Infant formula supplemented with milk fat globule membrane compared with standard infant formula for the cognitive development of healthy term-born formula-fed infants: protocol for a randomised controlled trial.

INTRODUCTION: Milk fat globule membrane (MFGM) is a complex lipid-protein structure in mammalian milk and human milk that is largely absent from breastmilk substitutes. The objective of this trial is to investigate whether providing infant formula enriched with MFGM versus standard infant formula improves cognitive development at 12 months of age in exclusively formula-fed full-term infants. METHODS AND ANALYSIS: This is a randomised, controlled, clinician-blinded, researcher-blinded and participant-blinded trial of two parallel formula-fed groups and a breastfed reference group that were recruited in the suburban Adelaide (Australia) community by a single study centre (a medical research institute). Healthy, exclusively formula-fed, singleton, term-born infants under 8 weeks of age were randomised to either an MFGM-supplemented formula (intervention) or standard infant formula (control) from enrolment until 12 months of age. The reference group was not provided with formula. The primary outcome is the Cognitive Scale of the Bayley Scales of Infant Development, Fourth Edition (Bayley-IV) at 12 months. Secondary outcomes are the Bayley-IV Cognitive Scale at 24 months, other Bayley-IV domains (language, motor, emotional and behavioural development) at 12 and 24 months of age, infant attention at 4 and 9 months of age, parent-rated language at 12 and 24 months of age, parent-rated development at 6 and 18 months of age as well as growth, tolerance and safety of the study formula. To ensure at least 80% power to detect a 5-point difference in the mean Bayley-IV cognitive score, >200 infants were recruited in each group. ETHICS AND DISSEMINATION: The Women's and Children Health Network Human Research Ethics Committee reviewed and approved the study (HREC/19/WCHN/140). Caregivers gave written informed consent prior to enrolling in the trial. Findings of this study will be disseminated through peer-reviewed publications and conference presentations. TRIAL REGISTRATION NUMBER: ACTRN12620000552987; Australian and New Zealand Clinical Trial Registry: anzctr.org.au.

High-Dose Docosahexaenoic Acid in Newborns Born at Less Than 29 Weeks' Gestation and Behavior at Age 5 Years: Follow-Up of a Randomized Clinical Trial.

Importance: Children born at less than 29 weeks' gestation are at risk of behavioral difficulties. This may be due in part to the lack of transplacental supply of docosahexaenoic acid (DHA), a key fatty acid with structural and functional roles in the brain. Objective: To determine whether meeting the neonatal DHA requirement through supplementation is associated with improved behavioral functioning of children born at less than 29 weeks' gestation. Design, Setting and Participants: This was a follow-up of children from 10 Australian participating centers in a multi-center, blinded, parallel group randomized clinical trial of infants born at less than 29 weeks' gestation conducted from June 2012 and September 2015, excluding those with additional fatty acid supplementation or major congenital or chromosomal abnormalities. Follow-up took place from August 2018 to May 2021. Parents of surviving children who had not withdrawn from the original trial were invited to complete questionnaires when the child turned 5 years' corrected age. Interventions: Infants were randomized to receive daily enteral emulsions providing 60 mg/kg/d of DHA or a soy-oil emulsion (with no DHA) from within the first 3 days of enteral feeding until 36 weeks' postmenstrual age or discharge home, whichever occurred first. Main Outcomes and Measures: The primary outcome of this follow-up was parent-rated behavior and emotional functioning as indicated by the Total Difficulties score of the Strengths and Difficulties Questionnaire. Parents also completed questionnaires about their child's behavioral manifestations of executive functioning, as well as a range of health outcomes to assess potential longer-term side effects of DHA intervention. Results: Primary outcome data were available for 731 children (76% of 958 surviving eligible children; 361 in the intervention group and 370 in the control group). Of these 731, 452 (47%) were female, and the mean (SD) corrected age at follow-up was 5.4 (0.5) years. Following imputation for missing data, the mean Total Difficulties score was the same in both groups (intervention group, n = 465; mean [SD], 11.8 [6.3]; control group, n = 493; mean [SD], 11.8 [6.0]; mean difference adjusted for sex, gestational age stratum, and hospital, 0.01; 95% CI, -0.87 to 0.89; P = .98). There was no evidence for differences between the groups in any secondary outcomes of behavior, executive functioning, or health. Conclusions and Relevance: In this follow-up of a randomized clinical trial, enteral DHA supplementation at the equivalent of the estimated in utero dose for infants born at less than 29 weeks' gestation did not improve behavioral functioning at age 5 years. There were no indications of adverse effects with DHA supplementation. Trial Registration: Australian New Zealand Clinical Trial Registry: ACTRN12612000503820.

