Effects of Omega-3-6-9 fatty acid supplementation on behavior and sleep in preterm toddlers with autism symptomatology: Secondary analysis of a randomized clinical trial.

BACKGROUND: Children born extremely preterm disproportionately experience sequelae of preterm birth compared to those born at later gestational ages, including higher prevalence of autism spectrum disorder (ASD) and associated behaviors. AIM: Explore effects of combined dietary docosahexaenoic acid, eicosapentaenoic acid, gamma-linolenic acid, and oleic acid (omega 3-6-9) on caregiver-reported behavior and sleep in toddlers born at ≤29 weeks' gestation who were exhibiting symptoms commonly seen with ASD. STUDY DESIGN: 90-day randomized (1:1), double blinded, placebo-controlled trial. SUBJECTS: Thirty-one children aged 18-38 months received omega 3-6-9 (n = 15) or canola oil placebo (n = 16). OUTCOME MEASURES: Mixed effects regression analyses followed intent to treat and explored treatment effects on measures of caregiver-reported behavior (Child Behavior Checklist 1.5-5, Toddler Behavior Assessment Questionnaire - Short Form, Vineland Adaptive Behavior Scales, 2nd Edition) and sleep (Children's Sleep Habits Questionnaire, Brief Infant Sleep Questionnaire). RESULTS: Twenty-nine of 31 (94%; ntx = 13, nplacebo = 16) children randomized had data available for at least one outcome measure, 27 (87%; ntx = 12, nplacebo = 15) had complete outcome data. Children randomized to omega 3-6-9 experienced a medium magnitude benefit of supplementation on anxious and depressed behaviors (ΔDifference = -1.27, d = -0.58, p = 0.049) and internalizing behaviors (ΔDifference = -3.41, d = -0.68, p = 0.05); and a large magnitude benefit on interpersonal relationship adaptive behaviors (ΔDifference = 7.50, d = 0.83, p = 0.01), compared to placebo. No effects were observed on other aspects of behavior or sleep. CONCLUSIONS: Findings provide preliminary support for further exploration of omega 3-6-9 during toddlerhood to improve socioemotional outcomes among children born preterm, especially for those showing early symptoms commonly seen with ASD. Results need to be replicated in a larger sample. TRIAL REGISTRATION: Registered with ClinicalTrials.gov: NCT01683565.

DHA Supplementation Attenuates Inflammation-Associated Gene Expression in the Mammary Gland of Lactating Mothers Who Deliver Preterm.

BACKGROUND: In a randomized trial of DHA supplementation to lactating mothers who delivered preterm, there were significant increases in DHA status in the mother and her infant. OBJECTIVES: Our objective here was to characterize the mammary gland transcriptomes from the above study. We hypothesized that proinflammatory gene expression would be attenuated in the increased DHA group compared with the standard DHA group. METHODS: In the original trial, mothers delivering at <29 wk gestation at the University of Cincinnati Medical Center and intending to express their milk were randomly assigned to supplementation with 200 mg/d DHA (standard group: STD) or 1000 mg/d DHA (experimental group: EXP) within 7 d of delivery. Here, we conducted RNA-seq transcriptome analysis of n = 5 EXP and n = 4 STD extracellular mammary mRNA samples extracted from the fat layer of milk samples obtained 4 wk postenrollment. Transcripts were assessed for differential expression (false discovery rate adjusted P value <0.05) and clustering between EXP compared with STD groups. Ontological analysis of all differentially expressed genes (DEGs) was performed with Toppcluster. RESULTS: There were 409 DEGs. We observed 5 main groups of biological processes that were upregulated, including those associated with improved immune regulation and management of oxidative stress; and 3 main groups of biological processes that were downregulated, including 1 associated with immune dysregulation. For example, we observed upregulation of inflammation-inhibiting genes including NFKB inhibitor alpha (NFKBIA; fold-change (FC), adjusted P value: FC = 1.70, P = 0.007) and interleukin-18 binding protein (IL18BP: FC = 2.2, adjusted P = 0.02); and downregulation of proinflammatory genes including interleukin 7 receptor (IL7R: FC = -1.9, adjusted P = 0.02) and interleukin 1 receptor like 1 (IL1RL1: FC = -13.0, adjusted P = 0.02). CONCLUSIONS: Increased DHA supplementation during lactation can modulate the expression of inflammation-related genes within the mammary gland. This might translate to milk composition with a more optimal inflammasome profile. Future research with a larger clinical trial and greater interrogation of clinical outcomes is warranted.

