Clinical Nutrition 42 (2023) 22e28

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Clinical Nutrition
journal homepage: http://www.elsevier.com/locate/clnu

Randomized Control Trials

Effect of arachidonic and docosahexaenoic acid supplementation on

respiratory outcomes and neonatal morbidities in preterm infants
Kristina Wendel a, c, *, Marlen Fossan Aas a, c, Gunnthorunn Gunnarsdottir b, c,
Madelaine Eloranta Rossholt a, d, Marianne Bratlie a, d, Tone Nordvik a, c,
Erlend Christoffer Sommer Landsend e, Drude Fugelseth a, c, Magnus Domello € f f,
g a, c a, c
Are Hugo Pripp , Tom Stiris , Sissel Jennifer Moltu
a
Department of Neonatal Intensive Care, Oslo University Hospital, Norway
b
Department of Pediatric Neurology, Oslo University Hospital, Norway
c
Institute of Clinical Medicine, University of Oslo, Norway
d
Department of Pediatrics and Adolescence Medicine, Oslo University Hospital, Norway
e
Department of Ophthalmology, Oslo University Hospital, Norway
f
Department of Clinical Sciences, Pediatrics, Umea University, Sweden
g
Oslo Centre of Biostatistics and Epidemiology, Oslo University Hospital, Norway

a r t i c l e i n f o s u m m a r y

Article history: Background & aims: Studies have suggested that supplementation with docosahexaenoic acid (DHA) to
Received 31 May 2022 preterm infants might be associated with an increased risk of bronchopulmonary dysplasia (BPD). Our
Accepted 11 November 2022 aim was to investigate the effect of enteral supplementation with arachidonic acid (ARA) and DHA on
short-term respiratory outcomes and neonatal morbidities in very preterm infants.
Keywords: Methods: This is a secondary analysis of data from the ImNuT (Immature, Nutrition Therapy) study, a
Preterm
randomized double blind clinical trial. Infants with gestational age less than 29 weeks were randomized
Fatty acid supplementation
to receive a daily enteral supplement with ARA 100 mg/kg and DHA 50 mg/kg (intervention) or medium
Arachidonic acid
Docosahexaenoic acid
chain triglycerides (MCT) oil (control), from second day of life to 36 weeks postmenstrual age. Study
Bronchopulmonary dysplasia outcomes included duration of respiratory support, incidence of BPD and other major morbidities
Neonatal morbidities associated with preterm birth.
Results: 120 infants with mean (SD) gestational age 26.4 (1.7) weeks were randomized and allocated to
either the intervention or control group. Supplementation with ARA and DHA led to a significant
reduction in number of days with respiratory support (mean (95% CI) 63.4 (56.6e71.3) vs 80.6 (72.4
e88.8); p ¼ 0.03) and a lower oxygen demand (FiO2) (mean (95% CI) 0.26 (0.25e0.28) vs 0.29 (0.27
e0.30); p ¼ 0.03) compared to control treatment. There were no clinically important differences in
incidence of BPD and other major morbidities between the treatment groups.
Conclusions: Supplementation with ARA and DHA to preterm infants was safe and might have a bene-
ficial effect on respiratory outcomes.
Clinical trial registration: The trial has been registered in www.clinicaltrials.gov, ID: NCT03555019.
© 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).

Abbreviations: ARA, Arachidonic acid; BPD, Bronchopulmonary dysplasia; CPAP, continuous positive airway pressure; CRIB, Clinical Risk Index for Babies; DHA, Doco-
sahexaenoic acid; DMC, Data Monitoring Committee; FA, Fatty acid; GA, Gestational age; HR, Hazard ratio; IVH, Intraventricular hemorrhage; LCPUFA, Long-chain poly-
unsaturated fatty acid; LOS, Late-onset septicemia; MRI, Magnetic Resonance Imaging; MCT, Medium chain triglycerides; MD, Mean difference; NEC, Necrotizing
enterocolitis; PDA, Patent ductus arteriosus; PMA, Postmenstrual age; PVL, Periventricular leukomalacia; ROP, Retinopathy of prematurity; RR, Respiratory rate or Risk ratio;
TPTEF/Te ratio, Time to peak tidal expiratory flow as a ratio of expiratory time; Vt, Tidal volume.
* Corresponding author. Department of Neonatal Intensive Care, Oslo University Hospital, Postboks 4950 Nydalen, 0424 Oslo, Norway.
E-mail addresses: krwend@ous-hf.no, kristinalawendel@gmail.com (K. Wendel).

