Docosahexaenoic Acid Supplementation of Mothers to Improve Preterm Infant Nutrition and Immune Homeostasis
Phase: PHASE1
Conditions: Docosohexaenoic Acid Supplementation of Mothers to
Sponsor: Children's Hospital Medical Center, Cincinnati

 
 
 
 
 
 
Official title: DHA Supplementation for Lactating Mothers (DHA-1) 
NCT number: NCT01732874 
Date of Protocol with SAP: 7/12/2013 

Ardythe Morrow PhD., 
The University of Cincinnati / Cincinnati Children’s Hospital Medical Center, 
Docosahexaenoic Acid Supplementation to Mothers to Improve Preterm Infant Nutrition and 
Immune Homeostasis 
July 12, 2013 
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                  Docosohexaenoic Acid Supplementation of Mothers to 
Improve Preterm Infant Nutrition and Immune Homeostasis 
 
Abstract 
 
Background: 
Preterm infants miss the last trimester of nutrient accretion.  Inflammatory morbidities are 
common in this population.  The American Academy of Pediatrics recommends intrauterine 
accretion for nutrient goals.  Docosahexaenoic (DHA) is an anti-inflammatory dietary fatty acid 
and is accreted over the last trimester at 65mg/day. 
Preliminary data:  We measured DHA levels in breast milk from 38 mothers and found lower 
concentrations than those recommended (0.1 mol wt %).  Dietary intake of DHA was lower than 
that recommended for nursing mothers (23 vs 200 mg/day, p<0.001).  When we supplemented 
with a placebo (n=5) vs. 1 gram of DHA (n=6) mother’s tolerated it well.  The daily intake by a 
preterm infant was improved to a concentration that mimicked intrauterine intake when the 
mother took 1 gram of DHA per day. 
Hypothesis:  We designed a prospective, randomized trial to test the hypotheses that DHA 
supplementation to human milk providers will increase DHA concentration in milk and infant 
blood levels; and that infants receiving milk from DHA-supplemented providers will be receiving 
a more appropriate enteral intake to better mimic intrauterine accretion.  In addition, we 
hypothesize that pro-inflammatory cytokines will be reduced in the mothers and infants receiving 
the higher DHA diet. 
Study Design:  Breast milk providers for enterally-fed preterm infants <29 weeks gestational 
age will be randomized to receive daily oral supplementation with DHA at 200mg or 1000mg.  
Based on our previous study with a placebo, it was apparent that even motivated mothers had a 
paucity of DHA in the diet and in their prenatal supplement therefore we felt it important to 
randomize to at least the baseline 200mg per day and then added the other dose as the target 
optimal dose for milk concentrations to achieve 0.8 mol weight percent.  This concentration of 
DHA would provide intrauterine accretion when fed at 150ml/kg per day in the NICU.  DHA 
concentration will be analyzed by gas chromatography in milk and infant cell membranes.  Milk 
and infant plasma cytokine levels and cytokine production capacity will be measured. All 
samples will be obtained at baseline (within first week of life), weeks 2, 4, and 8.  Inflammatory 
complications (BPD, NEC, and ROP) will be tracked.  Cytokine gene expression from RNA 
extracted from the Week 4 milk samples will also be examined. Stool samples will not only have 
fatty acids but also bacteria and yeast analyzed. 
 
Significance:  Our unique investigation of maternal supplementation to improve DHA delivery 
to preterm infants represents the first study of its kind. 
Purpose 
The objective of this project is to improve preterm infant intake of DHA that approximate the levels 
obtained by fetal accretion during the third trimester of pregnancy and determine if there are 
functional relationships to inflammation, growth, and development.  
 
The following specific aims will test the hypotheses that: 
 
Aim 1: Maternal DHA supplementation will improve plasma and milk DHA concentration in the 
mother and preterm infant  
Aim 2: Higher intakes of dietary DHA will decrease expression of pro-inflammatory cytokines and 
increase expression of anti-inflammatory cytokines in the plasma and milk samples of  

Ardythe Morrow PhD., 
The University of Cincinnati / Cincinnati Children’s Hospital Medical Center, 
Docosahexaenoic Acid Supplementation to Mothers to Improve Preterm Infant Nutrition and 
Immune Homeostasis 
July 12, 2013 
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breast feeding mother - infant dyads through the alteration of sRAGE signaling. This will result in 
an improved microbiome as evidence of bacteria and yeast in stool samples. 
 
