The Effect of Iron Supplements on the Gut Microbiome of Females of Reproductive Age: A Randomized Controlled Trial.

BACKGROUND: Iron deficiency is the most common nutritional deficiency worldwide, particularly for young children and females of reproductive age. Although oral iron supplements are routinely recommended and generally considered safe, iron supplementation has been shown to alter the fecal microbiota in low-income countries. Little is known about the effect of iron supplementation on the fecal microbiota in high-income settings. OBJECTIVES: To assess the effect of oral iron supplementation compared with placebo on the gut microbiome in nonpregnant females of reproductive age in a high-income country. METHODS: A 21-d prospective parallel design double-blind, randomized control trial conducted in South Australia, Australia. Females (18-45 y) were randomly assigned to either iron (65.7 mg ferrous fumarate) or placebo. Fecal samples were collected prior to commencing supplements and after 21 d of supplementation. The primary outcome was microbiota ß-diversity (paired-sample weighted unique fraction metric dissimilarity) between treatment and placebo groups after 21 d of supplementation. Exploratory outcomes included changes in the relative abundance of bacterial taxa. RESULTS: Of 82 females randomly assigned, 80 completed the trial. There was no significant difference between the groups for weighted unique fraction metric dissimilarity (mean difference: 0.003; 95% confidence interval: -0.007, 0.014; P = 0.52) or relative abundance of common bacterial taxa or Escherichia-Shigella (q > 0.05). CONCLUSIONS: Iron supplementation did not affect the microbiome of nonpregnant females of reproductive age in Australia. This trial was registered at clinicaltrials.gov as NCT05033483.

Too Much Too Little: Clarifying the Relationship Between Maternal Iodine Intake and Neurodevelopmental Outcomes.

BACKGROUND: In 2009, the Australian government mandated the fortification of bread salt with iodine. In 2010, pregnant and lactating women were also advised to take an iodine-containing supplement. Our assessment of this policy in an iodine-sufficient population showed that children whose mothers were in the highest and lowest quartiles of iodine intake performed more poorly on early childhood tests of cognition and language than those in the second quartile. However, we did not quantify the iodine intake associated with optimal neurodevelopment. OBJECTIVES: The aim was to establish the iodine intake range in pregnancy associated with optimal child neurodevelopment. METHODS: A prospective cohort study of pregnant women and their young children (n = 699). Iodine intake was assessed by a validated food frequency questionnaire at 16 and 28 wk of gestation. Child neurodevelopment at 18 mo of age was measured using the Bayley Scales of Infant and Toddler Development, Third Edition (Bayley-III). The relationship between average iodine intake during pregnancy and child neurodevelopment was assessed using linear regression with fractional polynomials and adjustment for confounders. RESULTS: Mean (SD) iodine intake was similar at study entry and 28 wk, 308 (120) µg/d, with 82% of women taking iodine supplements at study entry. The relationship between iodine intake during pregnancy and Bayley-III cognitive and language scores was curvilinear (P = 0.001 and P = 0.004, respectively), with the lowest Bayley-III scores observed at lower and higher iodine intakes. The inflection point that drove the association between lower iodine intake in pregnancy and poorer child neurodevelopment scores was around 185 µg/d; for the higher pregnancy iodine intakes, language and cognitive scores were negatively affected from ∼350 µg/d to 370 µg/d, respectively. Higher iodine intakes were being driven by supplement use. CONCLUSIONS: Targeted, not blanket, iodine supplementation may be needed for pregnant women with low-iodine intake from food.

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.

Predictors of compliance with higher dose omega-3 fatty acid supplementation during pregnancy and implications for the risk of prematurity: exploratory analysis of the ORIP randomised trial.

