The Role of Long-Chain Polyunsaturated Fatty Acids in Very Preterm Nutrition

Abstract

Infants born very preterm miss out on the in utero transfer of the omega-3 and omega-6 long-chain polyunsaturated fatty acids that occurs during the third trimester. A number of studies have explored the impact of increasing the enteral intakes of omega-3 +/- omega-6 long-chain polyunsaturated fatty acids to match fetal accretion rates in such infants. These studies have shown early transient improvements in vision and development with both strategies, but with the use of omega-3 supplementation alone appearing to increase the incidence of bronchopulmonary dysplasia. A recent study of omega-3 + omega-6 supplementation demonstrated a significant reduction in the incidence of severe retinopathy of prematurity in a high-risk population, without apparent adverse effects; a larger study is needed to confirm the observed benefits, to assess safety, and to determine long-term developmental outcomes of this strategy.

Introduction

The omega-3 (n-3) long-chain polyunsaturated fatty acids (LCPUFAs) eicosa-pentaenoic acid and docosahexaenoic acid (DHA) together with the related omega-6 (n-6) LCPUFA arachidonic acid (AA) are felt to play important roles with respect to brain development and function (particularly vision), immune modulation, and the initiation and resolution of inflammation. Recognition of the fact that current feeding practices in very preterm (VPT) infants (<32 weeks' gestation), using either formula or breast milk, fail to meet the intrauterine ac-cretion rates of LCPUFAs has prompted intense interest over the past 10-15 years as to whether LCPUFA supplementation aimed to match these accretion rates can improve neurodevelopmental outcomes and perhaps even favorably modify the diverse inflammatory morbidities of prematurity such as bronchopulmonary dysplasia (BPD), retinopathy of prematurity (ROP), necrotizing enterocolitis (NEC), and late-onset sepsis (LOS). This chapter reviews the results of randomized controlled trials that have addressed this issue.

Fats and Fatty Acids

Breast milk is the ideal food for infants. It has a high fat content which includes the essential n-3 and n-6 LCUPFA precursors, alpha linolenic acid (ALA, n-3), and linoleic acid (LA, n-6), respectively, as well as LCPUFAs such as DHA and AA. Breast milk DHA levels are largely determined by maternal diet, averaging 0.2-0.3% in mothers consuming a typical western diet, but increasing to >1% in populations with a high intake of fatty fish or DHA alone [1, 2]; on the other hand, breast milk AA levels are largely independent of maternal diet and average 0.4-0.5% of breast milk fats [1]. Although the essential fatty acids ALA and LA are present in breast milk and formula, and can be converted endogenously to their respective LCPUFAs, this process is thought to be limited in preterm infants, with the LCPUFAs thought to be conditionally essential.

The third trimester of pregnancy is a period of rapid growth, during which the fetal accretion rate of DHA via the placenta is estimated to be ~45 mg/kg/ day and that of AA to be ~210 mg/kg/day [3]. Babies born very preterm miss out on the full benefit of this transplacental supply and have markedly lower levels of both DHA and AA in the week following birth [4], with the levels being directly related to gestational age, and with the levels decreasing further during the postnatal period [5, 6]. In this regard, lower levels of DHA following birth in VPT infants have been associated with BPD [5], while low levels of AA have been associated with LOS [5] and with ROP [6].

Recognition of the importance of the omega-3 fats, particularly in relation to retinal function, prompted studies of DHA supplementation in formula-fed term and preterm infants through the 1990s and early 2000s. These studies, which tended to involve more mature preterm infants and to exclude those with the typical problems of prematurity, supported beneficial effects with respect to visual function and neurodevelopment in both term and preterm formula-fed infants [7, 8]. Based on these studies, infant formulas were supplemented with DHA to match the average DHA content of western breast milk (~0.3% fats) beyond the early 2000s. Note that this level of supplementation results in a daily intake of DHA of ~ 15—20 mg/kg/day at a milk intake of 150-160 mL/kg/day); the comparable daily intake of AA based on a breast milk AA content of ~0.4- 0.5% would be ~23-28 mg/kg/day. Obviously, these intake levels of both DHA and AA fall well short of their respective in utero accretion rates and likely explain the decrease in levels seen in VPT infants following birth and beyond.

