Vitamin D Supplementation During Pregnancy for Prevention of Asthma in Childhood (ABCvitaminD)

Vitamin D Supplementation During Pregnancy for Prevention of Asthma in Childhood: An Interventional Trial in the ABC (Asthma Begins in Childhood) Cohort

The aim of this study is to prevent asthma symptoms (recurrent wheeze) in childhood by supplementation with high dose vitamin D to the mother during pregnancy. Participants are mothers and children of the ABC (Asthma Begins in Childhood) cohort. Mothers are recruited during pregnancy and receive daily supplement with 2400 IU of Vitamin D3 or placebo from week 24 og gestation to 1 week after delivery. In addition all mothers are advised to take the recommended dose of 400 IU vitamin D daily. The mothers in ABC also participate in an interventional trial with fish oil supplementation, and the vitamin D randomization is stratified by fish oil treatment group. The child is followed with acute and planned vits at the research unit, and wheeze is diagnosed according to predefined algorithms.

Dietary supplement: 2400 IU Vitamin D3 (2 tablets of 1200 IU) from week 24 of gestation to 1 week after delivery

Placebo: 2 placebo tablets with no active substance, identical to the active tablets, from week 24 of gestation to 1 week after delivery

Age at onset of persistent wheeze diagnosed according to predefined algorithm of recurrent troublesome lung symptoms, response to treatment and relapse after withdrawal of treatment

Main analysis: • Number of lower respiratory tract infections registered in daily diaries Secondary analyses: Acute otitis media Number of upper respiratory tract infections Number of other infections Total number of infections

Age at onset of eczema diagnosed prospectively by research doctors according to predefined algorithm based upon Hanifin and Rajka criteria

Age at onset of severe asthma exacerbation diagnosed by predefined criteria of acute severe asthma requiring oral/high dose inhaled steroids or acute hospital contact

Main analysis: • Cognitive development assessed at 2½ years using the cognitive part of Bayley Scales of Infant and Toddler Development, third edition Secondary analyses: Milestone development monitored prospectively by the parents using a registration form based on The Denver Development Index and WHO milestones registration (combined assessment by principal component analysis) Language development assessed at 1 and 2 years of age with the Danish version of The MacArthur Bates Communicative Developmental Inventory (CDI) The child´s general development (language, fine and gross motor, social and problem solving) at 3 years of age assessed with Ages and stages Questioner, third edition (ASQ-3)

Main analysis: • Body composition (fat mass and bone mineral density) assessed by DEXA scan at 3 years of age Secondary analysis • Development of BMI from birth to 3 years assesses longitudinally in the research clinic

Main analysis Immune status at 18 months measured in stimulated whole blood as cytokine release (combined assessments by prinicipal component analyses) Secondary analyses Composition of immune cell subsets in whole blood at birth and at 18 months of age

Immune status measured in airway mucosal lining fluid at 4 weeks and 2 years of age (combined assessments by prinicipal component analyses for each age point)

In a secondary analyses, we will determine the effect of 17q21 genotype on the efficacy of vitamin D supplementation in the prevention of asthma/wheeze. We will compute hazard ratios for the reduction in asthma/wheeze risk associated with prenatal supplementation, stratified by rs12936231. rs12936231 is a functional SNP influencing expression of ORMDL3, and given the role of ORMDL3 as a key sphingolipid biosynthesis regulator, we will subsequently investigate the relative abundance of sphingolipids between those in the vitamin D Intervention arm and those in the placebo group, stratified by 17q21 genotype. Finally we will identify interactions between prenatal vitamin D supplementation, rs12936231 genotype and sphingolipid metabolism in the risk of asthma/wheeze by age three.

Caries and enamel defects (molar incisor hypomineralization, MIH) determined at a dental examination at age 6 years.

Asthma diagnosed from age 3 to 10 years based on the same predefined algorithm of recurrent troublesome lung symptoms, response to treatment after withdrawal of treatment, which was used for persistent wheeze at age 0-3 in phase 1 of the study. Primary outcome in phase 2 is current asthma at specific visits till age 10 years, which is diagnosed in children fulfilling the persistent wheeze algorithm at any point during the first 10 years of life and still needing inhaled corticosteroids at specific visits (3, 4, 5, 6, 8 and 10 years of age) to control the symptoms.