Randomized Controlled Trial of Omega-3 and -6 Fatty Acid Supplementation to Reduce Inflammatory Markers in Children with Autism Spectrum Disorder.

This double-blind, randomized controlled trial, tested fatty acid (FA) supplementation in children (ages 2- < 6 years) recently diagnosed with Autism Spectrum Disorder (ASD). Participants received daily oral FA supplement containing omega-3 and omega-6 FA, or a placebo for 90 days based on participant weight. Erythrocyte FAs and the cytokines, IL-1ß, IL-2, IFNγ, were measured in plasma obtained from serial blood collections. Treatment increased omega-3 and omega-6 FA levels (1.40 mol% for EPA and 1.62 mol% for DHA) and reduced IL-2 levels compared to placebo (- 0.17 pg/mL, 95% CI - 0.31, - 0.02, d = - 0.62). Omega 3-6 treatment was tolerable and adherence was greater than 70%. Future research will assess the effects of Omega 3-6 treatment on ASD symptoms. Registered on 06/08/2018 with ClinicalTrials.gov: NCT03550209.

Higher-Dose DHA Supplementation Modulates Immune Responses in Pregnancy and Is Associated with Decreased Preterm Birth.

Pregnancy and parturition involve extensive changes in the maternal immune system. In our randomized, multi-site, double-blind superiority trial using a Bayesian adaptive design, we demonstrated that 1000 mg/day of docosahexaenoic acid (DHA) was superior to 200 mg/day in preventing both early preterm birth (less than 34 weeks' gestation) and preterm birth (less than 37 weeks' gestation). The goal of this secondary study is to compare the effects of 1000 mg/day versus 200 mg/day on maternal inflammation, a possible mechanism by which DHA may prevent preterm birth. Maternal blood samples were collected at enrollment (12-20 weeks' gestation) and at delivery. Red blood cell DHA levels were measured by gas chromatography, and plasma concentrations of sRAGE, IL-6, IL-1ß, TNFα, and INFγ were measured by ELISA. Data were analyzed for associations with the DHA dose, gestational age at birth, and preterm birth (<37 weeks). Higher baseline and lower delivery levels of maternal sRAGE were associated with a greater probability of longer gestation and delivery at term gestation. Higher-dose DHA supplementation increased the probability of a smaller decrease in delivery sRAGE levels. Higher IL-6 concentrations at delivery were associated with the probability of delivering after 37 weeks, and higher-dose DHA supplementation increased the probability of greater increases in IL-6 concentrations between enrollment and delivery. These data provide a proposed mechanistic explanation of how a higher dose of DHA during pregnancy provides immunomodulatory regulation in the initiation of parturition by influencing sRAGE and IL-6 levels, which may explain its ability to reduce the risk of preterm birth.

Higher dose docosahexaenoic acid supplementation during pregnancy and early preterm birth: A randomised, double-blind, adaptive-design superiority trial.