Studies in VPT Infants Fed to Match in utero Accretion Rates of DHA +/- AA

Over the past 10-15 years, a number of randomized controlled trials in VPT infants have explored the impact of increasing the DHA +/- AA intake to approximate the in utero accretion rates seen in the third trimester. Outcomes of interest have included neurodevelopment and a number of the common morbidities seen in this population. These trials have included babies representative of the usual clinical profile for this group and have accommodated supplementation of both breast and formula-fed infants.

Henriksen et al. [9] randomized 141 infants born weighing <1,500 g to receive either mother's own or donor breast milk supplemented with 32 mg DHA and 31 mg AA per 100 mL compared to no supplementation. The intervention was started at 1 week after birth and continued until hospital discharge, with the primary outcome, cognitive development, being assessed at 6 months corrected age (CA) using the Ages and Stages Questionnaire. Subsequently, babies in the study had cognitive function tested at 20 months [10] and at 8 years of age [11]. In our DHA for Improvement of Neurodevelopmental Outcome (DINO) trial, we randomized 657 infants born <33 weeks' gestation to receive breast milk from their mothers who were supplemented with either 800 mg DHA per day (breast milk DHA ~ 1% of milk fats) or soy oil (breast milk DHA ~0.3% of milk fats); appropriate formula (1% DHA vs. 0.3% DHA, with no change in AA concentration) was available for use if mothers were unable to provide adequate amounts of breast milk [12]. The primary outcome, neurodevelopment, was assessed at 18 months [12] and at 7 years CA [13], with subgroups of children assessed for various functions at 4 months [14], 2 and 3-5 years [15], and at 7 years CA [16]. Moltu et al. [17] randomized 50 babies to milk supplemented to provide ~ 60 mg/kg/day DHA per day. However, this was part of a multicomponent supplementation strategy to increase energy, protein, vitamin A, and DHA intakes to reduce postnatal growth failure in infants weighing <1,500 g at birth; as such, it is difficult to tease out the effects, if any, of DHA alone in this study.

Our large N-3 Fatty Acids for Improvement in Respiratory Outcomes (N3RO) trial was specifically designed to determine the effect of DHA on BPD in infants born at <29 weeks' gestation [18]. The trial was designed to explore an observed secondary outcome of a reduction in the incidence of BPD (defined as an oxygen requirement at 36 weeks postmenstrual age; PMA) in boys (RR 0.67, 95% CI 0.47-0.96) and in infants with birth weight <1,250 g (RR 0.75, 95% CI 0.57-0.98) in the DINO study [19]. N3RO enrolled 1,273 infants and randomized them to receive either 60 mg/kg DHA per day via an enteral emulsion (n = 631) or a control (soy) emulsion without DHA (n = 642) from within 3 days of their first enteral feed and continuing up until 36 weeks' PMA or discharge home, whichever occurred first. The primary outcome was a diagnosis of physiological BPD at 36 weeks' PMA, as determined on the basis for supplemental oxygen or respiratory support with an assessment of oxygen saturation [20]. The parallel large Canadian study of Marc et al. [21], the Maternal Omega-3 Supplementation to Reduce Bronchopulmonary Dysplasia in Very Preterm Infants (MOBYDIck) trial, randomized breastfed babies <29 weeks' gestation to receive milk from their mothers who took supplements providing either 1,200 mg DHA per day or a 1:1 blend of soy and corn oils. The primary outcome was BPD-free survival at 36 weeks' PMA, with the diagnosis of BPD as determined in N3RO with the additional requirement for oxygen for at least 28 days. The MOBYDIck trial was terminated early, at 60% enrolment (high DHA n = 268; standard DHA n = 255), following publication of the N3RO results and an interim analysis of the MOBYDIck trial.

Two smaller Mexican studies of Bernabe-Garcia et al. [22, 23], explored the impact of DHA supplementation at 75 mg/kg/day compared to a sunflower oil control, given from day 1 of enteral feeding for 14 days, to babies with a birth weight of 1,000-1,499 g. In their first trial, 110 babies were randomized with a primary outcome of ROP incidence and severity [22], while in a later trial, 210 babies were randomized with a primary outcome of confirmed NEC [23]. Finally, the recent Mega Donna Mega study from the Swedish team of Hellstrom et al. [24] randomized 206 infants <28 weeks' gestation to receive either an enteral oil supplement containing 100 mg AA and 50 mg DHA/kg/day or no supplement from within 3 days of birth up until term, with the primary outcome being severe ROP defined as stage 3 and/or type 1 ROP.