Background:  
Preterm infants miss the last trimester of nutrient accretion and may be deficient in essential 
molecules.  These deficiencies may contribute to increased rates of infection and make these 
infants more vulnerable to disease [1].   Long chain omega-3 fatty acids, in particular 
docosahexaenoic acid (DHA), are preferentially transported across the placenta and provide 
components of membrane phospholipids that have been associated with improved markers of 
brain and retinal development [2-4].  Because the bulk of this transport occurs in the third 
trimester of pregnancy, premature infants do not benefit from this increase in DHA delivery.  In 
adults, DHA can be synthesized from its precursor, linolenic acid - an essential fatty acid - 
through a series of elongase and desaturase enzyme activities that may be limited in the 
preterm infant [5].  The preterm infant may be particularly vulnerable to biosynthesis of DHA 
because of dietary protein limitations and steroid use thereby making a preformed dietary DHA 
source imperative – figure 1 (Valentine, Adv. Nutr, 2012).   
 
 
Likewise, these DHA synthesizing enzymes are competitively shared by the omega-6 fatty acid 
series.  Both the omega 6 and the omega 3 fatty acids, provide substrates for a host of pro- and 
anti-inflammatory lipid mediators.  Omega 6 fatty acids are closely associated with the 
production of pro-inflammatory mediators and indirectly influence the production of tumor 
necrosis factor (TNF)-α.  Conversely, omega-3 fatty acids are more closely linked to 
suppression of inflammation and production of anti-inflammatory or pro-resolution molecules.  
Specifically, DHA has been shown to suppress NFkB activation which results in decreased 
inflammatory responses and increased production of anti-inflammatory mediators such as 
interleukin (IL)-10 [6]. The maternal murine model supplemented with DHA promotes less 
inflammatory mediators in the lung of the pup and results in significantly better survival.[7] 
Deficits in nutrient sources of omega-3 long chain fatty acids, specifically DHA, may influence 
the infant’s immune balance and contribute to development of disease [8, 9] [10] [11, 12].   
 
 

Ardythe Morrow PhD., 
The University of Cincinnati / Cincinnati Children’s Hospital Medical Center, 
Docosahexaenoic Acid Supplementation to Mothers to Improve Preterm Infant Nutrition and 
Immune Homeostasis 
July 12, 2013 
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Infants fed diets supplemented with DHA have higher levels of DHA in red blood cell 
phospholipids [13], and have demonstrated improved functional outcomes [14, 15].  While 
dietary sources of DHA may ultimately qualify as semi-essential for the preterm infant, DHA is 
currently lacking in the intravenous nutrition used in neonatal intensive care units (NICU) [16].  
Current estimates are that 67 mg/day of DHA is accumulated in utero during the last trimester 
[2] The American Academy of Pediatrics recommends that preterm infant feeding goals mimic 
intrauterine nutrient intake as the most logical approach to achieving  appropriate nutritional 
levels [17].  However, stable isotope studies indicate  a preterm intake of only 4 mg/day [18], far 
below intrauterine nutrient intake. Human milk is the preferred enteral nutrition for the preterm 
infant and contains vital anti-infective properties but milk DHA concentrations vary among 
mothers, dependent on maternal dietary intake [19-22].  Previous studies have demonstrated 
that fatty acid supplementation improves human milk fatty acid composition [23, 24] but levels 
of human milk DHA that would approximate in utero accretion for the human milk-fed 
preterm infant have never been targeted.  
 
Epidemiologic studies have demonstrated that maternal intakes of 1 gram of DHA correspond to 
1 mole weight % in human milk [25]. Dietary intake of DHA up to 3 grams per day are 
considered GRAS by the FDA [26].  A diet of human milk with 1% DHA at 150 ml/kg would 
better achieve intrauterine intake of 67 mg/day. Animal studies have shown reduced rates of 
necrotizing enterocolitis (NEC) when fed omega-3 fatty acid supplemented diets [27]. A dietary 
strategy that seeks to approximate intrauterine accretion values of DHA by improving the 
mother’s diet could therefore promote improved nutrient availability and more appropriate 
immune response in preterm infants. In our previous 2 years we have demonstrated that the 
current milk composition of DHA is less than optimal for the preterm infant [28] and that a RCT 
of 1 gram maternal dietary DHA intake vs. a placebo soy oil resulted in enhanced DHA  milk 
composition equal to intrauterine concentration provided to the NICU infant (Valentine, 2011 
manuscript in review)- figure 2.  
 