BACKGROUND: Several meta analyses have concluded n-3 fatty acids, including docosahexaenoic acid (DHA), reduce early preterm birth (EPB, < 34 weeks), however, the amount of DHA required is unclear. We hypothesized that 1000 mg DHA per day would be superior to 200 mg, the amount in most prenatal supplements. METHODS: This randomised, multicentre, double-blind, adaptive-design, superiority trial was conducted in three USA medical centres. Women with singleton pregnancies and 12 to 20 weeks gestation were eligible. randomization was generated in SAS® by site in blocks of 4. The planned adaptive design periodically generated allocation ratios favoring the better performing dose. Managing study personnel were blind to treatment until 30 days after the last birth. The primary outcome was EPB by dose and by enrolment DHA status (low/high). Bayesian posterior probabilities (pp) were determined for planned efficacy and safety outcomes using intention-to-treat. The study is registered with ClinicalTrials.gov (NCT02626299) and closed to enrolment. FINDINGS: Eleven hundred participants (1000 mg, n = 576; 200 mg, n = 524) were enrolled between June 8, 2016 and March 13, 2020 with the last birth September 5, 2020. 1032 (n = 540 and n = 492) were included in the primary analyses. The higher dose had a lower EPB rate [1.7% (9/540) vs 2.4% (12/492), pp=0.81] especially if participants had low DHA status at enrolment [2.0% (5/249) vs 4.1%, (9/219), pp=0.93]. Participants with high enrolment DHA status did not realize a dose effect [1000 mg: 1.4% (4/289); 200 mg: 1.1% (3/271), pp = 0.57]. The higher dose was associated with fewer serious adverse events (maternal: chorioamnionitis, premature rupture of membranes and pyelonephritis; neonatal: feeding, genitourinary and neurologic problems, all pp>0.90). INTERPRETATION: Clinicians could consider prescribing 1000 mg DHA daily during pregnancy to reduce EPB in women with low DHA status if they are able to screen for DHA. FUNDING: The National Institutes of Health Child Health and Human Development (NICHD) funded the study. Life's DHA™-S oil, DSM Nutritional Products LLC, Switzerland provided all capsules.

Perinatal inflammation alters histone 3 and histone 4 methylation patterns: Effects of MiR-29b supplementation.

Preterm birth is still a major health problem and maternal inflammation has been shown to play a role. The combination of maternal inflammation and neonatal hyperoxia contributes to epigenetic changes that influence gene expression and the development of bronchopulmonary dysplasia (BPD). We have previously demonstrated suppression of miR-29b and increases in DNA methylation in infants with severe BPD and in our mouse model of maternal inflammation and neonatal hyperoxia exposure. The present studies further explored epigenetic changes in the murine model to include histone methylation. We identified a global suppression of histone methylation in exposed mice and validated decreases in expression in well-defined histone modifications, specifically H3K4me3, H3K27me3, H3K36me2, H3K79me2, and H4K20me3. We further tested the hypothesis that restoration of miR-29b expression would restore the histone methylation marks. Using lipid nanoparticle delivery of miR-29b, partial to full methylation was reestablished for H3K4me3, H3K27me3, and H4K20me3; all tri-methylation marks. To identify the causes of decreased methylation in exposed mice, we measured commonly identified methylases and demethylases. We found a decreased expression of SUV40H2, a methylase primarily associated with H4K20me3. Further studies are needed to identify the causes for the decreased global histone methylation and potential therapeutic opportunities.

A Randomized Trial of Maternal Docosahexaenoic Acid Supplementation to Reduce Inflammation in Extremely Preterm Infants.

Maternal supplementation with 1000 mg/day docosahexaenoic acid (DHA) provides third trimester DHA accretion levels in breast milk for the preterm infant. We hypothesized that DHA supplementation to mothers providing breastmilk for extremely preterm infants would result in decreased inflammatory markers, in the infant. Mother/infant dyads (n = 27) were enrolled at birth and mothers were assigned to receive 200 or 1000 mg/day of DHA. Milk and plasma samples were analyzed for fatty acids and inflammatory markers. Decreases in inflammation were observed in both maternal and infant plasma and correlated with red blood cell (RBC) DHA levels. The fact that maternal DHA supplementation decreases infant markers of inflammation implies that DHA, delivered through breastmilk, has the potential to decrease inflammation in the infant.

Effect of Docosahexaenoic Acid Supplementation vs Placebo on Developmental Outcomes of Toddlers Born Preterm: A Randomized Clinical Trial.

