BF: Dietary Factors, Breast Milk and Infant Outcomes (2008)


Dunstan JA, Roper J, Mitoulas L, Hartmann PE, Simmer K and Prescott Sl. The effect of supplementation with fish oil during pregnancy on breast milk immunoglobulin A, soluble CD14, cytokine levels and fatty acid composition. Clin Exp Allergy 2004; 34:1,237-1,242.


PubMed ID: 15298564
Study Design:
Randomized controlled trial
A - Click here for explanation of classification scheme.
Quality Rating:
Neutral NEUTRAL: See Quality Criteria Checklist below.
Research Purpose:

To determine if changes in breast milk n-3 polyunsaturated fatty acid (n-3 PUFA) composition as a result of maternal dietary fish oil supplementation during pregnancy can modify levels of immunological parameters in breast milk.

Inclusion Criteria:

Pregnant atopic women in Western Australia were recruited because their babies were considered to be at high risk of allergic disease. All women had a history of doctor diagnosed allergic rhinitis (AR) or asthma and one or more positive skin prick test to common allergens (house dust mite, grass pollens, molds, cat, dog and cockroach extracts: Hollister Steir Laboratories, Spokane, Washington, USA).

Exclusion Criteria:

Women were ineligible for the study if they smoked, had other medical problems, complicated pregnancies, seafood allergy or their normal dietary intake exceeded two meals of fish per week.




Description of Study Protocol:


The recruitment techniques were not delineated.


Randomized controlled trial.

Blinding used

Double blind study.


The groups were blocked randomized according to parity (no previous term childbirth vs. one or more), pre-pregnancy body mass index (BMI), age and maternal allergy (AR or asthma). Women in the fish oil group (N=52) received four (1g) fish oil capsules per day (Ocean Nutrition, Halifax, Nova Acotia, Canada) comprising a total of 3.7g of n-3 PUFA with 56.0% as docosahexaenoic acid (DHA) and 27.7% as eicosapentaenoic acid (EPA), confirmed by gas liquid chromatography.

Supplementation was from 20 weeks gestation until delivery.  

Statistical Analysis

  • Only mothers of healthy infants (born >36 weeks gestation) were included in the data analysis (to avoid confounding effects of pre-maturity)
  • Fatty acids were expressed as mean percentage and standard deviation of the total fatty acids measured
  • Differences between the groups were determined by independent T-test
  • Cytokine data were analyzed as continuous data, described by the median and interquartile range, and as dichotomous data (detected or non-detected)
  • Differences between the groups were determined by Mann-Whitney test for non-parametric data
  • Differences between the two groups for dichotomous data were determined by x2. The two groups were also combined (N=73 with breast milk samples) to determine associations between individual fatty acid proportions and cytokine and Ig A levels using Spearman’s correlation. Factors with potential confounder effects were tested by Spearman’s correlation.
  • All statistical analysis were performed using SPSS software (Version 10 for Macintosh)
  • A P-value <0.05 was considered statistically significant for all analysis.     




Data Collection Summary:

Timing of Measurements 

Breast milk was collected three days post-partum and fatty acids were analyzed by gas liquid chromatography and IgaA, sCD14 and cytokines (IL-5, IL-6, IL-10, TNF-α and IFN-γ ) were quantified by ELISA or time resolved fluorescence (TRF).

Dependent Variables

  • Fatty Acid Composition
    • Lipids were extracted with chloroform-methanol (2:1) using butylatedhydroxyanisole as an antioxidant, as published previously
    • Fatty acids were identified based on retention time to authentic lipid standards (Nu-Chek-Prep Inc., Elysian, Minnesota, and USA).

  • Cytokines and Immunoglobulin A Levels
    • Total breast milk IgA levels were detected by Nephelometry (Beckman Coulterin, Immage, Fullerton, California, USA), measuring the intensity of scattered light particles suspended in a cuvette using a 670nm laser as the light source.