Anthropometrics: Clinical follow up on the development of weight in kg (calibrated digital weight scales), height in cm (Harpenden stadiometer), waist-, thorax- and head circumference in cm (using tape; 3 times each) at every visit till age 10 years assessed longitudinally in the research clinic.

Age at onset of eczema diagnosed prospectively by research doctors according to predefined algorithm based upon Hanifin and Rajka criteria

Allergic sensitization at 6 and 10 years of age assessed by skin prick test (ALK-Abelló, Denmark) and specific IgE in blood (ImmunoCAP, PHarmacia Diagnostics AB, Sweden).

Immune status measured in airway mucosal lining fluid at 3, 6 and 10 years of age (combined assessments by principal component analyses for each age point).

Allergic rhinitis will be diagnosed by combining allergic sensitization with symptom recording of troublesome congestion or sneezing or runny nose upon relevant exposure to allergens at age 3, 4, 5, 6, 8 and 10 years.

Age at onset and number of severe asthma exacerbation diagnosed by predefined criteria of acute severe asthma requiring oral/ high dose inhaled steroids or acute hospital contact

Multiple breath wash-out using SF6 and N2 as inert gasses to determined LCI, Scond and Sacin at ages 3, 4 and 5 years.

Provocative dose of methacholine leading to a 20% drop in FEV1 from baseline (PD20 value) at age 6 years.

Body composition measured as fat mass, lean mass, bone mineral content (BMC) and bone mineral density assessed (BMD) through DXA scans at 3 and 6 years of age. Body impedance measurements at 10 years of age.

A 2,5 hours cognitive evaluation at 10 years of age. Cognitive functions from a broad spectrum of cognitive domains will be assessed with subtests both using paper pencil tests and Cambridge Neuropsychological Test Automated Battery .

Behavioral and psychopathological dimensions will be assessed at 10 years of age. The childrens' potential psychiatric diagnoses will be ascertained through the semi-structured clinical interview Schedule for Affective Disorders and Schizophrenia for School-Age Children - Present and Lifetime Version (K-SADS-PL) first with a parent and next with the child.

The Strengths and Difficulties Questionnaire (SDQ) is a brief behavioural screening questionnaire about 3-16 year olds. Questionnaires will be administered to the parents to obtain their evaluation of their child's potential psychopathology and behavior problems. Scores will be calculated form the questionnaires in order validated methods

The ADHD Rating Scale obtains parent ratings regarding the frequency of each ADHD symptom based on DSM criteria. Questionnaires will be administered to the parents to obtain their evaluation of their child's potential psychopathology and behavior problems. Scores will be calculated form the questionnaires. Scores will be calculated form the questionnaires in order validated methods.

The SRS-2 identifies social impairment associated with autism spectrum disorders (ASDs) and quantifies its severity Questionnaires will be administered to the parents to obtain their evaluation of their child's potential psychopathology. Scores will be calculated form the questionnaires. Scores will be calculated form the questionnaires in order validated methods.

The Child Behavior Checklist (CBCL) is a widely used questionnaire to assess behavioral and emotional problems. Questionnaires will be administered to the parents to obtain their evaluation of their child's potential psychopathology and behavior problems. Scores will be calculated form the questionnaires. Scores will be calculated form the questionnaires in order validated methods.

The BRIEF-2 is a set of questionnaires for parents and teachers , designed to evaluate executive function from multiple perspectives. Questionnaires will be administered to the parents to obtain their evaluation of their child's potential psychopathology. Scores will be calculated form the questionnaires. Scores will be calculated form the questionnaires in order validated methods.

Inclusion Criteria: Mother: Pregnant in week 22-26 of gestation Participating in the ABC-cohort Living in Sealand, Denmark Fluent in Danish Willing to let the child participate in the study Exclusion Criteria: Mother: Intake of more than 400 IU of vitamin D during the last 6 months Endocrinological disease such as calcium metabolic disorder, parathyroid disorder, thyroid disorder or Diabetes type 1 Tuberculosis Sarcoidosis In need of diuretics or heart medication including calcium channel blockers

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