Prenatal Intravenous Magnesium at 30-34 Weeks' Gestation and Neurodevelopmental Outcomes in Offspring: The MAGENTA Randomized Clinical Trial.

Importance: Intravenous magnesium sulfate administered to pregnant individuals before birth at less than 30 weeks' gestation reduces the risk of death and cerebral palsy in their children. The effects at later gestational ages are unclear. Objective: To determine whether administration of magnesium sulfate at 30 to 34 weeks' gestation reduces death or cerebral palsy at 2 years. Design, Setting, and Participants: This randomized clinical trial enrolled pregnant individuals expected to deliver at 30 to 34 weeks' gestation and was conducted at 24 Australian and New Zealand hospitals between January 2012 and April 2018. Intervention: Intravenous magnesium sulfate (4 g) was compared with placebo. Main Outcomes and Measures: The primary outcome was death (stillbirth, death of a live-born infant before hospital discharge, or death after hospital discharge before 2 years' corrected age) or cerebral palsy (loss of motor function and abnormalities of muscle tone and power assessed by a pediatrician) at 2 years' corrected age. There were 36 secondary outcomes that assessed the health of the pregnant individual, infant, and child. Results: Of the 1433 pregnant individuals enrolled (mean age, 30.6 [SD, 6.6] years; 46 [3.2%] self-identified as Aboriginal or Torres Strait Islander, 237 [16.5%] as Asian, 82 [5.7%] as Maori, 61 [4.3%] as Pacific, and 966 [67.4%] as White) and their 1679 infants, 1365 (81%) offspring (691 in the magnesium group and 674 in the placebo group) were included in the primary outcome analysis. Death or cerebral palsy at 2 years' corrected age was not significantly different between the magnesium and placebo groups (3.3% [23 of 691 children] vs 2.7% [18 of 674 children], respectively; risk difference, 0.61% [95% CI, -1.27% to 2.50%]; adjusted relative risk [RR], 1.19 [95% CI, 0.65 to 2.18]). Components of the primary outcome did not differ between groups. Neonates in the magnesium group were less likely to have respiratory distress syndrome vs the placebo group (34% [294 of 858] vs 41% [334 of 821], respectively; adjusted RR, 0.85 [95% CI, 0.76 to 0.95]) and chronic lung disease (5.6% [48 of 858] vs 8.2% [67 of 821]; adjusted RR, 0.69 [95% CI, 0.48 to 0.99]) during the birth hospitalization. No serious adverse events occurred; however, adverse events were more likely in pregnant individuals who received magnesium vs placebo (77% [531 of 690] vs 20% [136 of 667], respectively; adjusted RR, 3.76 [95% CI, 3.22 to 4.39]). Fewer pregnant individuals in the magnesium group had a cesarean delivery vs the placebo group (56% [406 of 729] vs 61% [427 of 704], respectively; adjusted RR, 0.91 [95% CI, 0.84 to 0.99]), although more in the magnesium group had a major postpartum hemorrhage (3.4% [25 of 729] vs 1.7% [12 of 704] in the placebo group; adjusted RR, 1.98 [95% CI, 1.01 to 3.91]). Conclusions and Relevance: Administration of intravenous magnesium sulfate prior to preterm birth at 30 to 34 weeks' gestation did not improve child survival free of cerebral palsy at 2 years, although the study had limited power to detect small between-group differences. Trial Registration: anzctr.org.au Identifier: ACTRN12611000491965.

Enteral supplementation with high-dose docosahexaenoic acid on the risk of bronchopulmonary dysplasia in very preterm infants: a collaborative study protocol for an individual participant data meta-analysis.