 
 
 
 
 
 
Figure 2: Milk concentration of DHA in 
DHA supplemented women, Valentine 
C, Breastfeeding Medicine, 2012 

Ardythe Morrow PhD., 
The University of Cincinnati / Cincinnati Children’s Hospital Medical Center, 
Docosahexaenoic Acid Supplementation to Mothers to Improve Preterm Infant Nutrition and 
Immune Homeostasis 
July 12, 2013 
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Although the mechanisms by which DHA decreases inflammation are not understood, the anti-
inflammatory properties of long chain fatty acids include changes in membrane fluidity, effects 
on signaling pathways resulting in modified gene transcription, and enhanced production of anti-
inflammatory lipid mediators due to the availability of DHA as a substrate [29, 30]. Receptor for 
Advanced Glycation End Products (RAGE) is described as a “Damage Associated Molecular  
Pattern” receptor and as such is able to engage classes of unrelated molecules using tertiary 
structure for ligand recognition [31]. RAGE exists in multiple forms; a membrane bound form  
(mRAGE), a splice-variant soluble form (svRAGE), and a cleaved form (sRAGE) generated by 
protease activity from mRAGE. In the context of lung injury, the source of cleaved sRAGE 
protein is likely type I cells or alveolar macrophages, and greater levels of sRAGE in BAL or 
plasma are linked to the severity of lung injury [31] specifically capillary leak and fluid clearance 
[32].We hypothesize that DHA will suppress the production of inflammatory cytokines and 
chemokines in both the mother and the preterm infant through decreasing RAGE expression 
and signaling.  
 
Understanding this relationship will help determine if maternal dietary supplementation 
strategies with DHA are optimal or if infant direct supplementation is necessary to 
improve immune homeostasis. 
 
Study Design: We propose a prospective, randomized, trial of oral DHA supplementation of 
lactating mothers. Cincinnati Children’s Hospital research staff will screen all infants admitted to 
the Cincinnati University Hospital NICU on a weekly basis. Eligible participants will be identified 
by the Principle Investigator and the research coordinator. Potential participants (mothers) will 
be approached regarding the possibility of study participation by study staff trained in the 
consent process. Eligible participants will be screened and if eligible and interested, will be 
consented within one week of being admitted to Cincinnati University Hospital NICU.  Maternal 
consent will be obtained for the infant. Consent will be administered by Cincinnati Children’s 
Hospital Medical Center staff trained in the consent process and having completed all required 
CITI training.  Informed consent will be documented by an Informed Consent Process Note.  All 
data and infants biological sample collection will take place during the infants stay in Cincinnati 
University Hospital NICU. Mother’s biological samples will be collected by Cincinnati University 
Hospital staff, CCHMC study staff and Hoxworth outpatient lab. 
 
After informed consent, lactating mothers will be randomized by The Cincinnati University 
Hospital Investigational Pharmacy to receive DHA (DHA- Martek Biosciences, now known as 
DSM Nutritional Lipid- Expecta®) 200 mg, or 1000 mg/day.  The randomization schedule has 
been devised in collaboration with the Biostatistics expert, Dr. Ardythe Morrow, identified in 
these studies. We have used the Consolidated Standards of Reporting Trials 
(CONSORT)framework [33]. 
Participants who meet any exclusion criteria during the course of the study, are non-compliant 
or are lost to follow-up will be withdrawn.  Provided that there are at least 8 mother/baby pairs 
with evaluable data in each group, withdrawn participants will not be replaced.  Anticipated 
enrollment will take two years.   
  