BACKGROUND: Intention-to-treat analyses of the Omega-3 to Reduce the Incidence of Prematurity (ORIP) trial found that omega-3 (n-3) fatty acid supplementation reduces the risk of prematurity in the subgroup of women with a singleton pregnancy and low n-3 status early in pregnancy, but not overall. However, results may have been influenced by less-than-optimal compliance. OBJECTIVES: To identify predictors of compliance with n-3 supplementation and determine treatment effects among compliers. DESIGN: Exploratory analyses of a multicentre-blinded randomised trial. SETTING: 6 tertiary care centres in Australia. PARTICIPANTS: 5328 singleton pregnancies. INTERVENTIONS: Daily capsules containing 900 mg n-3 long-chain polyunsaturated fatty acids or vegetable oil, consumed from before 20 weeks gestation until 34 weeks gestation. OUTCOME MEASURES: Early preterm (<34 weeks gestation) and preterm birth (<37 weeks gestation). Women were considered compliant if they reported missing less than a third of their allocated capsules in the previous week during a mid-pregnancy appointment. RESULTS: Among 2654 singleton pregnancies in the n-3 intervention group, 1727 (65%) were deemed compliant with supplementation. Maternal characteristics associated with compliance included age, years of full-time education, consuming alcohol but not smoking in the 3 months leading up to pregnancy, fewer previous births and taking dietary supplements at enrolment. Based on complier average causal effects, n-3 supplementation reduced the risk of preterm birth in compliers (relative risk=0.76; 95% CI 0.60 to 0.97), but not early preterm birth (relative risk=0.80; 95% CI 0.44 to 1.46). Consistent with intention-to-treat analyses, the lack of an overall effect on early preterm birth in compliers appeared to be due to beneficial effects in women with low n-3 status at enrolment but not women with replete status. CONCLUSIONS: Results in compliers were similar to those from intention-to-treat analyses, suggesting that non-compliance was not a major factor in explaining outcomes from the ORIP trial. TRIAL REGISTRATION NUMBER: ACTRN12613001142729.

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.

Prenatal iodine supplementation and early childhood neurodevelopment: the PoppiE trial - study protocol for a multicentre randomised controlled trial.

INTRODUCTION: Observational studies suggest both low and high iodine intakes in pregnancy are associated with poorer neurodevelopmental outcomes in children. This raises concern that current universal iodine supplement recommendations for pregnant women in populations considered to be iodine sufficient may negatively impact child neurodevelopment. We aim to determine the effect of reducing iodine intake from supplements for women who have adequate iodine intake from food on the cognitive development of children at 24 months of age. METHODS AND ANALYSIS: A multicentre, randomised, controlled, clinician, researcher and participant blinded trial with two parallel groups. Using a hybrid decentralised clinical trial model, 754 women (377 per group) less than 13 weeks' gestation with an iodine intake of ≥165 µg/day from food will be randomised to receive either a low iodine (20 µg/day) multivitamin and mineral supplement or an identical supplement containing 200) µg/day (amount commonly used in prenatal supplements in Australia), from enrolment until delivery. The primary outcome is the developmental quotient of infants at 24 months of age assessed with the Cognitive Scale of the Bayley Scales of Infant Development, fourth edition. Secondary outcomes include infant language and motor development; behavioural and emotional development; maternal and infant clinical outcomes and health service utilisation of children. Cognitive scores will be compared between groups using linear regression, with adjustment for location of enrolment and the treatment effect described as a mean difference with 95% CI. ETHICS AND DISSEMINATION: Ethical approval has been granted from the Women's and Children's Health Network Research Ethics Committee (HREC/17/WCHN/187). The results of this trial will be presented at scientific conferences and published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT04586348.

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.

Identifying women who may benefit from higher dose omega-3 supplementation during pregnancy to reduce their risk of prematurity: exploratory analyses from the ORIP trial.

OBJECTIVES: The risk factors for prematurity are multifactorial and include low omega-3 status. Omega-3 supplementation in pregnancy has been found to reduce prematurity risk, particularly among women with low omega-3 levels. This study aimed to identify maternal characteristics that predict whether women with a singleton pregnancy will benefit from omega-3 supplementation to reduce their risk of prematurity. DESIGN: Exploratory analyses of a multicentre, double-blind randomised trial. SETTING: 6 tertiary care centres in four states in Australia. PARTICIPANTS: 5328 singleton pregnancies in 5305 women recruited before 20 weeks of gestation. INTERVENTIONS: Fish oil capsules containing 900 mg omega-3 long-chain polyunsaturated fatty acids per day versus vegetable oil capsules consumed from enrolment until 34 weeks' gestation. OUTCOME MEASURES: Early preterm birth (EPTB, <34 weeks' gestation) and preterm birth (PTB, <37 weeks' gestation) analysed using logistic regression models with interactions between treatment group and a range of maternal biological, clinical and demographic characteristics. RESULTS: Omega-3 supplementation reduced the odds of EPTB for women with low total omega-3 status in early pregnancy (OR=0.30, 95% CI 0.10-0.93). No additional maternal characteristics influenced whether omega-3 supplementation reduced the odds of EPTB. For PTB, women were more likely to benefit from omega-3 supplementation if they were multiparous (OR=0.65, 95% CI 0.49-0.87) or avoided alcohol in the lead up to pregnancy (OR=0.62, 95% CI 0.45-0.86). CONCLUSIONS: Our results support previous findings that women with low total omega-3 levels in early pregnancy are most likely to benefit from taking omega-3 supplements to reduce their risk of EPTB. Understanding how other maternal characteristics influence the effectiveness of omega-3 supplementation on reducing PTB requires further investigation. TRIAL REGISTRATION NUMBER: ACTRN12613001142729.