Importance: Intake of dietary docosahexaenoic acid (DHA) among toddlers is low. Supplementation may benefit developmental outcomes of toddlers who were born preterm. Objective: To determine whether 6 months of daily DHA supplementation improves developmental outcomes of toddlers who were born preterm. Design, Setting, and Participants: A randomized, fully masked, placebo-controlled trial was conducted from April 26, 2012, to March 24, 2017, at a large US pediatric academic center with 9 neonatal intensive care units. Children born at less than 35 weeks' gestation who were 10 to 16 months corrected age underwent 6 months of intervention. Of 2363 children assessed, 982 were eligible, 605 declined, and 377 enrolled and were randomized. Analyses were according to intent to treat. Interventions: One-to-one allocation to receive daily microencapsulated DHA, 200 mg, and arachidonic acid (AA), 200 mg (DHA+AA), or microencapsulated corn oil (placebo). Main Outcomes and Measures: The primary outcome specified a priori was Bayley Scales of Infant and Toddler Development, third edition (Bayley-III), cognitive composite score at 16 to 22 months corrected age. Secondary outcomes were Bayley-III language and motor composite scores and Infant Behavior Questionnaire-Revised and Early Childhood Behavior Questionnaire effortful control and activity level scores. Subgroup analyses defined a priori were by income, sex, and birth weight. Results: Among 377 children randomized and included in the analysis (182 girls and 195 boys; median corrected age, 15.7 months), 338 children (89.7%) had complete data on the primary outcome. Bayley-III cognitive scores did not differ between the DHA+AA and placebo groups (difference in change, 0.5 [95% CI, -1.8 to 2.8]; effect size, 0.05; P = .66). Assignment to the DHA+AA group had a small to medium negative effect on Bayley-III language scores among children with lower birth weights (eg, a child with a birth weight of 1000 g assigned to receive DHA+AA experienced a 4.1-point relative decrease, while a child assigned to placebo did not; P = .03 for interaction). Supplementation had a similar negative effect on effortful control scores among children with annual household incomes greater than $35 000 (difference in change, -0.3 [95% CI, -0.4 to -0.1]; effect size, -0.37; P = .01). Bayley-III motor scores and activity level scores were unaffected. Conclusions and Relevance: Daily supplementation with 200 mg of DHA and 200 mg of AA for 6 months resulted in no improvement in cognitive development and early measures of executive function vs placebo, and may have resulted in negative effects on language development and effortful control in certain subgroups of children. These findings do not support DHA supplementation in the second year of life for children who are born preterm. Trial Registration: ClinicalTrials.gov Identifier: NCT01576783.

ω-3 and ω-6 Fatty Acid Supplementation May Reduce Autism Symptoms Based on Parent Report in Preterm Toddlers.

Background: Children born preterm are at increased risk of autism spectrum disorder (ASD). n-3 (ω-3) Combined with n-6 (ω-6) fatty acids including γ-linolenic acid (GLA) may benefit children born preterm showing early signs of ASD. Previous trials have reported that docosahexaenoic acid (DHA) promotes cognitive development in preterm neonates and n-3 fatty acids combined with GLA improve attention-deficit-hyperactivity disorder. Objectives: The objectives of the pilot Preemie Tots Trial were 1) to confirm the feasibility of a full-scale trial in toddlers born very preterm and exhibiting ASD symptoms and 2) to explore the effects of supplementation on parent-reported ASD symptoms and related behaviors. Methods: This was a 90-d randomized, fully blinded, placebo-controlled trial in 31 children 18-38 mo of age who were born at ≤29 wk of gestation. One group was assigned to daily Omega-3-6-9 Junior (Nordic Naturals, Inc.) treatment (including 338 mg eicosapentaenoic acid, 225 mg DHA, and 83 mg GLA), and the other group received canola oil (124 mg palmitic acid, 39 mg stearic acid, 513 mg linoleic acid, 225 mg α-linolenic acid, and 1346 mg oleic acid). Mixed-effects regression analyses followed intent-to-treat analysis and explored effects on parent-reported ASD symptoms and related behaviors. Results: Of 31 children randomly assigned, 28 had complete outcome data. After accounting for baseline scores, those assigned to treatment exhibited a greater reduction in ASD symptoms per the Brief Infant Toddler Social Emotional Assessment ASD scale than did those assigned to placebo (difference in change = - 2.1 points; 95% CI: - 4.1, - 0.2 points; standardized effect size = - 0.71). No other outcome measure reflected a similar magnitude or a significant effect. Conclusions: This pilot trial confirmed adequate numbers of children enrolled and participated fully in the trial. No safety concerns were noted. It also found clinically-significant improvements in ASD symptoms for children randomly assigned to receive Omega-3-6-9 Junior, but effects were confined to one subscale. A future full-scale trial is warranted given the lack of effective treatments for this population. This trial was registered at www.clinicaltrials.gov as NCT01683565.