Inclusion criteria: Lactating mothers at Cincinnati University Hospital with infants born at < 29 
weeks gestation admitted to The Cincinnati University Hospital NICU 
 
 
 

Ardythe Morrow PhD., 
The University of Cincinnati / Cincinnati Children’s Hospital Medical Center, 
Docosahexaenoic Acid Supplementation to Mothers to Improve Preterm Infant Nutrition and 
Immune Homeostasis 
July 12, 2013 
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Exclusion criteria:  
 
 
Infants with the presence of congenital anomalies (trisomy 13, 18, 21, urethral, 
gastrointestinal and cardiac defects) that affect the standard course of care.   
 
 
mother’s <18 and  
 
mothers with known allergy to algeal source 
 
mothers with history of bleeding disorders or taking any other medication that may 
increase the risk of bleeding 
 
infants with bleeding disorders or receiving any other medication that may increase the 
risk of bleeding 
 
Study Procedures: 
   
Maternal Dietary intake and breastmilk samples will be collected and assessed at initiation of 
supplementation and at weeks 2, 4, and 8.  Maternal breast expression and breastfeeding 
patterns; anthropometry (weight, height, circumferences); exercise patterns; and smoking habits 
will be obtained at week 4. 
 
Maternal blood and buccal swabs (saliva) will be collected and assessed at baseline and at 
weeks 4, and 8.   
 
Infant Dietary intake, blood samplesbuccal swabs (saliva), and stool samples will be 
collected and assessed at baseline and at weeks 2, 4, and 8. – see Figure 3. 
 
Maternal blood collected will be 5 mL at each collection therefore a total of 15 mL whole blood 
collected for the study.  Infant blood collected will be ~ 1 mL at each collection therefore a total 
of ~5 mL whole blood collected. The infant blood will be collected during routine nutritional lab 
collections which are clustered with standard of care. 
The dietary and supplemental intake of the mother will be assessed at The Bionutrition Center 
using NDSR software and the dietary intake of the infant will be calculated by the Neonatal 
Dietitian. The fatty acid contents of the breastmilk, blood, and stool samples will be measured 
by gas chromatography in our laboratory using Bligh-Dyer extraction (Chloroform: Methanol 
(2:1))[34]. Cytokine (IL-10, IL 6, IL-8, IL 1B, TNF a, TGF, adiponectin, leptin) and sRAGE 
concentrations will be examined in maternal milk, and maternal and infant blood samples using 
a MSD SECTOR® Imager 2400A.  This instrument allows for the analyses of 40 individual 
samples in duplicate at one time using a 96-well format.  Infant growth, clinical outcome, and 
development data will be collected. Breastmilk samples will be analyzed for fatty acids and 
certain proteins as is routinely analyzed at Human milk banks of North America and clinically at 
Cincinnati Children’s Hospital Medical Center. Cytokine analysis will be performed on infant 
stool samples. Saliva samples will be analyzed for genetic polymorphisms.  Buccal swabs 
(saliva) will be analyzed for fatty acids. 
 An additional teaspoon of fresh milk will be collected at Week 4 and aliquoted into whole, 
aqueous, and fat layer fractions. Fat layer RNA will be isolated using a Maxwell™16 integrated 
system (Promega, Madison, WI). RNA quantity (target=1000 ng) and quality (A260:A280 target 
>1.9) will be confirmed prior to RNA sequencing. Na:K ratio, as a marker of mammary gland 
inflammation, will be assayed on the milk aqueous fraction.            
All biological samples will be collected within study time frames as feasible. 
 

Ardythe Morrow PhD., 
The University of Cincinnati / Cincinnati Children’s Hospital Medical Center, 
Docosahexaenoic Acid Supplementation to Mothers to Improve Preterm Infant Nutrition and 
Immune Homeostasis 
July 12, 2013 
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Stool will also have bacteria and yeast measured by using 16S ribosomal markers. 
Growth and Hospitalization 
Growth measures are standard of care at University NICU and include weekly weight, length, 
and head circumference measurements and will be extracted from the participant’s charts.  
At discharge we will obtain body composition on the infant using a validated isolette that has 
non-invasive, volume and mass measurement capability. Cincinnati University Hospital course 
records that could be pertinent to the study outcomes will be collected from both mother and 
infant. Compliance will be assessed as part of diet record evaluations; rate of compliance will 
not be assessed 
 
