birth in ELGANs
who were resuscitated with an initial FIO2 of either 90% or 30% and later developed BPD or not. B,
Urinary o-tyrosine/phenylalanine 1000 ratio (o-tyr/Phenyl ratio) and urinary 8-hydroxy-2deoxyguanosine/2-deoxyguanosine 100 ratio (8-oxodG/2dG ratio) in ELGANs who were resuscitated
with an initial FIO2 of either 90% or 30% and later developed BPD or not. a P .01.

We express our gratitude to Drs Neil N.

Finer and Tina A. Leone (University of
California, San Diego, CA) for their inestimable help in reviewing and editing this manuscript.

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Preterm Resuscitation With Low Oxygen Causes Less Oxidative Stress,

Inflammation, and Chronic Lung Disease
Maximo Vento, Manuel Moro, Raquel Escrig, Luis Arruza, Gema Villar, Isabel
Izquierdo, L. Jackson Roberts, II, Alessandro Arduini, Justo Javier Escobar, Juan
Sastre and Miguel A. Asensi
Pediatrics 2009;124;e439-e449; originally published online Aug 3, 2009;
DOI: 10.1542/peds.2009-0434
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