INTRODUCTION: Severe bronchopulmonary dysplasia (BPD) is a well-known factor consistently associated with impaired cognitive outcomes. Regarding reported benefits on long-term neurodevelopmental outcomes, the potential adverse effects of high-dose docosahexaenoic acid (DHA) supplementation on this short-term neonatal morbidity need further investigations in infants born very preterm. This study will determine whether high-dose DHA enteral supplementation during the neonatal period is associated with the risk of severe BPD at 36 weeks' postmenstrual age (PMA) compared with control, in contemporary cohorts of preterm infants born at less than 29 weeks of gestation. METHODS AND ANALYSIS: As part of an Australian-Canadian collaboration, we will conduct an individual participant data (IPD) meta-analysis of randomised controlled trials targeting infants born at less than 29 weeks of gestation and evaluating the effect of high-dose DHA enteral supplementation in the neonatal period compared with a control. Primary outcome will be severe grades of BPD (yes/no) at 36 weeks' PMA harmonised according to a recent definition that predicts early childhood morbidities. Other outcomes will be survival without severe BPD, death, BPD severity grades, serious brain injury, severe retinopathy of prematurity, patent ductus arteriosus and necrotising enterocolitis requiring surgery, sepsis, combined neonatal morbidities and growth. Severe BPD will be compared between groups using a multivariate generalised estimating equations log-binomial regression model. Subgroup analyses are planned for gestational age, sex, small-for-gestational age, presence of maternal chorioamnionitis and mode of delivery. ETHICS AND DISSEMINATION: The conduct of each trial was approved by institutional research ethics boards and written informed consent was obtained from participating parents. A collaboration and data sharing agreement will be signed between participating authors and institutions. This IPD meta-analysis will document the role of DHA in nutritional management of BPD. Findings will be disseminated through conferences, media interviews and publications to peer-reviewed journals. PROSPERO REGISTRATION NUMBER: CRD42023431063. TRIAL REGISTRATION NUMBER: NCT05915806.

Mediation Analysis to Untangle Opposing Associations of High-Dose Docosahexaenoic Acid With IQ and Bronchopulmonary Dysplasia in Children Born Preterm.

Importance: High-dose omega-3 docosahexaenoic acid (DHA) supplementation of children born at less than 29 weeks' gestation has been shown to improve IQ despite increasing the risk of bronchopulmonary dysplasia (BPD). Given that BPD is associated with poorer cognitive outcomes, it is unclear whether the increased risk of BPD with DHA supplementation is associated with decreased benefit to IQ. Objective: To investigate whether the increased risk of BPD with DHA supplementation was associated with diminished IQ benefit. Design, Setting, and Participants: This cohort study used data collected from a multicenter, blinded, randomized controlled trial of DHA supplementation in children born at less than 29 weeks' gestation. Participants were recruited from 2012 to 2015 and followed up until 5 years' corrected age. Data were analyzed from November 2022 to February 2023. Interventions: Enteral DHA emulsion (60 mg/kg/d, to match the estimated in-utero requirement) or a control emulsion from the first 3 days of enteral feeds until 36 weeks' postmenstrual age or discharge home. Main Outcomes and Measures: Physiological BPD was assessed at 36 weeks' postmenstrual age. IQ was assessed at 5 years' corrected age using the Wechsler Preschool and Primary Scale of Intelligence, 4th Edition; children from the 5 highest-recruiting Australian hospitals were assessed. The total effect of DHA supplementation on IQ was divided into direct and indirect effects using mediation analysis, with BPD as the presumed mediating variable. Results: Among 656 surviving children from hospitals involved in IQ follow-up (mean [SD] gestational age at birth, 26.8 [1.4] weeks; 346 males [52.7%]), there were 323 children with DHA supplementation and 333 children in the control group. Mean IQ was 3.45 points (95% CI, 0.38 to 6.53 points) higher in the DHA group than the control group, despite an increase in the risk of BPD (160 children [49.7%] vs 143 children [42.8%] with BPD). The indirect effect of DHA on IQ via BPD was not statistically significant (-0.17 points; 95% CI, -0.62 to 0.13 points), with most of the effect of DHA on IQ occurring independently of BPD (direct effect = 3.62 points; 95% CI, 0.55 to 6.81 points). Conclusions and Relevance: This study found that associations of DHA with BPD and IQ were largely independent. This finding suggests that if clinicians supplement children born preterm with high-dose DHA, any resulting increase in BPD risk would not be associated with meaningful reductions in the IQ benefit.

Growth of late preterm infants fed nutrient-enriched formula to 120 days corrected age-A randomized controlled trial.