Omega-3 and -6 fatty acid supplementation and sensory processing in toddlers with ASD symptomology born preterm: A randomized controlled trial.

BACKGROUND: Despite advances in the health and long-term survival of infants born preterm, they continue to face developmental challenges including higher risk for autism spectrum disorder (ASD) and atypical sensory processing patterns. AIMS: This secondary analysis aimed to describe sensory profiles and explore effects of combined dietary docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and gamma-linolenic acid (GLA) supplementation on parent-reported sensory processing in toddlers born preterm who were exhibiting ASD symptoms. STUDY DESIGN: 90-day randomized, double blinded, placebo-controlled trial. SUBJECTS: 31 children aged 18-38months who were born at ≤29weeks' gestation. OUTCOME MEASURE: Mixed effects regression analyses followed intent to treat and explored effects on parent-reported sensory processing measured by the Infant/Toddler Sensory Profile (ITSP). RESULTS: Baseline ITSP scores reflected atypical sensory processing, with the majority of atypical scores falling below the mean. Sensory processing sections: auditory (above=0%, below=65%), vestibular (above=13%, below=48%), tactile (above=3%, below=35%), oral sensory (above=10%; below=26%), visual (above=10%, below=16%); sensory processing quadrants: low registration (above=3%; below=71%), sensation avoiding (above=3%; below=39%), sensory sensitivity (above=3%; below=35%), and sensation seeking (above=10%; below=19%). Twenty-eight of 31 children randomized had complete outcome data. Although not statistically significant (p=0.13), the magnitude of the effect for reduction in behaviors associated with sensory sensitivity was medium to large (effect size=0.57). No other scales reflected a similar magnitude of effect size (range: 0.10 to 0.32). CONCLUSIONS: The findings provide support for larger randomized trials of omega fatty acid supplementation for children at risk of sensory processing difficulties, especially those born preterm.

Effect of Omega-3 and -6 Supplementation on Language in Preterm Toddlers Exhibiting Autism Spectrum Disorder Symptoms.

Delayed language development may be an early indicator of autism spectrum disorder (ASD). Early intervention is critical for children with ASD, and the present study presents pilot data on a clinical trial of omega-3 and -6 fatty acid supplementation and language development, a secondary trial outcome, in children at risk for ASD. We randomized 31 children to receive an omega-3 and -6 supplement or a placebo for 3 months, and measured their language abilities at baseline and after supplementation. Gesture use, but not word production, increased for children in the treatment group more than children in the placebo group. These results suggest possible effectiveness of omega-3 and -6 supplementation for language development in children at risk for ASD.

Lactational Stage of Pasteurized Human Donor Milk Contributes to Nutrient Limitations for Infants.