https://doi.org/10.1016/j.clnu.2022.11.012
0261-5614/© 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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K. Wendel, M.F. Aas, G. Gunnarsdottir et al.
1. Introduction
Bronchopulmonary dysplasia (BPD) is a common complication
of preterm birth, affecting more than 50% of infants born before 28
weeks gestational age (GA) [1]. BPD is mainly caused by arrested
pulmonary development and sustained postnatal inflammation
aggravated by mechanical ventilation and oxidative stress [2,3].
Approaches to reduce postnatal inflammation are numerous, but
the incidence of BPD has remained unchanged over the last decades
[4]. The long-chain polyunsaturated fatty acids (LCPUFAs) arach-
idonic acid (ARA) and docosahexaenoic acid (DHA) are important
structural lipids in the cell membrane, but they also act as signal
molecules attenuating the inflammatory cascade [5]. ARA and DHA
mainly influence the inflammatory process through the formation
of specialized pro-resolving lipid mediators; lipoxins derived from
ARA and protectins, maresins and d-series resolvins derived from
DHA [6,7]. DHA has additional immunomodulatory properties,
including an inhibiting effect on the production of pro-
inflammatory cytokines [8]. Fetal plasma concentrations of ARA
are physiologically high at the beginning of the third trimester, with
an ARA:DHA ratio around 3:1 [9]. Infants born before 29 weeks GA
are deprived of the fetal accretion of LCPUFAs occurring during the
third trimester and rapidly become deficient in ARA and DHA after
birth [10]. Postnatally, preterm infants rely on exogenous supply of
ARA and DHA since their capacity of endogenous synthesis from
linoleic acid and alfa-linolenic acid is low [11]. Human breast milk
provides ARA and DHA in a 2:1 ratio, but in insufficient amounts to
prevent a postnatal deficiency [12].
Low postnatal DHA concentrations are associated with an
increased risk of BPD [10]. However, studies on the effect of DHA
supplementation on respiratory outcomes in preterm infants have
shown conflicting results and there is a concern that DHA supple-
mentation may have adverse effects. Although a high-DHA diet
given to lactating mothers of premature infants reduced the inci-
dence of BPD in one study [13], a similar intervention study was
terminated early due to higher rates of BPD in the intervention
group [14]. Higher rates of BPD were also reported in a randomized
controlled trial with enteral DHA supplementation to infants less
than 29 weeks GA [15]. High doses of DHA without concomitant
ARA supplementation are associated with a significant reduction in
ARA blood concentrations and show less beneficial effects on
developmental outcomes in term infants compared to more mod-
erate DHA supplementation [16]. This suggests that a balanced
intake of ARA and DHA is important. Few studies have investigated
the effect of both ARA and DHA on respiratory outcomes and
neonatal morbidities in preterm infants.
The objective of ImNuT (Immature, Nutrition Therapy), a
double blind randomized controlled trial, was to determine the
effect of ARA and DHA supplementation on growth, inflamma-
tion and metabolism in premature infants with a gestational age
less than 29 weeks [17]. This study is a secondary analysis of data
from the ImNuT trial. We hypothesized that a balanced supple-
mentation of ARA and DHA would improve respiratory outcomes
and reduce morbidities associated with preterm birth. Further,
we hypothesized that supplementation with ARA and DHA would
result in increased LCPUFA blood concentrations during
hospitalization.
2. Material & methods
2.1. Study design
The ImNuT study was a double-blind randomized controlled
trial conducted at a tertiary neonatal intensive care unit at Oslo
University Hospital in Norway. The trial design has previously been
23
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Clinical Nutrition 42 (2023) 22e28
published [17]. The primary outcome in the ImNuT study was to
determine the effect of ARA and DHA supplementation on brain
growth and maturation in very preterm infants. Pre-specified sec-
ondary outcomes included respiratory outcomes, incidence of
neonatal morbidities, quality of growth, cardiovascular health and
neuro-development. The Norwegian Regional Ethics Committee
approved the study (2016-003700-31) and a data monitoring
committee (DMC) with access to reported adverse effects and
clinical outcomes reviewed the study for safety and efficacy. We
have followed the Consolidated Standards of Reporting Trials
(CONSORT) guideline.
2.2. Study participants
Infants born at Oslo University Hospital with GA less than 29
weeks were eligible to participate in the study. Exclusion criteria
were congenital malformations, chromosomal abnormalities or
critical illness with short life expectancy. Study participation
required written informed, parental consent within 48 h after
birth.
2.3. Randomization and blinding
We used a computer-generated list of random numbers for
allocation of the participants to one of the two treatment groups.
The block size was 4, 6 or 8, randomly alternated. To decrease the
risk of unbalanced baseline characteristics, we stratified the
randomization by growth status at birth (small for gestational age
or not) with an allocation ratio of 1:1 within each block. In case of
multiple births, the infants were allocated to the same treatment by
randomizing only the first infant. After randomization, the local
investigator contacted the study pharmacist for preparation of
allocated fatty acid intervention. All participants, investigators and
staff, except for the pharmacist were blinded to the fatty acid