Effect of Docosahexaenoic Acid (DHA) Supplementation of Preterm Infants on Growth, Body Composition, and Blood Pressure at 7-Years Corrected Age: Follow-Up of a Randomized Controlled Trial.

Aim: To determine if supplementation of infants born <33 weeks' gestation with higher dose docosahexaenoic acid (DHA) affects growth, body composition, and blood pressure at 7 y corrected age (CA) and if treatment effects differed by infant sex at birth and birth weight strata (<1250 and ≥1250 g). Methods: Seven-year follow-up of an Australian multicenter randomized controlled trial in which 657 infants were fed high-DHA (≈1% total fatty acids) enteral feeds or standard-DHA (≈0.3% total fatty acids) from age 2−4 d until term CA. Seven-year CA outcomes were growth (weight, height), body composition (lean body mass, fat mass, waist, and hip circumference), and blood pressure. Results: There was no effect of high-DHA enteral feeds compared with standard-DHA on growth, body composition, and blood pressure at 7-year CA either overall or in subgroup analysis by sex. There was a significant interaction between high-DHA and birthweight strata on height at 7-y CA (p = 0.03). However, the post-hoc analyses by birthweight strata did not reach significance (p > 0.1). High-DHA group infants were more likely to be classified as obese (relative risk 1.6 (95% CI 1.0, 2.6); p = 0.05). Conclusions: DHA supplementation of premature infants did not affect growth, body composition, or blood pressure at 7-year CA overall by sex and birthweight strata. The finding of a higher risk of obesity in children who receive high-DHA needs to be interpreted with caution due to the small number of children classified as obese.

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.).

Estimated Choline Intakes and Dietary Sources of Choline in Pregnant Australian Women.

(1) Background: Despite the postulated importance of choline during pregnancy, little is known about the choline intake of Australians during pregnancy. In this study, we estimated dietary intakes of choline in early and late pregnancy, compared those intakes to recommendations, and investigated food sources of choline in a group of pregnant women in Australia. (2) Methods: 103 pregnant women enrolled in a randomized controlled trial. In early pregnancy (12−16 weeks gestation) and late pregnancy (36 weeks gestation), women completed a food frequency questionnaire designed to assess dietary intake over the previous month. (3) Results: Choline intakes and sources were similar in early and late pregnancy. Median choline intake in early pregnancy was 362 mg/day. Of the women, 39% and 25% had choline intakes above the Australian National Health and Medical Research Council (NHMRC) adequate intake (AI) of >440 mg/day and the European Food Safety Authority (EFSA) AI of >480 mg/day for choline in pregnancy, respectively. Eggs, red meat, nuts, legumes, and dairy accounted for 50% of choline intake, with eggs being the most significant contributor at 17%. (4) Conclusions: Few pregnant women in our study met the AI recommended by the NHMRC and EFSA. In Australia, choline intake in pregnancy may need to be improved, but further work to define choline requirements in pregnancy is required.

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.

Translating n-3 polyunsaturated fatty acid status from whole blood to plasma and red blood cells during pregnancy: Translating n-3 status across blood fractions in pregnancy.

Women with low n-3 (omega-3) status in pregnancy can reduce their risk of early preterm birth (<34 weeks' gestation) through n-3 long chain polyunsaturated fatty acid (LCPUFA) supplementation. As investigators measure fatty acid status in different blood fractions, equations are needed to compare results across studies. Similarly, derived cut-points for defining low and replete n-3 status are needed to assist clinical interpretation during early pregnancy. Our aims were to develop equations to convert the percentage of total n-3 fatty acids, EPA+DHA and DHA between whole blood, plasma and red blood cells (RBC), and to derive cut-points for defining low and replete total n-3 fatty acid status in plasma and RBC from those already established in whole blood. Using blood samples from 457 pregnant women in a multicentre randomised controlled trial, equations for these interconversions were developed using simple linear regression models. Measures of n-3 fatty acid status in whole blood and plasma were strongly related (R2 > 0.85), while more moderate relationships were observed between measures in whole blood and RBC (R2 0.55 - 0.71), or plasma and RBC (R2 0.55 - 0.63). Using the conversion equations, established cut-points for low and replete n-3 status in whole blood (<4.2% and >4.9% of total fatty acids) converted to <3.7% and >4.3% of plasma total fatty acids, and to <7.3% and >8.1% of RBC total fatty acids. Agreement to define low and replete n-3 status was better between whole blood and plasma, rather than between whole blood and RBC. Our data also show that total n-3 fatty acids in plasma and serum are interchangeable. We conclude that either whole blood or plasma total n-3 fatty acids can be used to define low status in pregnancy and identify women who will most benefit from n-3 LCPUFA supplementation to reduce their risk of early birth. Further research is needed to determine the clinical utility of other fatty acid measures in various blood lipid fractions.

