Study Design

In this single-center, double-blind, placebo-controlled, parallel-group trial, pregnant women between 22 and 26 weeks of gestation were recruited into the Copenhagen Prospective Studies on Asthma in Childhood 2010 (COPSAC 2010 ) pregnancy cohort.11 Women taking more than 600 IU of vitamin D per day and women with any endocrine, heart, or kidney disorder were excluded. The trial was approved by the local ethics committee and the Danish Data Protection Agency. Both parents of each child provided oral and written informed consent before enrollment.

At week 24 of pregnancy, women were randomly assigned in a 1:1 ratio to receive 2.4 g per day of n−3 LCPUFA (55% EPA and 37% DHA) in triacylglycerol form (Incromega TG33/22, Croda Health Care) or placebo (in the form of olive oil, containing 72% n–9 oleic acid and 12% n−6 linoleic acid [Pharma-Tech A/S]). (For complete information on the components of Incromega TG33/22, see Table S1 in the Supplementary Appendix, available with the full text of this article at NEJM.org. The trial protocol is also available at NEJM.org.) Both n−3 LCPUFA and placebo were administered in four identical 1-g capsules, with both investigators and participants unaware of group assignments. Neither the fish oil nor the placebo nor any medications, diagnostic equipment, or other materials used in the trial were donated. Similarly, no funding agency played any role in the design or conduct of the trial, the collection, management, or interpretation of the data, the preparation, review, or approval of the manuscript for publication, or the decision to submit the manuscript for publication. In addition, no pharmaceutical company and no company that produces n−3 LCPUFA was involved in the trial. The intervention was funded solely by COPSAC.

Supplementation continued until 1 week after delivery, and both investigators and participants remained unaware of group assignments until the youngest child in the trial reached 3 years of age. The trial was continued for an additional 2 years, during which the investigators determined whether children with persistent wheeze had asthma. Follow-up is still ongoing, with the investigators only (not the participants) remaining unaware of group assignments. A subgroup of 623 women also participated in a trial with a nested, factorial design in which they took 2400 IU of vitamin D 3 per day during the third trimester of pregnancy,12 a subgroup of 51 women participated in a trial in which they were vaccinated against influenza A during pregnancy,11,13 and a subgroup of 72 children with persistent wheeze participated in a trial in which they took azithromycin or placebo during asthmatic episodes.14

The participating women completed a validated 360-item food frequency questionnaire so that their dietary intake in the 4 weeks before randomization could be assessed.15-17 Maternal whole-blood levels of EPA and DHA were assessed at the time of randomization and 1 week after birth.18,19 Samples of breast milk were obtained 1 month after birth and were analyzed for EPA and DHA levels. Maternal variation in the genes encoding fatty acid desaturase (FADS) was tagged by genotyping the single-nucleotide polymorphism (SNP) rs1535 in mothers of European descent (with genotyping performed by LGC Group). Adherence to the intervention was assessed by comparing the number of capsules returned with the number expected.

End Points

Pediatricians collected information during clinical visits scheduled at 1 week after birth, at 1, 3, 6, 12, 18, 24, 30, and 36 months after birth, and yearly thereafter. Additional visits for acute care were arranged whenever a child had symptoms related to the lungs, an allergy, or the skin. Asthma, allergy, and eczema were diagnosed and treated by COPSAC pediatricians in accordance with predefined algorithms.11

Diary cards were completed daily by parents from the birth of their child for the purpose of monitoring troublesome lung-related symptoms, including cough, wheeze, and dyspnea that severely affected the well-being of the child,11 skin-related symptoms, and symptoms related to infections of the lower respiratory tract. The diary cards were reviewed by COPSAC pediatricians, and the data were checked after being entered into the database.

Persistent wheeze or asthma (the primary end point) was diagnosed on the basis of a previously described quantitative diagnostic algorithm20,21 that included diary recordings of five episodes of troublesome lung symptoms within the preceding 6 months, each lasting for at least 3 consecutive days; symptoms typical of asthma; the rescue use of inhaled beta 2 -agonist; and response to a 3-month course of inhaled glucocorticoids followed by relapse after the end of treatment.20 Remission was defined as a period of 12 months without relapse. The diagnosis was termed “persistent wheeze” until a child reached 3 years of age and was termed “asthma” thereafter.

Infection of the lower respiratory tract was defined as a diagnosis of pneumonia or bronchiolitis on the basis of symptoms and clinical presentation (i.e., without confirmation by means of pathogen identification or radiologic or laboratory findings).22-24 Allergic sensitization was determined at 6 months and 18 months as a wheal larger than 2 mm in response to any skin-prick test (ALK-Abelló) or a specific IgE level of 0.35 kU per liter or higher against milk, egg, dog, or cat allergens (ImmunoCAP tests, Thermo Fisher Scientific).11 Allergic rhinoconjunctivitis was diagnosed longitudinally by COPSAC pediatricians on the basis of systematic interviews and was defined as allergic rhinitis, allergic conjunctivitis, or both. Lung function at 5 years of age was assessed by means of spirometry, specific airway resistance by means of whole-body plethysmography, and the lung-clearance index by means of multiple-breath washout.11 A diagnosis of eczema was based on the criteria defined by Hanifin and Rajka.25-27

Statistical Analysis