assignment.
2.4. Trial intervention
Infants randomized to the ARA:DHA group received a fatty
acid supplement containing ARA and DHA in a 2:1 ratio (For-
mulaid™, DSM Nutritional Products Inc.). Infants randomized to
the control group received medium chain triglycerides (MCT-
oil™, Nutricia) (product details are found in Supplement,
Appendix 1). MCT-oil is currently the standard lipid supple-
ment in our department to enhance enteral energy supply and
does not contain ARA or DHA. Lipid supplementation in a dosage
of 0.2 ml/kg was started on the second day of life and advanced
to target 0.4 ml/kg from day four and onwards, resulting in an
intake of 100 mg/kg of ARA and 50 mg/kg of DHA in the inter-
vention group. The dose was weekly weight-adjusted, but weight
at birth was used until birth weight was regained. The enteral
fatty acid supplementation was administered in the feeding tube
as a daily bolus until 36 weeks postmenstrual age (PMA). Study
participants transferred to local hospitals continued to receive
the fatty acid supplementation. All study participants followed a
standardized nutritional protocol to accommodate international
recommendations [18] (Supplement, eTable 1). The intravenous
solution used for parenteral nutrition (PN) contained a composite
lipid emulsion of soybean and olive oil emulsion (Clinoleic®,
Baxter). This emulsion has a small amount of ARA, but no DHA.
Full enteral nutrition was defined as  150 mL/kg/day of human
milk fortified with PreNAN FM 85® (Nestle ) 3 g per100 mL. The
human milk fortifier contained 1.6 mg DHA per gram and no
ARA.
K. Wendel, M.F. Aas, G. Gunnarsdottir et al.
2.5. Study outcomes
We registered respiratory outcomes from birth up to 40 weeks
PMA, including number of days requiring respiratory support
(mechanical ventilation, continuous positive airway pressure
(CPAP), high flow nasal cannula 3 L/min or oxygen therapy),
mean oxygen demand (FiO2) during respiratory support, and use
of postnatal steroids. BPD was defined by need of respiratory
support or oxygen demand at 36 weeks PMA [19]. The target range
for oxygen saturation during oxygen supplementation was
90e94%.
Lung function was assessed at 36 ± 2 weeks PMA by a tidal
breathing flow-volume test [20]. The test procedure is described in
supplement (Appendix 2). Tidal breathing parameters included in
the analysis were respiratory rate (RR), tidal volume (Vt)/kg, and
time to peak tidal expiratory flow as a ratio of expiratory time
(TPTEF/Te ratio). Infants who needed respiratory support or were
transferred to local hospitals before the time of evaluation were
excluded from the lung function test.
We also assessed the occurrence of neonatal morbidities
including retinopathy of prematurity (ROP), necrotizing enteroco-
litis (NEC), intraventricular hemorrhage (IVH), patent ductus arte-
riosus (PDA) requiring treatment, and late-onset septicemia (LOS).
For detailed definitions, see supplement (Appendix 3). Major
morbidity was defined as the presence of at least one of the
following morbidities: any BPD, severe brain injury (IVH grade 3
or periventricular leukomalacia (PVL)) or ROP stage 3 [21].
We registered the total intake (mg/kg/d) of ARA and DHA from
additional sources (including intravenous PN, human milk, fortifier
and formula) up to 36 weeks PMA [22]. To determine postnatal
blood concentrations of ARA and DHA, we collected whole blood
samples on filter sticks (10 mL) at inclusion and then at day 3, 7, 14,
21, 28, and week 36 PMA. ARA and DHA concentrations were
measured as separate fatty acids in dry blood spots; the analytic
method is described in supplement (Appendix 4).
Fig. 1. Study flow diagram.
24
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Clinical Nutrition 42 (2023) 22e28
2.6. Statistical methods
The sample size calculation was based on the primary
outcome in the ImNuT trial; 120 infants would provide 80% po-
wer to detect a 0.04 difference in mean diffusivity (mm2/sec) in
major white matter tracts on magnetic resonance imaging (MRI)
at term equivalent age. We performed efficacy analyses on the
intention-to-treat (ITT) population and for pre-specified study
outcomes on infants who survived up to 36 weeks PMA. Cumu-
lative days of respiratory support was described by using the
Kaplan-Meyer technique and tested with a log-rank test. Dropout
and death were considered as non-events and censored at the
time of discontinuation. For analysis of days with mechanical
ventilation, we used quantile regression since the data was not
normally distributed. Other normally distributed continuous
outcomes were analyzed by an independent t-test. The effect size
for dichotomous outcomes was estimated by risk ratio using a
chi-square test or Fishers exact test as appropriate. For the
analysis of ARA and DHA blood concentrations, we used mixed
models for repeated measures with a subject-specific random
intercept. Day 3 was used as baseline, whereas fixed effects were
fatty acid blood concentrations at day 3, treatment group, time
and the interaction between group and time. All results are
presented un-adjusted. To adjust for baseline imbalances, a post-
hoc sensitivity analysis was performed using cox regression and
multiple regression. For all analyses, we considered a p-value of
<0.05 as statistically significant. SPSS version 26.0 and STATA
statistical software were used in the statistical analyses.
3. Results
3.1. Study participants
From April 2018 to January 2021, a total of 121 infants born before
GA 29 weeks were randomized. Sixty-one infants were allocated to
K. Wendel, M.F. Aas, G. Gunnarsdottir et al. Clinical Nutrition 42 (2023) 22e28