Statistical Plan: All data analyses will employ intention to treat principles. In our primary analysis, 
ANOVA will be used to compare DHA levels at four weeks in the breast milk of mothers  
assigned to the different concentrations of DHA supplements. As in any prospective study, If we 
experience substantial loss to follow-up (e.g., > 10% of subjects), we will fit a linear mixed 
model [35]  We will use two sample t tests to compare infant blood DHA concentrations and 
TNF-alpha and IL-10 levels at 8 weeks of infants randomized to DHA-supplemented milk.  The 
comparison of DHA concentrations will be performed at a significance level of 0.05 while the 
comparison of TNF-alpha and IL-10 levels will be performed at a significance level of 0.025 to 
adjust for the two outcomes.  All analyses will be performed using SAS (SAS Inc., Cary, NC). 
Our sample size determination to detect a two-fold difference in the DHA levels at four weeks in  
expressed milk of mothers assigned DHA treatments, we will need data on a total of 16 mothers 
at week 4 (8 per group).  This sample size calculation was based on 90% power, a two-sided 
significance level of 0.025, and a coefficient of variation (CV) of 30%; the CV is based on a  
previous study [36]. Assuming a conservative 20% loss to follow-up before week 4, we will 
enroll enough mother-infant pairs to achieve our desired sample size of 16 at 4 weeks. It is 
expected that evaluable data at week 4 from at least sixteen of the mother/baby pairs (8 pairs 
per group) will be obtained. The University Hospital Investigational Pharmacy will randomize 
participants per the Consolidated Standards of Reporting Trials (CONSORT) framework.  
 Secondary outcomes will use linear mixed models and Bonferroni correction. 
  Among participants with adequate quality milk fat layer RNA at Week 4, five pairs will be 
randomly selected from each group for sequencing (matched on gestational week of delivery,  
milk DHA concentration at baseline, and Week 4 breast milk output level (exceeds, at, or below 
infant’s needs). The remaining will be set aside for future studies. RNA integrity (RIN >7) will be 
confirmed at the microarray core and then sent to the gene discovery core for RNA-Seq library 
preparation (Illumina TruSeq RNA kit) and sequencing (Illumina Hi-Seq2500). Libraries will be 
multiplexed in batches of 10 with at least 30 million 150bp paired-end reads per sample. 
Sequence reads will be trimmed for quality and then aligned to the human genome using 
TopHat. We will use Cufflinks to determine gene expression levels, normalized to Fragments 
Per Kilobase of transcript per Million mapped reads (fpkm). We will identify differentially 
expressed genes using DESeq and intersect this gene list with known cytokine signaling genes. 
We will also conduct Gene Set Enrichment Analysis. Significant biomarker genes will be 
assayed for all participants using TaqMan™ qPCR. Power: N=5 pairs per group enables 90% 
power (=0.05) to detect 1.5-fold differences between DHA versus placebo for moderate to high 
abundance genes (FPKM > 10) and 3.0-fold differences for low abundance genes (FPKM ~1.0). 
 
 
 
 
 

Ardythe Morrow PhD., 
The University of Cincinnati / Cincinnati Children’s Hospital Medical Center, 
Docosahexaenoic Acid Supplementation to Mothers to Improve Preterm Infant Nutrition and 
Immune Homeostasis 
July 12, 2013 
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Safety: All records will be kept according to IRB and regulatory standards. All PHI will be stored 
in a password protected database. All biological sample data will be kept in a separate database 
from PHI and study identifiers. Written research records, participant contact information, and  
signed consent forms will be kept in a locked file cabinet near the study coordinator’s work 
station.  All data will be entered from source documents into a Redcap database using a  
password protected, secure server. Individually identifiable information will not be included in 
the data set or in any reports. 
Laboratory samples will be de-identified and stored at Cincinnati Children’s Hospital Medical 
Center and Nationwide Children’s in Columbus Ohio.. 
All supplements will be stored in the Investigational pharmacy at Cincinnati University Hospital.  
FDA Investigator IND was accepted and in addition regards DHA as a generally regarded as 
safe (GRAS) category with levels < 3 grams per day in the adult diet.  Our supplement strategy 
is well below this level.   
 
Risk/Benefit Analysis: The risk is very negligible since the product has been on the market 
and women have been taking up to 3 grams per day without adverse effects in cardiovascular 
prevention trials.  The benefit is great to the pregnant and lactating woman to improve health.  
 