Objectives: We aimed to compare the effects of nutrient-enriched formula with standard term formula on rate of body weight gain of late preterm infants appropriately grown for gestational age. Study design: A multi-center, randomized, controlled trial. Late preterm infants (34-37 weeks' gestation), with weight appropriate for gestational age (AGA), were randomized to nutrient enriched formula (NEF) with increased calories (22 kcal/30 ml) from protein, added bovine milk fat globule membrane, vitamin D and butyrate or standard term formula 20 kcal/30 ml (STF). Breastfed term infants were enrolled as an observational reference group (BFR). Primary outcome was rate of body weight gain from enrollment to 120 days corrected age (d/CA). Planned sample size was 100 infants per group. Secondary outcomes included body composition, weight, head circumference and length gain, and medically confirmed adverse events to 365 d/CA. Results: The trial was terminated early due to recruitment challenges and sample size was substantially reduced. 40 infants were randomized to NEF (n = 22) and STF (n = 18). 39 infants were enrolled in the BFR group. At 120 d/CA there was no evidence of a difference in weight gain between randomized groups (mean difference 1.77 g/day, 95% CI, -1.63 to 5.18, P = 0.31). Secondary outcomes showed a significant reduction in risk of infectious illness in the NEF group at 120 d/CA [relative risk 0.37 (95% CI, 0.16-0.85), P = 0.02]. Conclusion: We saw no difference in rate of body weight gain between AGA late preterm infants fed NEF compared to STF. Results should be interpreted with caution due to small sample size. Clinical Trial Registration: The Australia New Zealand Clinical Trials Registry (ACTRN 12618000092291). "mailto:maria.makrides@sahmri.com" maria.makrides@sahmri.com.

Protocol for a multicentre prospective observational study of families with full-term infants on postnatal wards and in the community to capture feeding practices across the first year of life: the Mother Infant Lactation Questionnaire (MILQ) study.

INTRODUCTION: Breastmilk is considered the gold standard for infant nutrition. Breast feeding is recommended as the sole source of nutrition between birth until around 6 months of age and should be continued beyond this age as complementary foods are introduced. While breast feeding initiation is generally high in developed countries, continuation of breast feeding appears to drop rapidly. This is a prospective observational study of life that aims to characterise a current picture of infant feeding practices across the first year, and motivations for feeding practices, and to identify barriers and enablers for breast feeding. METHODS AND ANALYSIS: Caregivers with newborn singleton infants of normal birth weight are approached on the postnatal units of three hospitals in South Australia, or through targeted online advertising campaigns promoting the study. Caregivers are asked to complete surveys when their infant reaches 3, 5 and 7 weeks', and at 3, 4, 5, 6, 9 and 12 months of age. Initially, baseline characteristics, intentions and preferences for infant milk feeds, as well as reasons for preferences are captured. Latter surveys query how infants are being fed, difficulties or barriers to breast feeding, as well as any enablers (if breast feeding). Once infants reach 5 months of age, surveys capture complementary feeding. A large opportunistic sample from the Adelaide community with a minimum of 1000 mother-infant pairs will be enrolled. The data will be analysed descriptively and using regression models. ETHICS AND DISSEMINATION: Women's and Children's Health Network Human Research Ethics Committee reviewed and approved the study (approval no HREC/19/WCHN/140, approval date: 22 November 2019). Study results will be disseminated through academic meetings, peer-reviewed journals, in-services for postnatal healthcare services, results letters for participants and social media. TRIAL REGISTRATION NUMBER: ACTRN12620000529943.

Neonatal Docosahexaenoic Acid in Preterm Infants and Intelligence at 5 Years.

BACKGROUND: Docosahexaenoic acid (DHA) is a component of neural tissue. Because its accretion into the brain is greatest during the final trimester of pregnancy, infants born before 29 weeks' gestation do not receive the normal supply of DHA. The effect of this deficiency on subsequent cognitive development is not well understood. METHODS: We assessed general intelligence at 5 years in children who had been enrolled in a trial of neonatal DHA supplementation to prevent bronchopulmonary dysplasia. In the previous trial, infants born before 29 weeks' gestation had been randomly assigned in a 1:1 ratio to receive an enteral emulsion that provided 60 mg of DHA per kilogram of body weight per day or a control emulsion from the first 3 days of enteral feeds until 36 weeks of postmenstrual age or discharge home, whichever occurred first. Children from 5 of the 13 centers in the original trial were invited to undergo assessment with the Wechsler Preschool and Primary Scale of Intelligence (WPPSI) at 5 years of corrected age. The primary outcome was the full-scale intelligence quotient (FSIQ) score. Secondary outcomes included the components of WPPSI. RESULTS: A total of 1273 infants underwent randomization in the original trial; of the 656 surviving children who had undergone randomization at the centers included in this follow-up study, 480 (73%) had an FSIQ score available - 241 in the DHA group and 239 in the control group. After imputation of missing data, the mean (±SD) FSIQ scores were 95.4±17.3 in the DHA group and 91.9±19.1 in the control group (adjusted difference, 3.45; 95% confidence interval, 0.38 to 6.53; P = 0.03). The results for secondary outcomes generally did not support that obtained for the primary outcome. Adverse events were similar in the two groups. CONCLUSIONS: In infants born before 29 weeks' gestation who had been enrolled in a trial to assess the effect of DHA supplementation on bronchopulmonary dysplasia, the use of an enteral DHA emulsion until 36 weeks of postmenstrual age was associated with modestly higher FSIQ scores at 5 years of age than control feeding. (Funded by the Australian National Health and Medical Research Council and Nu-Mega Ingredients; N3RO Australian New Zealand Clinical Trials Registry number, ACTRN12612000503820.).