Outcomes

Effect on Neurodevelopment and Visual Development

In the studies assessing neurodevelopmental and/or visual outcomes, some benefit of supplementation was seen in infancy, but not beyond 2 years. Thus, at 6 months of age (corrected for prematurity; CA), with 75% follow-up, the DHA/ AA-supplemented infants of Henriksen et al. [9] showed better problem solving, while at 20 months, with a 65% follow-up, a significant improvement in attention was reported [10]; however, by 7-8 years of age, with 70% follow-up, no differences in cognitive outcomes were seen [11].

In the DINO study [12], although no difference overall was found in devel-opmental quotient (DQ) scores at 18 months CA (follow-up 93%), less babies in the high DHA group had significant cognitive delay (DQ <70) (RR 0.50; 95% CI 0.26-0.93). In addition, girls randomized to the high DHA group had higher scores compared to controls (MD 4.5, 95% CI 0.5-8.5), and were less likely to have mild (DQ <85; RR 0.43, 95% CI 0.23-0.80) and significant cognitive delay (DQ <70; RR 0.17, 95% CI 0.04-0.72). In infants born <1,250 g, the incidence of mild cognitive delay was reduced in the high DHA group (RR 0.57, 95% CI 0.36-0.91). In exploratory post hoc analyses of the effect of maternal education and occupation, children in the high-dose DHA group whose mothers did not complete secondary education had significantly higher DQ scores (MD 5.3, SE 2.7; p = 0.047), as did those whose mothers were in trade, semi- or unskilled occupations (MD 5.6, SE 2.5; p = 0.02) [25].

In a subgroup of the DINO infants, there was no impact of high DHA on lan-guage development assessed at 26 months or behavior at 3-5 years CA [15]. Vi-sual acuity in this group was better at 4 months CA [14], but in a different group of the DINO cohort, no long-term visual benefit was seen at 7 years [16]. By 7 years' CA, any early effect of high-dose DHA had washed out, though girls had poorer measures of executive function and behavior as reported by parents, but not as determined by objective psychological testing [13]. Inasmuch as there were only 200 babies <29 weeks' gestation in the DINO trial, the results of the neurodevelopmental follow-up of babies in the N3RO trial (at 5 years) and the MOBYDIck trial (at 2 years), will provide valuable information regarding the effect of high-dose DHA on cognitive function in those most at risk; these results are pending. Note however that in a small subgroup of the N3RO trial, assessment of attention at 18 months CA did not show any benefit attributable to high- dose DHA [26].

Effect on Mortality and Conditions of Prematurity

Mortality

There was no difference in mortality in any of the studies.

Growth

Although the Moltu study showed less growth failure in the group supplemented with DHA, the group was also supplemented with higher calories and protein, which likely explains the effect [27]. There was no effect of high-dose DHA (= /- AA) on growth up to 36 or 40 weeks' postmenstrual age in any of the studies that addressed this issue. With respect to growth beyond the neonatal period, babies in the high DHA group in the DINO study were significantly longer at 18 months corrected (82.8 cm SD 5.2; 81.7 SD 4.7, ratio of means 0.9 cm, 95% CI 0.2-1.2 cm), though with no effect on weight or head circumference [25]. In addition, those babies <1,250 g birth weight in the high DHA arm of the DINO study were longer at 4 months, heavier and longer at 12 and 18 months, and had a greater rate of head growth to 18 months CA compared to those with birth weight <1,250 g in the control group. Note however that the difference in head growth was small (0.017 cm/week, 95% CI 0.003-0.03 cm) [25]. By 7 years, there were no differences in any measure of growth or body composition between groups in the DINO study [13].
Bronchopulmonary Dysplasia
Although the DINO trial had suggested a reduction in BPD with an increased intake of DHA, the results of the N3RO study showed instead that high-dose DHA increased the incidence of physiological BPD in babies <29 weeks' gestation (RR 1.13, 95% CI 1.02-1.25, p = 0.02) [18]. Moreover, a similar finding was seen in the MOBYDIck study (RR 1.36; 95% CI 1.07-1.73, p = 0.01) [20], though no difference in the incidence of BPD was seen in the other, smaller studies, involving high-dose DHA (+/- AA). The mechanism via which high dose DHA may increase the risk of BPD is unclear, though studies in a neonatal murine model of BPD suggest that both the DHA metabolite, resolvin D1 and the AA metabolite, lipoxin A4 are beneficial in ameliorating lung injury [28]. Inasmuch as high-dose DHA supplementation results in a modest decrease in AA levels [18], it is possible that this limits the availability of AA metabolites, thus decreasing any benefit attributable to DHA. A pending analysis of LCPUFA metabolites from the N3RO study may help to clarify this issue.