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.

Maternal Late-Pregnancy Serum Unmetabolized Folic Acid Concentrations Are Not Associated with Infant Allergic Disease: A Prospective Cohort Study.

BACKGROUND: The increase in childhood allergic disease in recent decades has coincided with increased folic acid intakes during pregnancy. Circulating unmetabolized folic acid (UMFA) has been proposed as a biomarker of excessive folic acid intake. OBJECTIVE: We aimed to determine if late-pregnancy serum UMFA and total folate concentrations were associated with allergic disease risk in the offspring at 1 y of age in a population at high risk of allergy. METHODS: The cohort consisted of 561 mother-infant pairs from Western Australia. To be eligible the infant had to have a first-degree relative (mother, father, or sibling) with a history of medically diagnosed allergic disease. Maternal venous blood was collected between 36 and 40 wk of gestation. Serum UMFA was measured by LC-tandem MS. Serum total folate was determined using a microbiological method with chloramphenicol-resistant Lactobacillus rhamnosus as the test organism, and was collected between 36 and 40 wk of gestation. UMFA concentrations were measured by tandem MS using stable isotope dilution; folate concentrations were determined using the microbiological method with standardized kits. Infant allergic disease outcomes of medically diagnosed eczema, steroid-treated eczema, atopic eczema, IgE-mediated food allergy, allergen sensitization, and medically diagnosed wheeze were assessed at 1 y of age. RESULTS: Median (IQR) concentrations for UMFA and serum folate were 1.6 (0.6-4.7) and 53.2 (32.6-74.5) nmol/L, respectively. Of the infants, 34.6% had medically diagnosed eczema, 26.4% allergen sensitization, and 14.9% had an IgE-mediated food allergy. In both adjusted and unadjusted models there was little evidence of association between UMFA or serum folate and any of the infant allergy outcomes. CONCLUSIONS: In this cohort of children at high risk of allergic disease there was no association between maternal UMFA or serum folate concentrations measured in late pregnancy and allergic disease outcomes at 1 y of age.

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.

Study protocol for a randomised controlled trial evaluating the effect of folic acid supplementation beyond the first trimester on maternal plasma unmetabolised folic acid in late gestation.

INTRODUCTION: Taking folic acid containing supplements prior to and during early pregnancy reduces the risk of neural tube defects. Neural tube defects occur prior to 28 days postconception, after which, there is no proven benefit of continuing to take folic acid. However, many women continue to take folic acid containing supplements throughout the pregnancy. At higher intakes, folic acid is not converted to its active form and accumulates in circulation as unmetabolised folic acid (UMFA). Recently, concerns have been raised about possible links between late gestation folic acid supplementation and childhood allergy, metabolic disease and autism spectrum disorders. We aim to determine if removing folic acid from prenatal micronutrient supplements after 12 weeks gestation reduces circulating levels of maternal UMFA at 36 weeks gestation. METHODS AND ANALYSIS: This is a parallel-design, double-blinded randomised controlled trial. Women ≥12 and <16 weeks' gestation with a singleton pregnancy and able to give informed consent are eligible to participate. Women (n=100; 50 per group) will be randomised to receive either a micronutrient supplement containing 0.8 mg of folic acid or a micronutrient supplement without folic acid daily from enrolment until delivery. The primary outcome is plasma UMFA concentration at 36 weeks gestation. Secondary outcomes include red blood cell folate and total plasma folate concentration. We will assess whether there is a difference in mean UMFA levels at 36 weeks gestation between groups using linear regression with adjustment for baseline UMFA levels and gestational age at trial entry. The treatment effect will be described as a mean difference with 95% CI. ETHICS AND DISSEMINATION: Ethical approval has been granted from the Women's and Children's Health Network Research Ethics Committee (HREC/19/WCHN/018). The results of this trial will be presented at scientific conferences and published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: ACTRN12619001511123.

Red Blood Cell Folate Likely Overestimated in Australian National Survey: Implications for Neural Tube Defect Risk.