the control group and 60 infants to the ARA:DHA group. One patient 3.2. Respiratory outcomes
randomized to the control group was later diagnosed with chro-
mosomal abnormality and therefore excluded from the trial. The ITT Infants in the ARA:DHA group had significantly fewer days with
population included 120 infants (Fig. 1). Neonatal outcome data at 36 respiratory support (63.4 vs 80.6 days; p ¼ 0.03) and a lower mean
weeks PMA was available for 110 of the study participants. Ninety oxygen demand (FiO2 0.27 vs. 0.29; p ¼ 0.03), compared to the
four percent of ordered fatty acid doses were given to the ARA:DHA control group. There was no significant differences in incidence of
group and 92.6% to the control group. One patient in the ARA:DHA BPD, need of respiratory support after 40 weeks PMA or use of
group received 38% of planned doses due to complicated NEC. postnatal steroids. The tidal breathing test at 36 weeks PMA
Baseline characteristics of the study population were similar in the showed that infants in the ARA:DHA group had significantly higher
treatment groups, except for number of infants who had received tidal volumes than the control group (6.26 vs. 5.17 ml/kg; p ¼ 0.02),
two doses of antenatal steroids and Apgar score at 5 min (Table 1). but respiratory rate and TPTEF/Te ratio did not differ (Table 2).
Adjustments for baseline imbalances (number of doses of antenatal
steroids and 5 min Apgar score) in post-hoc sensitivity analysis,
Table 1 resulted in no longer significant differences in days with respiratory
Baseline characteristics (Intention-to-Treat Population). support (hazard ratio (95% CI); 0.72 (0.46e1.13), p ¼ 0.15) and ox-
ygen demand (FiO2 mean difference (MD) (95% CI); 0.016 (0.007-
Control ARA:DHA
(n ¼ 60) (n ¼ 60) 0.40), p ¼ 0.17) between the treatment groups. The difference in
tidal volume remained significant after adjustments (MD (95% CI)
Mothers
Antenatal glucocorticoids any dose, n 60 (100) 60 (100)
ml/kg; 0.99 (0.05e1.93), p ¼ 0.04).
(%)
Antenatal glucocorticoids 2 doses, n (%) 35 (58.3) 48 (80.0)
Cesarean delivery, n (%) 38 (63.3) 29 (48.3) 3.3. Neonatal morbidities
Infants
Gestational age, weeks There were no clinically important differences in incidence of
Mean (SD) 26.2 (1.6) 26.6 (1.7) ROP, NEC, IVH, PDA requiring treatment, or late-onset-septicemia
Median (range) 26.7 (23.0e28.9) 27.0 (22.9e28.9)
Birth weight, g
between the study groups. There was no significant difference in
Mean (SD) 833 (255) 879 (241) number of infants diagnosed with one or more major morbidities,
Median (range) 763 (470e1485) 824 (444e1370) 26 infants in the ARA:DHA group compared to 32 infants in the
Small for gestational age, n (%) 12 (20.0) 11 (18.3) control group. None of the study participants suffered from three
Male sex, n (%) 31 (51.7) 35 (58.3)
major morbidities (Table 3).
Plurality, n (%)
Single 47 (78.3) 44 (73.3)
Twin 10 (16.7) 16 (26.7)
Triplet 3 (5) e
3.4. Intake estimates and blood concentrations of ARA and DHA
Apgar score at 5 min, median (IQR) 7 (6e8) 8 (7e9)
Mechanically ventilated within first 42 (70.0) 35 (58.3) The mean (SD) mg/kg/d intake of ARA was 110.6 (9.4) in the
48 h of life, n (%) ARA:DHA group and 23.8 (1.6) in the control group, while the mean
Surfactant administration, n (%) 56 (93.3) 51 (85.0)
(SD) mg/kg/d intake of DHA was 63.0 (5.7) in the ARA:DHA group
CRIB score, median (IQR) 7 (2e10) 5 (2e9)
Whole blood ARA concentration at 13.0 (3.5) 13.4 (4.5) and 18.4 (2.2) in the control group. The overall ARA concentrations
inclusion mol%, mean (SD) from baseline to 36 weeks PMA did not differ significantly between
Whole blood DHA concentrations at 2.6 (0.8) 2.5 (0.8) the treatment groups, but the DHA blood concentrations was
inclusion mol%, mean (SD) significantly higher in the ARA:DHA group compared to the control
ARA, arachidonic acid; CRIB, Clinical Risk Index for Babies; DHA, docosahexaenoic group with a MD (95% CI) of 0.35 (0.09e0.61) mol%, p ¼ 0.01 at 36
acid; IQR, interquartile range; SD, standard deviation. weeks PMA (Fig. 2).