Collaboration: Dr. Morrow will collaborate with the original author of this work Christina J. 
Valentine MD, MS, RD who is now at Mead Johnson Nutrition to complete and write the 
manuscript. Dr. Henry Akinbi will provide medical oversight as a Sub-Investigator. 
Dr. Laurie Nommsen-Rivers will collaborate in collecting the lactation history data and capturing 
mammary cell RNA from the milk fat layer and conducting transcriptome analysis on the RNA-
sequenced samples. 
 
 
Potential Risks, Discomforts, Inconveniences and Precautions 
Minimal pain or bruising may occur at the blood draw site. This occurrence is minimized by 
having blood drawn by trained and experienced nurses and phlebotomists. 
All infant blood will be drawn during standard of care routine nutritional labs therefore 
eliminating an additional venipuncture. 
 
Because of the longevity of the product on the market and its use in infant formulas we do not 
anticipate any unknown or unforeseeable risks associated with this study. 
 
 
The mothers are sent home with an AE form to collect any adverse events that occur from their 
first dose of the investigational agent until the end of the study. This form will be checked at all 
study time points by study staff. 
Since infants in this study are premature, it is anticipated that adverse changes in clinical status 
will occur related to their prematurity.  The following common, serious neonatal morbidities as 
defined in the NRN Generic Data Base (GDB) will also be collected. These include data on in-
hospital growth, the incidence and severity of intraventricular hemorrhage, seizures, patent  
ductus arteriosus (PDA) and its treatment, nosocomial sepsis (and organisms), hearing 
impairment, and pneumothorax. 
 
 
 
 
 

Ardythe Morrow PhD., 
The University of Cincinnati / Cincinnati Children’s Hospital Medical Center, 
Docosahexaenoic Acid Supplementation to Mothers to Improve Preterm Infant Nutrition and 
Immune Homeostasis 
July 12, 2013 
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Data And Safety Monitoring Plan 
Independent review of the ongoing data, patient reports, and adverse events will be reviewed 
routinely every quarter to determine on going safety. Dr. Kurt Schibler, The Medical Director of 
Clinical research will be our member on the DSMB. 
All serious adverse events that are determined to be related to the study intervention and that 
are unexpected will be reported in an expedited fashion to the FDA, IRB and study DSMB.  
Serious adverse events that are deemed related will be reported within 7 calendar days of the 
sponsor’s initial receipt of the information. Serious adverse events that are deemed related but  
not fatal or life-threatening will be reported within 15 calendar days.  
On-going Reporting:  Serious adverse events not meeting the criteria for expedited reporting 
and all other adverse events will be reported annually per federal and institutional regulations 
and guidelines and to the study member of the DSMB per the Data and Safety Monitoring Plan. 
Pregnancy and fetal special circumstances will be given through the IRB review. The informed 
consent is reviewed by the IRB. Quarterly data surveillance for safety will be done by a 
Neonatologist in the Division of Neonatology and Pulmonary Biology, Kurt Schibler, MD. 
 
 
 
 
Current financial support: 
Divisional Funds are the primary source of funding 
 
The PI in the study does receive 8% salary support from a collaboration with: 
R01AT006880-01    (PI: LK Rogers)                    7/1/2011- 6/30/2015                     NCCAM/ODS 
DHA Attenuates Inflammatory Responses through Altering RAGE Signaling  
The goal of this grant is to identify the mechanisms associated with decreased inflammation in 
response to DHA supplementation, specifically through RAGE-mediated signaling. Funding is 
also provided by The Division of Neonatology/ Perinatal Research Institute at Cincinnati 
Children’s Hospital, Ohio. No extra cost outside of standard of practice will be incurred to the 
patient.  
 
Based on admissions at University Hospital, the anticipated time frame for the study will be 1 
year and then a following year for data analysis and manuscript preparation. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Ardythe Morrow PhD., 
The University of Cincinnati / Cincinnati Children’s Hospital Medical Center, 
Docosahexaenoic Acid Supplementation to Mothers to Improve Preterm Infant Nutrition and 
Immune Homeostasis 
July 12, 2013 
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Figure 3. Study time table for recruitment, sample, and data collection 
 
 
 
Summary and Significance:  
Premature birth is responsible for 75% of perinatal death [37]. Inflammatory conditions such as 
necrotizing enterocolitis (NEC), bronchopulmonary dysplasia (BPD), and retinopathy of 
prematurity (ROP) in the preterm infant are a tremendous source of morbidity and mortality.  
DHA is a key regulator of inflammation. Breast milk is the ideal delivery vehicle for DHA based 
upon the ability of the breast to provide balanced fatty acid composition as well as anti-infective 
molecules. Further understanding of the mechanisms could ensure our nutritional strategy is 
optimal.   
 