Breastfeeding outcomes in late preterm infants: A multi-centre prospective cohort study.

OBJECTIVES: To describe (1) infant feeding practices during initial hospitalisation and up to 6 months corrected age (CA) in infants born late preterm with mothers intending to breastfeed, (2) the impact of early feeding practices on hospital length of stay and (3) maternal and infant factors associated with duration of breastfeeding. METHODS: We conducted a prospective cohort study of infants born at 34+0 to 36+6 weeks gestational age during 2018-2020. Families were followed up until the infant reached 6 months of age (corrected for prematurity). Feeding practices during the birth hospitalisation, length of initial hospital stay, and the prevalence of exclusive or any breastfeeding at 6 weeks, 3 months, and 6 months CA were examined. Associations between maternal and infant characteristics and breastfeeding at 6 weeks, 3 months and 6 months CA were assessed using multivariable logistic regression models. RESULTS: 270 infants were enrolled, of these, 30% were multiple births. Overall, 78% of infants received only breastmilk as their first feed, and 83% received formula during the hospitalisation. Seventy-four per cent of infants were exclusively breastfed at discharge, 41% at 6 weeks CA, 35% at 3 months CA, and 29% at 6 months CA. The corresponding combined exclusive and partial breastfeeding rates (any breastfeeding) were 72%, 64%, and 53% of babies at 6 weeks CA, 3 months CA, and 6 months CA, respectively. The mean duration of hospitalisation was 2.9 days longer (95% confidence interval (CI) 0.31, 5.43 days) in infants who received any formula compared with those receiving only breastmilk (adjusted for GA, maternal age, multiple birth, site, and neonatal intensive care unit admission). In multivariable models, receipt of formula as the first milk feed was associated with a reduction in exclusive breastfeeding at 6 weeks CA (odds ratio = 0.22; 95% CI 0.09 to 0.53) and intention to breastfeed >6 months with an increase (odds ratio = 4.98; 95% CI 2.39 to 10.40). Intention to breastfeed >6 months remained an important predictor of exclusive breastfeeding at 3 and 6 months CA. CONCLUSIONS: Our study demonstrates that long-term exclusive breastfeeding rates were low in a cohort of women intending to provide breastmilk to their late preterm infants, with approximately half providing any breastmilk at 6 months CA. Formula as the first milk feed and intention to breastfeed >6 months were significant predictors of breastfeeding duration. Improving breastfeeding outcomes may require strategies to support early lactation and a better understanding of the ongoing support needs of this population.

Tailoring Human Milk Oligosaccharides to Prevent Necrotising Enterocolitis Among Preterm Infants.

Necrotising enterocolitis (NEC) is a devastating disease affecting preterm infants, with little improvement in mortality rates and treatment strategies in the last 30 years. Human milk oligosaccharides (HMOs) are emerging as a potential preventive therapy, with multiple protective functions postulated. Our aim is to summarise the evidence concerning the role of HMOs in NEC development and emerging strategies to tailor the delivery of HMOs to preterm infants. Most research efforts to date have focused on supplementing preterm infants with simple oligosaccharides, which are structurally different to HMOs and derived mainly from plants. Clinical trials demonstrate limited benefits for NEC prevention arising from the use of these supplements. Alternative strategies under investigation include optimising HMOs for infants receiving donor human milk, concentrating oligosaccharides from donor human milk and from animal milks, as well as more sophisticated synthetic oligosaccharide production strategies. Critically, high quality evidence to support implementation of any of these approaches in the neonatal unit is lacking. Whether it is a specific HMO alone or a combination of HMOs that exert protective effects remains to be elucidated. Further challenges include how best to manufacture and administer oligosaccharides whilst retaining bioactivity and safety, including evaluation of the long-term effects of altering the balance of HMOs and gut microbiota in preterm infants. While several human clinical trials are underway, further research is needed to understand whether a tailored approach to oligosaccharide supplementation is beneficial for preterm infants.

Cord Blood T Cells Expressing High and Low PKCζ Levels Develop into Cells with a Propensity to Display Th1 and Th9 Cytokine Profiles, Respectively.