  • Cytokine and Soluble CD14 analysis.
    • Levels of SCD14, IL 13, TNF-α and IL-5 were measured by ELISA as previously described. IL-10, IL-6 and IFN-γ were quantified using time resolved fluorometry (TRF) (DELPHIA, Perkin-Elmer, Life Sciences, Boston, Massachusetts, USA)

Independent Variables

  • The control group (N=46) received four (1g) capsule of olive oil per day (containing 66.6% n-69 oleic acid, and less than 1% n-3 PUFA (Pan Laboratories, Moore Bank, NSW, Australia). This amount was chosen as approximately equivalent to one fatty-fish meal per day.  
  • Compliance was monitored by measuring the incorporation of DHA and EPA into the cell membranes of erythrocytes.







Description of Actual Data Sample:


Initial N


Attrition (final N)



Not delineated.


Not delineated.

Other relevant demographics


Perth, Australia.


Summary of Results:


Table 1. Detection of IgA, sCD14 and cytokines in breast milk in women supplemented with fish oil (N=33) compared with the control group (N=40).


Parameters in Breast Milk Control Group
Fish Oil  P-value 



























30(90.9 %) 


The number (and proportion) of samples with detectable levels are shown. P-values were determined by x2 and P<0.05 was considered statistically significant.

There was no significance difference in the level of free cytokines or IgA between the two groups.

Table 2. Levels of IgA, sCD14 and cytokines in breast milk collected three days post-partum from women supplemented with fish oil (N=33) during pregnancy compared with control group (N=40).


  Control Group Fish Oil  
Cytokine Median IQR Median IQR P-value
1.0 0.7-1.4 1.2 0.8-1.6  0.409
sCD14 (µg/ml)
16.9 15.6-19.1 18.2 14.7-20.2 0.270
Il-13 (pg/ml)
6.7 0.14.4 6.3 0-15.3 0.973
0 0.3.4 0 0.42 0.196
IL-6 (pg/ml)
38.7 15.6-81.7 45.7 24.6-89.7 0.323

Median levels and interquartile ranges (IQR) are shown. Groups were compared using the Mann-Whitney parametric test. A P-value of <0.05 was considered statistically significant.

When the two groups were combined there were significant correlation between IgA and individual proportions of fatty acids in breast milk.

Table 3. The relationship (correlation) between immunological parameters (IgA, sCD14 and cytokines) and fatty acids in breast milk.


  BM Fatty Acids
 0.190 0.072  0.235
 0.197 0.023 0.182
 0.023 0.087 -0.002
 0.100 0.154 0.194
 0.221 -0.029 0.190

The correlation coefficients are shown for the whole study population (n=73), and we calculated using the Spearman correlation test.

Significance levels are annotated where *P<0.05 and **P <0.01

  • IgA was positively associated with DHA (22:6n-3) (r=-0.235), P=0.046) and 22:5n-3 r=0.344), P=0.003) and inversely associated with linoleic acid (LA) (18:2n-6) (r=0.249, P=0.034)
  • Breast milk levels of sCD14 were positively correlated with 22:5n-3 (r=0.306, P=0.009
  • IGA levels in the breast milk samples were positively correlated with levels of IL-10 (r=0.459, P<0.001), IL-6 (r=0.288, P=0.013, IL-13 (r=0.375, P=0.001) and sCD14 (r=0.335, P=0.004), in the samples.

Other Findings

Breast milk concentrations of DHA (22:6n-3), 22:5n-3 and EPA (20:5n-3) were significantly higher (P<0.001) in fish oil supplemented mothers compared with controls.

There was no significance difference in detection of free cytokines or IgA between the two groups.