 
 
0 baseline {within first 
week of life} 
*Enroll & Consent   
Maternal Milk -  
fatty acids 
cytokines  
Maternal blood – fatty 
acids 
Diet analysis 
Buccal swabs (saliva) 
 
Infant blood - fatty 
acids, cytokines, and 
sRAGE 
Nutrition labs 
Growth measures: 
Weight, length, head 
circumference 
Diet analysis 
Stool from diaper 
Buccal swabs (saliva) 
2 week 
Maternal Milk - 
fatty acids, 
cytokines,  
Diet analysis 
 
Infant blood - fatty 
acids and 
cytokines 
labs, sRAGE 
Stool from diaper 
Buccal swabs 
(saliva) 
Growth measures  
4 week 
Maternal - Milk 
fatty acids, 
cytokines, , RNA, 
Na:K ratio 
 
Maternal blood – 
fatty acids 
Diet analysis  
Exercise and 
Smoking habits 
Breast milk 
expression and 
breastfeeding 
patterns and 
concerns 
Anthropometry 
(weight, height and 
circumferences) 
  
Buccal swabs 
(saliva) 
 
Infant blood  fatty 
acids and 
cytokines 
labs, sRAGE 
Stool from diaper 
Buccal swabs 
(saliva) 
Growth measures 
8 week/Discharge 
Maternal - Milk 
fatty acids, cytokines,  
Maternal blood – fatty 
acids 
Diet analysis 
Buccal swabs (saliva) 
 
Infant blood - fatty 
acids and cytokines 
labs, sRAGE 
Buccal swabs (saliva) 
Stool from diaper 
Growth measures 
Body composition 
using the Pea Pod ® 
air displacement 
isolette 

Ardythe Morrow PhD., 
The University of Cincinnati / Cincinnati Children’s Hospital Medical Center, 
Docosahexaenoic Acid Supplementation to Mothers to Improve Preterm Infant Nutrition and 
Immune Homeostasis 
July 12, 2013 
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References: 
1. 
Fanaroff, A.A., et al., Trends in neonatal morbidity and mortality for very low 
birthweight infants. Am J Obstet Gynecol, 2007. 196(2): p. 147 e1-8. 
2. 
Innis, S.M., Essential Fatty Acid Transfer & Fetal Develoopment. Placenta, 2005. Suppl 
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3. 
Turner, N., P.L. Else, and A.J. Hulbert, Docosahexaenoic acid (DHA) content of 
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4. 
SanGiovanni, J.P., et al., Meta-analysis of dietary essential fatty acids and long-chain 
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Innis, S.M., Perinatal biochemistry and physiology of long-chain polyunsaturated fatty 
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Rogers, L.K., Valentine CJ et al., Maternal docosahexaenoic acid supplementation 
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12. 
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Carlson, S.E., Docosahexaenoic acid supplementation in pregnancy and lactation. Am J 
Clin Nutr, 2009. 89(2): p. 678S-84S. 
14. 
Henriksen, C., et al., Improved cognitive development among preterm infants attributable 
to early supplementation of human milk with docosahexaenoic acid and arachidonic 
acid. Pediatrics, 2008. 121(6): p. 1137-45. 
15. 
Carlson, S.E., et al., Lower incidence of necrotizing enterocolitis in infants fed a preterm 
formula with egg phospholipids. Pediatr Res, 1998. 44(4): p. 491-8. 
16. 
Cooke, R.J., P. Zee, and Y.Y. Yeh, Essential fatty acid status of the premature infant 
during short-term fat-free parenteral nutrition. J Pediatr Gastroenterol Nutr, 1984. 3(3): 
p. 446-9. 
17. 
Pediatrics, A.A.o., Nutritional needs of preterm infants. Pediatric Nutrition Handbook  
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Ardythe Morrow PhD., 
The University of Cincinnati / Cincinnati Children’s Hospital Medical Center, 
Docosahexaenoic Acid Supplementation to Mothers to Improve Preterm Infant Nutrition and 
Immune Homeostasis 
July 12, 2013 
DHA-1 
 
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