Low Protein Kinase C zeta (PKCζ) levels in cord blood T cells (CBTC) have been shown to correlate with the development of allergic sensitization in childhood. However, little is known about the mechanisms responsible. We have examined the relationship between the expression of different levels of PKCζ in CBTC and their development into mature T cell cytokine producers that relate to allergy or anti-allergy promoting cells. Maturation of naïve CBTC was initiated with anti-CD3/-CD28 antibodies and recombinant human interleukin-2 (rhIL-2). To stimulate lymphocyte proliferation and cytokine production the cells were treated with Phytohaemagglutinin (PHA) and Phorbol myristate acetate (PMA). Irrespective of the PKCζ levels expressed, immature CBTC showed no difference in lymphocyte proliferation and the production of T helper 2 (Th2) cytokine interleukin-4 (IL-4) and Th1 cytokine, interferon-gamma (IFN-γ), and influenced neither their maturation from CD45RA+ to CD45RO+ cells nor cell viability/apoptosis. However, upon maturation the low PKCζ expressing cells produced low levels of the Th1 cytokines, IFN-γ, IL-2 and tumour necrosis factor-alpha (TNF), no changes to levels of the Th2 cytokines, IL-4, IL-5 and IL-13, and an increase in the Th9 cytokine, IL-9. Other cytokines, lymphotoxin-α (LT-α), IL-10, IL-17, IL-21, IL-22 and Transforming growth factor-beta (TGF-ß) were not significantly different. The findings support the view that low CBTC PKCζ levels relate to the increased risk of developing allergic diseases.

Protocol for assessing if behavioural functioning of infants born <29 weeks' gestation is improved by omega-3 long-chain polyunsaturated fatty acids: follow-up of a randomised controlled trial.

INTRODUCTION: During the last trimester of pregnancy, the fetal brain undergoes a rapid growth spurt and accumulates essential nutrients including docosahexaenoic acid (DHA). This takes place ex-utero for infants born <29 weeks' gestation, without the in-utero provisions of DHA. Infants born <29 weeks' are more likely to experience behavioural and emotional difficulties than their term-born counterparts. It has been hypothesised that supplementing preterm infants with dietary DHA may alleviate insufficiency and subsequently prevent or minimise behavioural problems. This protocol describes a follow-up of infants born <29 weeks gestation who were enrolled in a randomised controlled trial (RCT) of DHA supplementation. We aim to determine whether DHA supplementation improves the behaviour, and general health of these infants. METHODS AND ANALYSIS: Infants born <29 weeks' gestation were enrolled in a multicentre blinded RCT of enteral DHA supplementation. Infants were randomised to receive an enteral emulsion that provided 60 mg/kg/day of DHA or a control emulsion commenced within the first 3 days of enteral feeding, until 36 weeks' postmenstrual age or discharge home, whichever occurred first. Families of surviving children (excluding those who withdrew from the study) from the Australian sites (up to 955) will be invited to complete a survey. The survey will include questions regarding child behavioural and emotional functioning, executive functioning, respiratory health and general health. We hypothesise that the DHA intervention will have a benefit on the primary outcome, parent-rated behaviour and emotional status as measured using the Total Difficulties score of the Strengths and Difficulties Questionnaire. Detecting a 2-point difference between groups (small effect size of 0.25 SD) with 90% power will require follow-up of 676 participants. ETHICS AND DISSEMINATION: The Women's and Children Health Network Human Research Ethics Committee reviewed and approved the study (HREC/16/WCHN/184). Results will be disseminated in peer-reviewed publications and conference presentations. TRIAL REGISTRATION NUMBER: ACTRN12612000503820.

Vitamin D upregulates the macrophage complement receptor immunoglobulin in innate immunity to microbial pathogens.

Vitamin D deficiency remains a global concern. This 'sunshine' vitamin is converted through a multistep process to active 1,25-dihydroxyvitamin D3 (1,25D), the final step of which can occur in macrophages. Here we demonstrate a role for vitamin D in innate immunity. The expression of the complement receptor immunoglobulin (CRIg), which plays an important role in innate immunity, is upregulated by 1,25D in human macrophages. Monocytes cultured in 1,25D differentiated into macrophages displaying increased CRIg mRNA, protein and cell surface expression but not in classical complement receptors, CR3 and CR4. This was associated with increases in phagocytosis of complement opsonised Staphylococcus aureus and Candida albicans. Treating macrophages with 1,25D for 24 h also increases CRIg expression. While treating macrophages with 25-hydroxyvitamin D3 does not increase CRIg expression, added together with the toll like receptor 2 agonist, triacylated lipopeptide, Pam3CSK4, which promotes the conversion of 25-hydroxyvitamin D3 to 1,25D, leads to an increase in CRIg expression and increases in CYP27B1 mRNA. These findings suggest that macrophages harbour a vitamin D-primed innate defence mechanism, involving CRIg.