BACKGROUND: Mother's own milk is the first choice for feeding preterm infants, but when not available, pasteurized human donor milk (PDM) is often used. Infants fed PDM have difficulties maintaining appropriate growth velocities. To assess the most basic elements of nutrition, we tested the hypotheses that fatty acid and amino acid composition of PDM is highly variable and standard pooling practices attenuate variability; however, total nutrients may be limiting without supplementation due to late lactational stage of the milk. METHODS: A prospective cross-sectional sampling of milk was obtained from five donor milk banks located in Ohio, Michigan, Colorado, Texas-Ft Worth, and California. Milk samples were collected after Institutional Review Board (#07-0035) approval and informed consent. Fatty acid and amino acid contents were measured in milk from individual donors and donor pools (pooled per Human Milk Banking Association of North America guidelines). Statistical comparisons were performed using Kruskal-Wallis, Spearman's, or Multivariate Regression analyses with center as the fixed factor and lactational stage as co-variate. RESULTS: Ten of the fourteen fatty acids and seventeen of the nineteen amino acids analyzed differed across Banks in the individual milk samples. Pooling minimized these differences in amino acid and fatty acid contents. Concentrations of lysine and docosahexaenoic acid (DHA) were not different across Banks, but concentrations were low compared to recommended levels. CONCLUSIONS: Individual donor milk fatty acid and amino acid contents are highly variable. Standardized pooling practice reduces this variability. Lysine and DHA concentrations were consistently low across geographic regions in North America due to lactational stage of the milk, and thus not adequately addressed by pooling. Targeted supplementation is needed to optimize PDM, especially for the preterm or volume restricted infant.

Assessment of DHA on reducing early preterm birth: the ADORE randomized controlled trial protocol.

BACKGROUND: Preterm birth contributes to 0.5 million deliveries in the United States (one of eight pregnancies) and poses a huge burden on public health with costs in the billions. Of particular concern is that the rate of earliest preterm birth (<34 weeks) (ePTB), which has decreased little since 1990 and has the greatest impact on the overall infant mortality, resulting in the greatest cost to society. Docosahexaenoic acid (DHA) supplementation provides a potential high yield, low risk strategy to reduce early preterm delivery in the US by up to 75%. We propose a Phase III Clinical Trial (randomized to low or high dose DHA, double-blinded) to examine the efficacy and safety of high dose DHA supplementation to reduce ePTB. We also plan for a secondary pregnancy efficacy analysis to determine if there is a subset of pregnancies most likely to benefit from DHA supplementation. METHODS: Between 900 and 1200 pregnant women who are ≥ 18 years old and between 12 and 20 weeks gestation will be recruited from three trial experienced academic medical institutions. Participants will be randomly assigned to two daily capsules of algal oil (totaling 800 mg DHA) or soybean and corn oil (0 mg DHA). Both groups will receive a commercially available prenatal supplement containing 200 mg DHA. Therefore, the experimental group will receive 1000 mg DHA/d and the control group 200 mg DHA/d. We will then employ a novel Bayesian response adaptive randomization design that assigns more subjects to the "winning" group and potentially allows for substantially smaller sample size while providing a stronger conclusion regarding the most effective group. The study has an overall Type I error rate of 5% and a power of 90%. Participants are followed throughout pregnancy and delivery for safety and delivery outcomes. DISCUSSION: We hypothesize that DHA will decrease the frequency of ePTB <34 weeks. Reducing ePTB is clinically important as these earliest preterm deliveries carry the highest risk of neonatal morbidity, as well as contribute significant stress for families and post a large societal burden. TRIAL REGISTRATION: This trial was registered with ClinicalTrials.gov (identifier: NCT02626299 ) on December 8, 2015. Additional summary details may be found in Table 1.

Alterative Expression and Localization of Profilin 1/VASPpS157 and Cofilin 1/VASPpS239 Regulates Metastatic Growth and Is Modified by DHA Supplementation.