Author Conclusion:
  • N-3 docosahexaenoic acid (DHA; 22:6n-3) and eicosapentaenoic acid (EPA:20:5n-3) levels were significantly higher (P<0.001) in breast milk from women supplemented with fish oil (N=33) DHA mean 1.15%, SD 0.47%) and EPA mean 0.16%, SD 0.07% than in samples from the control group (N=40), DHA mean 0.50%, SD 0.17% and EPA  mean 0.05%, SD 0.02 %).
  • Breast milk arachidonic acid (AA;20;4n-6) levels were significantly lower (P=0.045) in the fish oil group (mean 0.55%, SD 0.12%) compared with control group (mean 0.61%, SD 0.14%)
  • Breast milk IgA was positively correlated with DHA (P= 0.046) and 22:5n-3 (P=0.003), but inversely correlated with linoleic acid (LA; 18-2n-6) (P=0034). Levels of sCD14 were positively correlated with 22:5n-3 (P=0.009).
  • Cytokines involved in IgA synthesis (IL-10 and IL-6) were also significantly correlated both IgA and n-3 PUFA levels, although there were no differences in the levels of breast milk IgA,sCD14 or cytokines between study groups.
  • Supplementation with fish oil during pregnancy significantly alters early post-partum breast milk fatty acid composition. N-3 PUFA levels were positively associated with IgA and sCD14 levels, suggesting a relationship between fatty acid status and mucosal immune function.
  • Although the relation between breast milk IgA levels and the development of allergic disease is unclear, several studies have suggested that both total IgA and cow’s milk specific IgA in breast milk levels are associated with reduced risk of developing cow’s milk allergy in children
  • It remains to be seen if these modest changes in maternal IgA in breast milk have any clinical effects on the infants
  • Although the significance is unclear, there have also been some reports that milk from allergic mothers is different from that of non-allergic mothers with regard to cytokine levels and composition
  • Although the process involved in normal oral tolerance are still poorly understood, a number of bystander factors may modify responses to allergens including enteric flora. 
Funding Source:
University/Hospital: Schol of Paediatrics and Child Health and iomedical and Chemical Sciences, University of Western Australia
Reviewer Comments:

Randomized Clinical Trials’ (RCT) are considered the ‘gold standard’ of research practice and are accepted as producing the most reliable scientific evidence. The use of the RCT methodology contributes to improving the overall quality and rigor of the research. Within an RCT, participants are allocated at random to either a ‘control’ or ‘experimental’, group, thereby eliminating all forms of spurious or additional causality. This ensures that both groups are 'statistically equivalent’ enabling valid conclusions to be drawn regarding the effect of a treatment or intervention on the experimental group. 

The limitations and critique of the study, as stated by the authors appear to be very appropriate.