Protocol for assessing whether cognition of preterm infants <29 weeks' gestation can be improved by an intervention with the omega-3 long-chain polyunsaturated fatty acid docosahexaenoic acid (DHA): a follow-up of a randomised controlled trial.

INTRODUCTION: Docosahexaenoic acid (DHA) is an omega-3 (n-3) fatty acid that accumulates into neural tissue during the last trimester of pregnancy, as the fetal brain is undergoing a growth spurt. Infants born <29 weeks' gestation are deprived the normal in utero supply of DHA during this period of rapid brain development. Insufficient dietary DHA postnatally may contribute to the cognitive impairments common among this population. This follow-up of the N-3 fatty acids for improvement in respiratory outcomes (N3RO) randomised controlled trial aims to determine if enteral DHA supplementation in infants born <29 weeks' gestation during the first months of life improves cognitive development at 5 years of age corrected for prematurity. METHODS AND ANALYSIS: N3RO was a randomised controlled trial of enteral DHA supplementation (60 mg/kg/day) or a control emulsion (without DHA) in 1273 infants born <29 weeks' gestation to determine the effect on bronchopulmonary dysplasia (BPD). We showed that DHA supplementation did not reduce the risk of BPD and may have increased the risk.In this follow-up at 5 years' corrected age, a predefined subset (n=655) of children from five Australian sites will be invited to attend a cognitive assessment with a psychologist. Children will be administered the Wechsler Preschool and Primary Scale of Intelligence (fourth edition) and a measure of inhibitory control (fruit stroop), while height, weight and head circumference will be measured.The primary outcome is full-scale IQ. To ensure 90% power, a minimum of 592 children are needed to detect a four-point difference in IQ between the groups.Research personnel and families remain blinded to group assignment. ETHICS AND DISSEMINATION: The Women's and Children Health Network Human Research Ethics Committee reviewed and approved the study (HREC/17/WCHN/187). Caregivers will give informed consent prior to taking part in this follow-up study. Findings of this study will be disseminated through peer-reviewed publications and conference presentations. TRIAL REGISTRATION NUMBER: ACTRN12612000503820.

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

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.

Effect of parenteral lipid emulsion on preterm infant PUFAs and their downstream metabolites.

OBJECTIVE: Oxylipins synthesized by oxidation of long-chain polyunsaturated fatty acids (LCPUFAs) are bioactive downstream lipid mediators. The aim of this study was to describe oxylipin levels in preterm infants born 30 to 33 weeks' gestation who were enrolled in a randomized controlled trial in which peripheral parenteral nutrition (P-PN), including lipid emulsion (containing soy, medium chain triglyceride, olive and fish oil), was compared with 10% glucose on growth during the transition to enteral feeds. METHODS: Of the 92 infants randomized to the P-PN study, the first 72 (P-PN n = 34, control n = 38) had blood taken for fatty acid analyses. P-PN infants received parenteral nutrition including 3% protein, 8% glucose and 17% SMOFlipid® lipid (containing soy, medium chain triglyceride, olive and fish oil), and control infants 10% glucose. Both groups commenced enteral feeds when clinically stable. 32 oxylipins and 5 free fatty acids were screened (using ultra-high-performance liquid chromatography-tandem mass spectrometry), and 5 total LCPUFA were measured (using gas chromatography), on study days 1 (baseline), 2, 4, 7, 14 and 21. RESULTS: Both total and free LA, ALA and EPA were significantly higher in the P-PN group compared with control over the first week of life. Whereas total AA was significantly lower and free DHA significantly higher over the same time period. All LA, ALA, EPA and four DHA derived oxylipins detected were significantly higher in the P-PN group compared with the control group during the first week of life, with three AA derived oxylipins significantly lower and one significantly higher. CONCLUSIONS: Parenteral lipid emulsion resulted in a change in total and free fatty acids and related oxylipins with the profiles suggesting increased omega-6 driven inflammation. Further studies to investigate the association between the oxylipin levels and nutrition and to determine whether the oxylipin profiles influence the clinical outcome in preterm infants are warranted.

DHA supplementation in infants born preterm and the effect on attention at 18 months' corrected age: follow-up of a subset of the N3RO randomised controlled trial.