Retinopathy of Prematurity

The Mega Donna Mega study was designed with severe ROP (defined as either stage 3 or zone 1 ROP) as its primary outcome [24] based on knowledge that the DHA metabolite resolvin D1 inhibits neovascularization in a neonatal mouse model of ROP [29] and an association between low levels of AA in VPT and ROP [6]. The study showed a significant reduction in the incidence of this serious eye problem - RR 0.50; 95% CI 0.28-0.91, p = 0.02. High-dose DHA (+/- AA) had no impact on ROP in any of the other studies, apart from a lower rate of diagnosed grade 3 ROP with DHA supplementation reported in the study of Bernabe-Garcia et al. [22], though with no difference in the incidence of ROP overall and no difference in the need for intervention.

Other Conditions of Prematurity

There was no difference in the reported incidence of NEC in any of the studies apart from the 2021 study of Bernabe-Garcia et al. [23] in which high DHA sup-plementation for 2 weeks was associated with a reduction in the risk of confirmed NEC in babies with birth weight 1,000-1,499 g (RR 0.93; 95% CI 0.88-0.98). It is difficult to reconcile these results, in a relatively low risk population, to those from studies with much larger numbers of smaller and more immature babies. With respect to severe intraventricular hemorrhage (IVH) (defined as grade 3 or 4), high-dose DHA in the MOBYDIck trial was associated with a lower risk of severe IVH (RR 0.48; 95% CI 0.29-0.08) [21]; however, this is likely a chance observation, given that severe IVH typically has its onset in the first 3-5 days, yet the trial intervention was only just starting at this time in the study. There was no evidence of an impact of high DHA (+/- AA) on IVH in any of the other studies. Finally, there was an increase in the rate of LOS in the DHA- supplemented group in the study by Moltu et al. [17], though the authors attributed this to the higher dose of amino acids in this group. There was no difference in the incidence of LOS in any of the other studies in which it was recorded.

Implications for Clinical Practice and Future Research

Based on this review of studies aimed at providing DHA (+/- AA) intakes to VPT infants that approximate calculated in utero accretion rates, there appears to be consistent evidence of early developmental benefits, though with these not evident beyond 2-3 years of age and with the suggestion of an adverse impact of high DHA exposure on executive function in girls at 7 years of age. Long-term developmental follow-up, particularly of the large N3RO and MOBYDIck studies, may help to clarify this issue. High intakes of DHA alone appear to increase the risk of BPD, though do not appear to have any effect on mortality, ROP, IVH, LOS, or long-term growth. The impressive reduction in the incidence of severe ROP seen with the combined DHA + AA strategy in the Mega Donna Mega study requires confirmation with a larger study, which would also help to address safety issues, particularly with respect to BPD, and to assess long-term developmental outcomes.

Acknowledgements

Supported by Australian National Health and Medical Research Council Fellowships (Translating Research into Practice Fellowship [AAP1132596] to Dr. Collins, Senior Principal Research Fellowship [APP1046207] to Dr. Gibson, and Principal Research Fel-lowship [APP1154912] to Dr. Makrides).

Conflict of Interest Statement

All authors report grants from the Australian National Health and Medical Research Council (NHMRC). The views expressed herein are solely the responsibility of the authors and do not reflect the views of the NH and MRC. Dr. Gibson has a patent - “Stabilising and Analysing Fatty Acids in a Biological Sample Stored on Solid Media” licensed to Adelaide Research and Innovation, The University of Adelaide.

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