In 2009, the Australian government mandated the addition of folic acid to bread flour to reduce the incidence of neural tube defects (NTD)-affected pregnancies. In 2011-2012, the Australian Health Measures Survey (AHMS) reported a mean red blood cell (RBC) folate in women of reproductive age (16-44 y) of 1647 nmol/L. Over 99% of women had an RBC folate ≥ 906 nmol/L, a concentration consistent with a very low risk of NTDs if a woman became pregnant. However, RBC folate was measured using an immunoassay, which is not a recommended method due to questionable accuracy. The microbiological assay is the preferred method for RBC folate measurement. To determine whether the immunoassay method may have led to spurious conclusions about the folate status of Australian women, we collected fasting blood samples from 74 healthy non-pregnant, non-lactating women (18-44 y) and measured RBC folate using both the immunoassay and microbiological methods. Mean RBC folate (95% confidence interval) concentration measured with the immunoassay method was 1735 (1666, 1804) nmol/L compared with 942 (887, 1012) nmol/L using the microbiological method. No woman had an RBC folate < 906 nmol/L using the immunoassay method, whereas 46% of women had an RBC folate < 906 nmol/L using the microbiological method. The NTD risk was estimated to be 0.06% using the immunoassay method and 0.14% using the microbiological method. RBC folate using AHMS survey may have underestimated NTD risk in Australian women.

DNA extraction approaches substantially influence the assessment of the human breast milk microbiome.

In addition to providing nutritional and bioactive factors necessary for infant development, human breast milk contains bacteria that contribute to the establishment of commensal microbiota in the infant. However, the composition of this bacterial community differs considerably between studies. We hypothesised that bacterial DNA extraction methodology from breast milk samples are a substantial contributor to these inter-study differences. We tested this hypothesis by applying five widely employed methodologies to a mock breast milk sample and four individual human breast milk samples. Significant differences in DNA yield and purity were observed between methods (P < 0.05). Microbiota composition, assessed by 16S rRNA gene amplicon sequencing, also differed significantly with extraction methodology (P < 0.05), including in the contribution of contaminant signal. Concerningly, many of the bacterial taxa identified here as contaminants have been reported as components of the breast milk microbiome in other studies. These findings highlight the importance of using stringent, well-validated, DNA extraction methodologies for analysis of the breast milk microbiome, and exercising caution interpreting microbiota data from low-biomass contexts.

Docosahexaenoic acid supplementation of preterm infants and parent-reported symptoms of allergic disease at 7 years corrected age: follow-up of a randomized controlled trial.

BACKGROUND: Docosahexaenoic acid (DHA, 22:6n-3) supplementation in the prenatal period is associated with a reduction in the incidence of some symptoms of allergic disease. Infants born preterm are at increased risk of allergic disease, but it is unknown if DHA supplementation reduces the risk of childhood allergies. OBJECTIVES: The aim of this study was to determine if supplementation of infants born at <33 wk gestation with high-DHA compared with standard-DHA enteral feeds decreases the incidence and severity of parent-reported allergic disease symptoms at a corrected age (CA) of 7 y. METHODS: This study was a follow-up of an Australian multicenter randomized controlled trial. Infants were given high-DHA (∼1% total fatty acids) or standard-DHA (∼0.3% total fatty acids) enteral feeds from 2-4 d of postnatal age until 40 wk postmenstrual age. Parent-reported incidence of respiratory allergic disease symptoms including wheeze and rhinitis at 7 y CA were the main outcomes. Other outcomes included the incidence of eczema symptoms; severity of any symptoms; and the incidence of wheeze, rhinitis, rhinoconjunctivitis, and eczema from birth to 7 y CA. RESULTS: Data were available for 569 of 657 (87%) children originally randomized. Symptoms of wheeze or rhinitis at 7 y CA did not differ between high- and standard-DHA groups [wheeze: RR: 1.10; 95% CI: 0.73, 1.65; P = 0.66; rhinitis: RR: 1.09; 95% CI: 0.81, 1.46; P = 0.59]. There was no difference in other allergic disease symptoms at 7 y CA or in the severity of symptoms. Parent-reported symptoms of wheeze, rhinitis, rhinoconjunctivitis, or eczema from birth to 7 y CA did not differ between the groups. CONCLUSIONS: High-dose DHA supplementation of infants born at <33 wk gestation did not alter allergic disease symptoms or severity at 7 y CA, or from birth to 7 y CA compared with standard-dose DHA. This trial was registered with the Australian New Zealand Clinical Trials Registry as ANZCTR 12606000327583 (http://www.anzctr.org.au).