Infants born preterm miss out on the peak period of in utero DHA accretion to the brain during the last trimester of pregnancy which is hypothesised to contribute to the increased prevalence of neurodevelopmental deficits in this population. This study aimed to determine whether DHA supplementation in infants born preterm improves attention at 18 months' corrected age. This is a follow-up of a subset of infants who participated in the N3RO randomised controlled trial. Infants were randomised to receive an enteral emulsion of high-dose DHA (60 mg/kg per d) or no DHA (soya oil - control) from within the first days of birth until 36 weeks' post-menstrual age. The assessment of attention involved three tasks requiring the child to maintain attention on toy/s in either the presence or absence of competition or a distractor. The primary outcome was the child's latency of distractibility when attention was focused on a toy. The primary outcome was available for seventy-three of the 120 infants that were eligible to participate. There was no evidence of a difference between groups in the latency of distractibility (adjusted mean difference: 0·08 s, 95 % CI -0·81, 0·97; P = 0·86). Enteral DHA supplementation did not result in improved attention in infants born preterm at 18 months' corrected age.

The efficacy and safety of peripheral intravenous parenteral nutrition vs 10% glucose in preterm infants born 30 to 33 weeks' gestation: a randomised controlled trial.

BACKGROUND: Preterm infants born 30 to 33 weeks' gestation often require early support with intravenous fluids because of respiratory distress, hypoglycemia or feed intolerance. When full feeds are anticipated to be reached within the first week, risks associated with intravenous delivery mode and type must be carefully considered. Recommendations are for parenteral nutrition to be infused via central venous lines (because of the high osmolarity), however, given the risks associated with central lines, clinicians may opt for 10% glucose via peripheral venous catheter when the need is short-term. We therefore compare a low osmolarity peripheral intravenous parenteral nutrition (P-PN) solution with peripheral intravenous 10% glucose on growth rate in preterm infants born 30 to 33 weeks' gestation. METHODS: In this parallel group, single centre, superiority, non-blinded, randomised controlled trial, 92 (P-PN 42, control 50) infants born 30+ 0 to 33+ 6 weeks' gestation, were randomised within 24 h of age, to receive either P-PN (8% glucose, 30 g/L amino acids, 500 IU/L heparin and SMOFlipid®) or a control of peripheral intravenous 10% glucose. Both groups received enteral feeds according to hospital protocol. The primary outcome was rate of weight gain from birth to 21 days of age. RESULTS: The rate of weight gain was significantly increased in P-PN infants compared with control (P-PN, n = 42, 18.7, SD 6.6 g/d vs control, n = 50, 14.8, SD 6.0 g/d; adjusted mean difference 3.9 g/d, 95% CI 1.3 to 6.6; P = 0.004), with the effect maintained to discharge home. Days to regain birthweight were significantly reduced and length gain significantly increased in P-PN infants. One infant in the P-PN group had a stage 3 extravasation which rapidly resolved. Blood urea nitrogen and triglyceride levels were significantly higher in the P-PN group in the first week of life, but there were no instances of abnormally high levels. There were no significant differences in any other clinical or biochemical outcomes. CONCLUSION: P-PN improves the rate of weight gain to discharge home in preterm infants born 30 to 33 weeks gestation compared with peripheral intravenous 10% glucose. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12616000925448 . Registered 12 July 2016.

Free Fatty Acid Concentration in Expressed Breast Milk Used in Neonatal Intensive Care Units.

Preterm and sick term infants are commonly fed with expressed breast milk (EBM) that has been subjected to various storage and handling conditions before feeding that may cause lipase-mediated elevation of free fatty acids (FFA). This study was designed to describe the variation, between mothers' and within the same mother over time, in the concentration of FFA in EBM used in an Australian neonatal unit. A total of 256 EBM samples, 149 freshly expressed in the unit cot-side and 87 expressed at home and brought in to the unit, were collected from 32 mothers with an infant admitted to the neonatal intensive and/or special care units at the Women's and Children's Hospital, Adelaide. Among the fresh EBM samples collected cot-side, the average total fat content was 29.78 ± 9.28 mg/mL, and the FFA concentration was 1.70% of total fats (interquartile range [IQR]: 1.17-2.37%). Among the 10 mothers who provided fresh EBM at different stages of lactation, the concentration of FFA remained low overall, with some day-to-day variation (min 0.58% and max 5.0% of total fats within the same mother). The average total fat content of home collected EBM was similar to the cot-side collected samples, at 27.37 ± 8.23 mg/mL, and the FFA concentration was slightly higher at 2.49% of total fats (IQR: 1.74-3.29%). Overall, the FFA concentration of breast milk in the neonatal unit before and even after a short period of cold storage and handling is universally low.