Profilin 1, cofilin 1, and vasodialator-stimulated phosphoprotein (VASP) are actin-binding proteins (ABP) that regulate actin remodeling and facilitate cancer cell metastases. miR-17-92 is highly expressed in metastatic tumors and profilin1 and cofilin1 are predicted targets. Docosahexaenoic acid (DHA) inhibits cancer cell proliferation and adhesion. These studies tested the hypothesis that the metastatic phenotype is driven by changes in ABPs including alternative phosphorylation and/or changes in subcellular localization. In addition, we tested the efficacy of DHA supplementation to attenuate or inhibit these changes. Human lung cancer tissue sections were analyzed for F-actin content and expression and cellular localization of profilin1, cofilin1, and VASP (S157 or S239 phosphorylation). The metastatic phenotype was investigated in A549 and MLE12 cells lines using 8 Br-cAMP as a metastasis inducer and DHA as a therapeutic agent. Migration was assessed by wound assay and expression measured by Western blot and confocal analysis. miR-17-92 expression was measured by qRT-PCR. Results indicated increased expression and altered cellular distribution of profilin1/VASP(pS157), but no changes in cofilin1/VASP(pS239) in the human malignant tissues compared with normal tissues. In A549 and MLE12 cells, the expression patterns of profilin1/VASP(pS157) or cofilin1/VASP(pS239) suggested an interaction in regulation of actin dynamics. Furthermore, DHA inhibited cancer cell migration and viability, ABP expression and cellular localization, and modulated expression of miR-17-92 in A549 cells with minimal effects in MLE12 cells. Further investigations are warranted to understand ABP interactions, changes in cellular localization, regulation by miR-17-92, and DHA as a novel therapeutic. Mol Cancer Ther; 15(9); 2220-31. ©2016 AACR.

DHA Suppresses Primary Macrophage Inflammatory Responses via Notch 1/ Jagged 1 Signaling.

Persistent macrophages were observed in the lungs of murine offspring exposed to maternal LPS and neonatal hyperoxia. Maternal docosahexaenoic acid (DHA) supplementation prevented the accumulation of macrophages and improved lung development. We hypothesized that these macrophages are responsible for pathologies observed in this model and the effects of DHA supplementation. Primary macrophages were isolated from adult mice fed standard chow, control diets, or DHA supplemented diets. Macrophages were exposed to hyperoxia (O2) for 24 h and LPS for 6 h or 24 h. Our data demonstrate significant attenuation of Notch 1 and Jagged 1 protein levels in response to DHA supplementation in vivo but similar results were not evident in macrophages isolated from mice fed standard chow and supplemented with DHA in vitro. Co-culture of activated macrophages with MLE12 epithelial cells resulted in the release of high mobility group box 1 and leukotriene B4 from the epithelial cells and this release was attenuated by DHA supplementation. Collectively, our data indicate that long term supplementation with DHA as observed in vivo, resulted in deceased Notch 1/Jagged 1 protein expression however, DHA supplementation in vitro was sufficient to suppress release LTB4 and to protect epithelial cells in co-culture.

DHA suppresses chronic apoptosis in the lung caused by perinatal inflammation.

We have previously shown that an adverse perinatal environment significantly alters lung growth and development and results in persistently altered cardiopulmonary physiology in adulthood. Our model of maternal LPS treatment followed by 14 days of neonatal hyperoxia exposure causes severe pulmonary disease characterized by permanent decreases in alveolarization and diffuse interstitial fibrosis. The current investigations tested the hypothesis that dysregulation of Notch signaling pathways contributes to the permanently altered lung phenotype in our model and that the improvements we have observed previously with maternal docosahexaenoic acid (DHA) supplementation are mediated through normalization of Notch-related protein expression. Results indicated that inflammation (IL-6 levels) and oxidation (F2a-isoprostanes) persisted through 8 wk of life in mice exposed to LPS/O2 perinatally. These changes were attenuated by maternal DHA supplementation. Modest but inconsistent differences were observed in Notch-pathway proteins Jagged 1, DLL 1, PEN2, and presenilin-2. We detected substantial increases in markers of apoptosis including PARP-1, APAF-1, caspase-9, BCL2, and HMGB1, and these increases were attenuated in mice that were nursed by DHA-supplemented dams during the perinatal period. Although Notch signaling is not significantly altered at 8 wk of age in mice with perinatal exposure to LPS/O2, our findings indicate that persistent apoptosis continues to occur at 8 wk of age. We speculate that ongoing apoptosis may contribute to persistently altered lung development and may further enhance susceptibility to additional pulmonary disease. Finally, we found that maternal DHA supplementation prevented sustained inflammation, oxidation, and apoptosis in our model.

