This study will encompass an analysis of an eventual association between vitamin D status (maternal during pregnancy, at birth or at 5 years) and hand grip strength at 5 years in children from Odense Child Cohort. Odense Child Cohort is a large-scale, prospective, population-based, follow-up study. Mothers and their children born from January 2010 to December 2012 and resident in the Municipality of Odense, Denmark, are followed from early pregnancy up to adulthood. Data is obtained from questionnaires and medical records and venous blood samples were drawn and stored at a biological bank.

Low muscle strength in children has been linked to adiposity, cardiovascular disease and metabolic risk factors and low muscle strength in adolescence has been shown to be a risk factor of early adulthood mortality. There are not many studies on muscle strength in small children. There is some evidence of vitamin D concentrations in the blood having a positive correlation to upper body muscle strength in adolescent girls.

Hypovitaminosis D defined as serum 25-hydroxyvitamin D <50nmol/L was evident in 27,8% of the pregnant women and 47,7% of the new-born children in Odense Child Cohort.

Animal studies have shown an effect of vitamin D on regulation of muscle function and development. Studies on humans adults have shown that vitamin D deficiency can lead to myopathy. Myopathy in children as a result of hypovitaminosis D is not well-studied.

Given the high prevalence of hypovitaminosis D, mild or severe vitamin D-associated myopathy may be prevalent in preschool children.

The objectives of this study are 1) to create reference values and determine predictors of hand grip strength at five years, 2) to analyze the associations between vitamin D at different time points and hand grip strength at 5 years.

Background The hand grip test has previously been used to study muscle strength in children (1). The test has also been found to be a reliable alternative in population-based studies to the gold standard isokinetic dynamometry (2).

There is not much research on hand grip strength in preschoolers and no consensus about which variables explain the variance in the normal distribution (3-5).

S-25-hydroxyvitamin D (s-25OHD) <50 nmol/L is prevalent in Denmark particularly in the winter due to the lack of ultraviolet B exposure and insufficient dietary intake (6). Vitamin D deficiency can lead to vitamin D-associated myopathy before developing actual bone disease (7). Little is known about vitamin-D-associated myopathy in children. Given the high prevalence of hypovitaminosis D, vitamin D-associated myopathy may be prevalent in preschool children.

The main objectives of this study were to: 1) establish a normative data-set of hand grip measurements for five-year-old children and determine which variables (during pregnancy, at birth and current) are associated to hand grip strength at five years of age and 2) investigate the association between vitamin-D-status (at 5 years, at birth, in early and in late pregnancy) and hand grip strength at the age of five.

Methods As many 5-year old children from Odense Child Cohort (OCC)as possible will be included during the study period (January 2016-June 2017). Expected number of children investigated by study completion is 1200. The exclusion criteria are multiple birth, preterm birth (GA<259 days), chronic disease and missing hand grip data at five years.

Questionnaires, blood samples for s-25OHD analysis and examinations of height, weight, skinfold thickness and 5-year hand grip measurement data will be needed for this study. When available, questionnaire data will be validated using medical records.

Blood samples Blood samples were obtained from mothers during early and late pregnancy, from offspring cord at birth and from the children at 5-years. The blood samples were stored at -80o C. S-25(OH)D2+3 analyses will be performed by liquid chromatography mass spectrometry (LC-MS/MS) as previously described (8, 9)

Explanatory Variables Child sex, age, height, weight, Body mass index (BMI), triceps skinfold and subscapular skinfold will be examined as continuous variables and examination season, vitamin D supplements, parents' education, ethnicity, mother's parity, smoking during pregnancy, birth weight, birth length, organized sports, physical activity compared to peers, motor skills compared to peers and child choice of free time activities as categorical variables.

Primary association The primary exposure is 5-year s-25OHD and the primary outcome is hand grip strength at five years. A Sub-analysis split by sex will be done.

Other associations

• S-25OHD in early and late pregnancy and chord s-25OHD and hand grip.
• Prediction of myopathy defined as hand grip <10th percentile vs. >10th percentile.

Statistics:

Descriptive statistics for baseline data, exposures and outcomes will be presented as mean (standard deviation) for parametric data and median (inter-quartile range) for non-parametric data. The students t-test for parametric and Mann-Whitney test for non-parametric data will be used to test the differences between the sexes and the difference between participants and non-participants. Univariate and multivariate regression models will be used to analyze the obtained data and to control for co-variates affecting the associations. Multiple regression variables will be decided upon based on appearance in the existing literature and a univariate p-value <0.10 will determine addition of further variables. All data will be investigated in total and for each of the sexes separately. Linear models will be used to predict hand grip strength and logistic regression will be used to assess myopathy defined as hand grip <10th percentile vs. >10th percentile. S-25OHD will be investigated both as a continuous and as a categorical parameter split by quartiles and the routine cut offs 25, 50 and 75 nmol/L. Model goodness-off-fit will be checked for all final models. Likelihood ratio test will be used to assess interaction between sex and all other variables and between s-25OHD and other variables in the models. In case of missing data, models will be reanalyzed with complete data of variables in larger models. A p-value <0.05 will be considered significant and p<0.10 will be considered a trend. Power calculation (primary outcome): Expected N=1200, 1 SD hand grip 18.4 N, 1SD of s-25OHD 22 nmol/L, alpha=0.05 and beta=0.80. It is calculated that our study will be able to detect a true difference of 0.68 N per 10 nmol/L change in s-25OHD (univariate association).

Ethics The OCC vitamin D project is an observational study, carried out according to the Helsinki II declaration and approved by the Regional Scientific Ethical Committee for Southern Denmark, no. S-20090130 and the Danish Data Protection Agency, no. 16/42833. Hand grip examination is an already approved part of the standard examination of 5-y children in OCC as part of the vitamin D project. All parents signed consent forms to receive information and invitations to follow-up examinations from OCC. Both parents' and children's permissions will be obtained before blood is drawn at five years and the children will be free to refuse any part of the examinations.

Inclusion Criteria:

• 5-year-old children participating in Odense Child Cohort.

Exclusion Criteria:

• Multiple and/or preterm birth (gestational age (GA) <259 days)
• Chronic disease
• No available hand grip data at five years.

This study was conducted using data from the Odense Child Cohort. Mothers and their children born from January 2010 to December 2012 and resident in the Municipality of Odense, Denmark, were followed from early pregnancy up to five years at present. Approximately 2500 children were included.