Table 2
Respiratory outcomes.

Respiratory outcomes Control group (n ¼ 60) ARA:DHA group (n ¼ 60) Comparison: ARA:DHA vs p-value
control group (95%CI)

BPDa, No./total (%) 30/55 (54.5) 23/55 (41.8) RR, 0.77 (0.52e1.14) 0.18
Mild BPD 7/55 (12.7) 6/55 (10.9) RR, 0.86 (0.31e2.39) 0.78
Moderate BPD 14/55 (25.5) 11/55 (20.0) RR, 0.79 (0.39e1.58) 0.49
Severe BPD 9/55 (16.4) 6/55 (10.9) RR, 0.67 (0.25e1.75) 0.41
Days with mechanical ventilation, median (IQR) 8 (1e24) 3 (0e18) MD, 5 (2e12) 0.15
Cumulative days with respiratory supportb, mean (95% CI) 80.6 (72.4e88.8) 63.4 (56.6e71.3) HR, 0.62 (0.40e0.95) 0.03
Oxygen demand (fiO2)c, mean (95% CI) 0.29 (0.27e0.30) 0.26 (0.25e0.28) MD, 0.024 (0.002e0.047) 0.03
Need of respiratory support > 40 weeks PMAd, No./total (%) 17/55 (30.9) 9/55 (16.3) RR, 0.53 (0.26e1.08) 0.07
Postnatal steroids, No./total (%) 28/60 (46.7) 21/60 (35.0) RR, 0.75 (0.48e1.16) 0.19
Tidal breathing parameters at 36 weeks PMAe, No./total (%) 24/60 (40.0) 27/60 (45.0)
Tidal volume (ml/kg), mean (95% CI) 5.17 (4.50e5.83) 6.26 (5.64e6.88) MD, 1.09 (0.21e1.98) 0.02
Respiratory rate (RR/min), mean (95% CI) 77 (70e83) 74 (67e81) MD, 3 (6e13) 0.5
TPTEF/Te ratio, mean (95% CI) 0.35 (0.32e0.39) 0.35 (0.31e0.40) MD, 0.00 (0.05-0.06) 0.93

Abbrevations: ARA, arachidonic acid: DHA, docosahexaenoic acid; BPD, bronchopulmonary dysplasia; PMA, post menstrual age; RR, relative risk; HR, hazard ratio; MD, mean
difference; IQR, interquartile range; TPTEF/Te ratio, time to peak tidal expiratory flow as a ratio of expiratory time.
a
Data are missing for patients who died or withdrawn before 36 weeks PMA.
b
Mechanical ventilation, nCPAP, HFNC 3 L/min or low-flow oxygen.
c
Oxygen demand during need of respiratory support.
d
Data are missing for patients who died or withdrawn before 40 weeks PMA.
e
Data are missing for infants who died, withdrawn, were transferred to local hospitals or needed respiratory support at 36 weeks PMA.

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Table 3
Neonatal morbidities.

Outcomes Control group No./total (%) ARA:DHA group No./total (%) Comparison ARA:DHA vs. p-value
control group Relative risk (95% CI)

ROPa
No ROP 32/55 (58.2) 38/55 (69.1) 1.18 (0.89e1.58) 0.23
Stage  3 7/55 (12.7) 3/55 (5.4) 0.43 (0.12e1.57) 0.32
NEC 2/60 (3.3) 2/60 (3.3) 1.0 (0.37e2.71) 1.0
IVH
No IVH 48/60 (80.0) 47/60 (78.3) 0.98 (0.82e1.18) 0.82
Grade III-IV 4/60 (6.7) 7/60 (11.7) 1.75 (0.54e5.67) 0.53
PDA requiring treatmentb 27/58 (46.6) 24/57 (42.1) 0.90 (0.60e1.36) 0.63
Late-onset-sepsisc 27/57 (47.4) 23/57 (40.4) 0.85 (0.56e1.29) 0.45
Major morbidity in survivors 32/55 (55.2) 26/55 (44.8) 0.81 (0.57e1.16) 0.25
at 36 weeks PMAd

Abbrevations: ROP, retinopathy of prematurity; NEC, necrotizing enterocolitis; IVH, intraventricular hemorrhage; PDA, patent ductus arteriosus; PVL, periventricular leu-
komalacia; PMA, postmenstrual age.
a
Data are missing for infants who died or withdrawn before first ROP-screening.
b
Data are missing for infants who died or withdrawn before first echocardiography at 3e5 days after birth.
c
Infants who died or withdrawn before 72 h were not included in the analysis.
d
Major morbidity included bronchopulmonary dysplasia, severe brain injury (IVH grade III-IV or cystic PVL) or ROP stage 3.

Fig. 2. Postnatal blood concentrations (mol%) of arachidonic acid (ARA) and docosahexaenoic acid (DHA) in the intervention (ARA:DHA) group and control group (Intention-To-
Treat population).