Maternal dietary docosahexaenoic acid supplementation attenuates fetal growth restriction and enhances pulmonary function in a newborn mouse model of perinatal inflammation.

The preterm infant is often exposed to maternal and neonatal inflammatory stimuli and is born with immature lungs, resulting in a need for oxygen therapy. Nutritional intervention with docosahexaenoic acid (DHA; 6.3 g/kg of diet) has been shown to attenuate inflammation in various human diseases. Previous studies demonstrated that maternal DHA supplementation during late gestation and lactation attenuated hyperoxic lung injury in newborn mouse pups. In the present studies, we tested the hypothesis that DHA supplementation to the dam would reduce hyperoxic lung injury and growth deficits in a more severe model of systemic maternal inflammation, including lipopolysaccharide (LPS) and neonatal hyperoxia exposure. On embryonic day 16, dams were placed on DHA (6.3 g DHA/kg diet) or control diets and injected with saline or LPS. Diets were maintained through weaning. At birth, pups were placed in room air or hyperoxia for 14 d. Improvements in birth weight (P < 0.01), alveolarization (P ≤ 0.01), and pulmonary function (P ≤ 0.03) at 2 and 8 wk of age were observed in pups exposed to perinatal inflammation and born to DHA-supplemented dams compared with control diet-exposed pups. These improvements were associated with decreases in tissue macrophage numbers (P < 0.01), monocyte chemoattractant protein-1 expression (P ≤ 0.05), and decreases in soluble receptor for advanced glycation end products concentrations (P < 0.01) at 2 and 8 wk. Furthermore, DHA supplementation attenuated pulmonary fibrosis, which was associated with the reduction of matrix metalloproteinases 2, 3, and 8 (P ≤ 0.03) and collagen mRNA (P ≤ 0.05), and decreased collagen (P < 0.01) and vimentin (P ≤ 0.03) protein concentrations. In a model of severe inflammation, maternal DHA supplementation lessened inflammation and improved lung growth in the offspring. Maternal supplementation with DHA may be a therapeutic strategy to reduce neonatal inflammation.

Randomized controlled trial of docosahexaenoic acid supplementation in midwestern U.S. human milk donors.

BACKGROUND: Docosahexaenoic acid (DHA) is a long-chain polyunsaturated fatty acid important for neonatal neurodevelopment and immune homeostasis. Preterm infants fed donor milk from a Midwestern source receive only 20% of the intrauterine accretion of DHA. We tested the hypothesis that DHA supplementation of donor mothers would provide preterm infants with DHA intake equivalent to fetal accretion. SUBJECTS AND METHODS: After Institutional Review Board approval and informed consent, human milk donors to the Mother's Milk Bank of Ohio were randomized to receive 1 g of DHA (Martek(®) [now DSM Nutritional Lipids, Columbia, MD]) or placebo soy oil. Dietary intake data were collected and analyzed by a registered dietitian. Fatty acids were measured by gas chromatography/flame ionization detection. Statistical analysis used linear mixed models. RESULTS: Twenty-one mothers were randomly assigned to either the DHA group (n=10) or the placebo group (n=11). Donor age was a median of 31 years in both groups with a mean lactational stage of 19 weeks. Dietary intake of DHA at baseline in both groups was a median of 23 mg/day (range, 0-194 mg), significantly (p<0.0001) less than the minimum recommended intake of 200 mg/day. The DHA content of milk increased in the DHA-supplemented group (p<0.05). CONCLUSIONS: The women enrolled in this study had low dietary DHA intake. Supplementation with preformed DHA at 1 g/day resulted in increased DHA concentrations in the donor milk with no adverse outcomes. Infants fed donor milk from supplemented women receive dietary DHA levels that closely mimic normal intrauterine accretion during the third trimester.