Quality Criteria Checklist: Primary Research
Relevance Questions
  1. Would implementing the studied intervention or procedure (if found successful) result in improved outcomes for the patients/clients/population group? (Not Applicable for some epidemiological studies) Yes
  2. Did the authors study an outcome (dependent variable) or topic that the patients/clients/population group would care about? Yes
  3. Is the focus of the intervention or procedure (independent variable) or topic of study a common issue of concern to dieteticspractice? Yes
  4. Is the intervention or procedure feasible? (NA for some epidemiological studies) Yes
Validity Questions
1. Was the research question clearly stated? Yes
  1.1. Was (were) the specific intervention(s) or procedure(s) [independent variable(s)] identified? Yes
  1.2. Was (were) the outcome(s) [dependent variable(s)] clearly indicated? Yes
  1.3. Were the target population and setting specified? Yes
2. Was the selection of study subjects/patients free from bias? Yes
  2.1. Were inclusion/exclusion criteria specified (e.g., risk, point in disease progression, diagnostic or prognosis criteria), and with sufficient detail and without omitting criteria critical to the study? Yes
  2.2. Were criteria applied equally to all study groups? Yes
  2.3. Were health, demographics, and other characteristics of subjects described? Yes
  2.4. Were the subjects/patients a representative sample of the relevant population? Yes
3. Were study groups comparable? Yes
  3.1. Was the method of assigning subjects/patients to groups described and unbiased? (Method of randomization identified if RCT) Yes
  3.2. Were distribution of disease status, prognostic factors, and other factors (e.g., demographics) similar across study groups at baseline? Yes
  3.3. Were concurrent controls or comparisons used? (Concurrent preferred over historical control or comparison groups.) Yes
  3.4. If cohort study or cross-sectional study, were groups comparable on important confounding factors and/or were preexisting differences accounted for by using appropriate adjustments in statistical analysis? N/A
  3.5. If case control study, were potential confounding factors comparable for cases and controls? (If case series or trial with subjects serving as own control, this criterion is not applicable.) N/A
  3.6. If diagnostic test, was there an independent blind comparison with an appropriate reference standard (e.g., "gold standard")? N/A
4. Was method of handling withdrawals described? No
  4.1. Were follow-up methods described and the same for all groups? No
  4.2. Was the number, characteristics of withdrawals (i.e., dropouts, lost to follow up, attrition rate) and/or response rate (cross-sectional studies) described for each group? (Follow up goal for a strong study is 80%.) No
  4.3. Were all enrolled subjects/patients (in the original sample) accounted for? No
  4.4. Were reasons for withdrawals similar across groups? ???
  4.5. If diagnostic test, was decision to perform reference test not dependent on results of test under study? N/A
5. Was blinding used to prevent introduction of bias? Yes
  5.1. In intervention study, were subjects, clinicians/practitioners, and investigators blinded to treatment group, as appropriate? Yes
  5.2. Were data collectors blinded for outcomes assessment? (If outcome is measured using an objective test, such as a lab value, this criterion is assumed to be met.) Yes
  5.3. In cohort study or cross-sectional study, were measurements of outcomes and risk factors blinded? N/A
  5.4. In case control study, was case definition explicit and case ascertainment not influenced by exposure status? N/A
  5.5. In diagnostic study, were test results blinded to patient history and other test results? N/A
6. Were intervention/therapeutic regimens/exposure factor or procedure and any comparison(s) described in detail? Were interveningfactors described? Yes
  6.1. In RCT or other intervention trial, were protocols described for all regimens studied? Yes
  6.2. In observational study, were interventions, study settings, and clinicians/provider described? Yes
  6.3. Was the intensity and duration of the intervention or exposure factor sufficient to produce a meaningful effect? Yes
  6.4. Was the amount of exposure and, if relevant, subject/patient compliance measured? Yes
  6.5. Were co-interventions (e.g., ancillary treatments, other therapies) described? Yes
  6.6. Were extra or unplanned treatments described? Yes
  6.7. Was the information for 6.4, 6.5, and 6.6 assessed the same way for all groups? Yes
  6.8. In diagnostic study, were details of test administration and replication sufficient? N/A
7. Were outcomes clearly defined and the measurements valid and reliable? Yes
  7.1. Were primary and secondary endpoints described and relevant to the question? Yes
  7.2. Were nutrition measures appropriate to question and outcomes of concern? Yes
  7.3. Was the period of follow-up long enough for important outcome(s) to occur? Yes
  7.4. Were the observations and measurements based on standard, valid, and reliable data collection instruments/tests/procedures? Yes
  7.5. Was the measurement of effect at an appropriate level of precision? Yes
  7.6. Were other factors accounted for (measured) that could affect outcomes? Yes
  7.7. Were the measurements conducted consistently across groups? Yes
8. Was the statistical analysis appropriate for the study design and type of outcome indicators? Yes
  8.1. Were statistical analyses adequately described and the results reported appropriately? Yes
  8.2. Were correct statistical tests used and assumptions of test not violated? Yes
  8.3. Were statistics reported with levels of significance and/or confidence intervals? Yes
  8.4. Was "intent to treat" analysis of outcomes done (and as appropriate, was there an analysis of outcomes for those maximally exposed or a dose-response analysis)? Yes
  8.5. Were adequate adjustments made for effects of confounding factors that might have affected the outcomes (e.g., multivariate analyses)? Yes
  8.6. Was clinical significance as well as statistical significance reported? Yes
  8.7. If negative findings, was a power calculation reported to address type 2 error? N/A
9. Are conclusions supported by results with biases and limitations taken into consideration? Yes
  9.1. Is there a discussion of findings? Yes
  9.2. Are biases and study limitations identified and discussed? Yes
10. Is bias due to study's funding or sponsorship unlikely? Yes
  10.1. Were sources of funding and investigators' affiliations described? Yes
  10.2. Was the study free from apparent conflict of interest? Yes