3.5. Safety mainly evaluated the effect of DHA supplementation alone, we

There were similar numbers of reported adverse events in the
ARA:DHA and control group (Supplement, eTable 2). Serious
adverse events occurred in 13.3% of infants in the ARA:DHA group
and 8.3% in the control group (Supplement, eTable 3). A total of five
study participants died during the study period, 3 (5.0%) in the
ARA:DHA group and 2 (3.2%) infants in the control group. The in-
dividual causes of death are described in Supplement (eTable 4).
4. Discussion
4.1. Respiratory outcomes
This secondary analysis of a randomized, double-blind
controlled trial showed that enteral supplementation with ARA
and DHA to preterm infants was safe and possibly associated with
improved respiratory outcomes compared to control treatment.
While earlier studies on respiratory outcomes in preterm infants
provided a daily dose of fatty acid supplementation containing
100 mg/kg ARA and 50 mg/kg DHA. In a large RCT, Collins et al.
found an increased risk of BPD after enteral supplementation with
60 mg/kg of DHA to infants less than 29 weeks GA, compared to
supplementation with a soy emulsion [15]. Similarly, an observa-
tional study of 477 infants with birthweight <1250 g reported a
lower oxygen saturation to FiO2 ratio at 36 weeks PMA after
administration of intravenous lipid emulsions containing DHA
compared to lipid emulsions without DHA [23]. In contrast to these
studies, we found that enteral supplementation with ARA and DHA
in a 2:1 ratio resulted in reduced duration of respiratory support
and lower oxygen demand during hospitalization. The importance
of a balanced intake of ARA and DHA is supported by the study of
Bernhard et al., which found that a low postnatal ARA:DHA ratio in
blood samples of extremely preterm infants was associated with a
higher risk of developing BPD [24]. The possible role of ARA in
preventing lung injury may be explained by its capacity to initiate
resolution of the inflammatory cascade, mainly mediated by ARA-

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K. Wendel, M.F. Aas, G. Gunnarsdottir et al.
derived lipoxins [25]. In a murine model of BPD, injection of Lipoxin
A4 led to reduced alveolar simplification and normalized septal
thickness after exposure to hyperoxia [26].
4.2. Adjustments of baseline imbalances
After adjustments for baseline imbalances (number of doses of
antenatal steroids and 5 min Apgar score), the differences in days
with respiratory support and oxygen demand between the treat-
ment groups were no longer significant. Apgar score at 5 min
explained most of the variance, while number of doses of antenatal
steroids did not change the validity of the results. The prognostic
value of Apgar score on neonatal outcomes in extremely preterm
infants has shown conflicting results [27e29]. Of interest, the
Clinical Risk Index for Babies (CRIB) score, a widely used tool for
prediction of mortality and morbidity in very low birth weight
infants [30], did not differ significantly between the ARA:DHA and
control group.
4.3. Tidal breathing parameters
The tidal breathing test was performed in infants with no need
of respiratory support at 36 ± 2 weeks PMA and consequently did
not include infants with moderate and severe BPD. The clinical
importance of the observed difference in tidal volumes between
the treatment groups is uncertain, since earlier studies on lung
function in premature infants are contradictory regarding the cor-
relation between tidal volumes at term and development of chronic
lung disease [31,32].
4.4. Neonatal morbidities
Low postnatal blood concentrations of ARA are not only asso-
ciated with the development of BPD, but also with an increased risk
of late-onset septicemia [10] and severe ROP [33]. Several studies
have indicated that supplementation with DHA reduced the risk of
ROP in premature infants [34e37]. Hellstrom et al. recently
confirmed this in a multi-center RCT of 207 infants with GA< 28
weeks. They reported a 50% reduction in severe ROP in infants
randomized to enteral supplementation with 100 mg/kg ARA and
50 mg/kg DHA, compared to standard treatment [38]. In our study,
5.4% in the ARA:DHA group and 12.7% in the control group were
diagnosed with ROP Stage 3. The difference was however not
statistically significant, probably due to the low number of infants
with ROP Stage 3 in the study population.
4.5. ARA and DHA blood concentrations
The DHA concentrations increased from third week of life in the
ARA:DHA group and became significantly higher compared to the
control group at the end of the study period, but interestingly the
change in ARA concentrations over time did not differ between the
treatment groups. This is in contrast to two similar RCTs that
showed a significant increase in blood concentrations of both ARA
and DHA after enteral supplementation to extremely preterm in-
fants [38,39]. The knowledge about uptake and distribution of ARA
and DHA in extremely preterm infants is scarce. Multiple factors
may explain the relatively low LCPUFA blood concentrations in the
ARA:DHA group, such as adherence of the fatty acid supplement to
the feeding tube, impaired intestinal absorption related to the use
of a non-emulsified supplementation, the unstructured timing of
blood sampling in relation to fatty acid administration, or the high
number of blood transfusions. An antagonistic regulation of ARA
and DHA, in which DHA supplementation alone suppresses ARA
concentrations, seems to be a factor of importance [40]. Makrides
27
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Clinical Nutrition 42 (2023) 22e28
et al. demonstrated that feeding term infants with formula con-
taining DHA and not ARA, resulted in lower concentration of ARA in
erythrocytes compared with a control formula without DHA [41].
Dietary supplementation with ARA and DHA in a 2:1 ratio to pre-
term baboons improved DHA concentrations but not ARA concen-
trations in brain tissue [42], indicating that ARA concentrations are
tighter regulated than DHA. This might be explained by the role of
ARA as a precursor of potent mediators in intracellular signaling,
implying that the concentration of free ARA must be maintained at
precise levels within the cell [43].
4.6. Limitations and strengths
This study has limitations. The sample size calculation of the
trial was based on the primary outcome, thus the study was not
powered to detect differences in respiratory outcomes and
neonatal morbidities. The relative low number of study participants
affected the robustness of the results. The major strength is that our
study was a double-blind randomized controlled trial, which
means that investigators, caregivers and parents were unaware of
treatment assignment.
5. Conclusions
Enteral supplementation with 100 mg/kg ARA and 50 mg/kg
DHA to preterm infants born before 29 weeks of gestation was safe
and may have beneficial effects on short-term respiratory out-
comes. Further studies are needed to confirm these findings.
Funding statement
The study was sponsored by funds from the Research Council of
Norway, with additional contribution from the public foundations
the South-Eastern Norway Regional Health Authority (Helse Sør-
Øst RHF) and Barnestiftelsen, Oslo University Hospital. DSM
Nutritional Products Inc. sponsored the investigational nutrition
product, Formulaid™. This was an investigator-initiated study. The
DSM Nutritional Products Inc. had no role in the design or conduct
of the study.
Author contributions
Dr Wendel designed the study, carried out the initial analyses,
drafted the initial manuscript, and reviewed and revised the
manuscript. Dr Fossan Aas, Dr Gunnarsdottir and Dr Nordvik
included study participants, collected data and reviewed the
manuscript. Rossholt and Bratlie collected nutritional data, fol-
lowed up study participants transferred to local hospitals and
reviewed the manuscript. Dr Sommer Landsend participated in
screening of retinopathy of prematurity, collected data and
reviewed the manuscript. Pripp supervised and conducted statis-
tical analysis, and critically reviewed the manuscript. Prof Fugelseth
diagnosed the patency of ductus arteriosus with Doppler echocar-
diography and critically reviewed the manuscript for important
intellectual content. Prof Domello €f and Prof Stiris made important
contributions to the study design and critically reviewed the
manuscript. Dr Moltu conceptualized and designed the study, co-
ordinated and supervised data collection, and critically reviewed
and revised the manuscript. All authors approved the final manu-
script as submitted and agreed to be accountable for all aspects of
the work.
Conflicts of interest
The authors have no conflicts of interest to disclose.
K. Wendel, M.F. Aas, G. Gunnarsdottir et al.
Acknowledgement
We would like to thank the Data Monitoring Committee mem-
bers: Inge Christoffer Olsen, Ketil Stordal and Trond Markestad; Dr
Helene Caroline Dale Osterholt for conducting Doppler echocardi-
ography; Cathrine Nygaard Espeland for supervising the study; the
neonatal nurses and doctors at Oslo University Hospital for their
support; and, most importantly the participating families for their
commitment to the study.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.clnu.2022.11.012.
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