S TATISTICAL ANALYSIS

All statistical analyses were performed using STATA versions 13 and 14 (StataCorp.

2013, StataCorp. 2015). For publications I and II, the statistical package SPSS version 22 was also used.

Data were described as frequencies and risk estimates with confidence intervals (CIs).

Potential confounding factors were chosen a priori as described above. For publication III, directed acyclic graphs were also used to minimize potential bias from intermediate variables when studying the association between potential paternal determinants and adequate maternal folic acid supplementation. Inclusions of confounders in the statistical models are discussed in more detail below, and information about the variables used is presented in Table 2.

3.6.1 Folic acid and maternal cancer risk (publication I)

In our study, the risk of total cancer and subtypes of cancer among women using folic acid in successive pregnancies compared to women using no folic acid were

estimated as hazard ratios (HRs) with 95% confidence intervals (CIs) using multivariate, time-dependent Cox proportional hazard regression models (Cox and Oakes 1984). Time since the first childbirth from 1999 through 2010 was used as the time variable. The Cox models were applied when there was an underlying

assumption of proportionality. Tests for linear trends over the categories of folic acid supplementation were calculated. All analyses were adjusted for maternal age at first childbirth (age at cohort entry), maternal year of birth, marital status, birth order, education, occupation, and smoking at time of birth. For total cancer and breast cancer only, we also adjusted for age at the woman’s first childbirth.

In this publication, we also used multiple imputations on missing smoking status at the time of birth due to 16% of the births missing data on smoking. The imputed analyses were performed following the recommendation by White and Royston (White and Royston 2009).

3.6.2 Folic acid and childhood cancer risk (publication II) Cancer risk in children exposed to maternal folic acid and/or multivitamin

supplements was compared with cancer risk in unexposed children and expressed as HRs with 95% CIs, using Cox proportional hazards regression models when there was an underlying assumption of proportionality. Time since birth was used as the time variable, and all analyses were adjusted for a priori selected covariates

associated with maternal folic acid use and childhood cancer risk; that is, birth order, maternal smoking, maternal and paternal age, and maternal and paternal education.

Maternal periconceptional folic acid and/or multivitamin use was divided into four exposure levels: 0 mg, approximately 0.2 mg, 0.4 mg and approximately 0.6 mg.

A test for linear trends was calculated by treating the four exposure levels as continuous in the models (Table 2).

3.6.3 Paternal characteristics associated with maternal periconceptional folic acid supplementation (publication III) To determine the associations between paternal determinants (age, education, occupation, country of origin) and maternal folic acid supplement use, crude and adjusted relative risks (RRs) with corresponding 95% CIs were calculated by log binomial regression models with the log-link function in STATA version 14 (StataCorp. 2015). RR is the ratio of the risk of adequate maternal folic acid use for different categories of paternal determinants and the risk of adequate maternal folic acid use for the reference category of paternal determinants. Our analyses included robust variance estimation of the 95% CIs with the sandwich estimator, to correct for the intra-individual correlation in women with more than one pregnancy during the study period (Cameron and Miller 2015). All analyses were adjusted according to the description in Table 2. P-values for overall difference between the categories of paternal determinants were calculated using likelihood ratio tests. Effect modification of the association between paternal education and adequate maternal folic acid supplementation by maternal education was evaluated by stratification, and tested with likelihood ratio tests.

Table 2: Overview of the outcomes, exposure variables, adjustment variables, design, statistical models and statistical software used in this study

Publication I Publication II Publication III

Main outcome Maternal cancer (ICD-10)¹

Total cancer

Colorectal cancer (C18–21) Lung cancer (C33–34) Melanoma of the skin (C43) Non-melanoma skin cancer (C44) Breast cancer (C50)

Cancer of the uterine cervix (C53) Ovarian cancer (C56)

Central nervous system tumors (C70–72, D42–43)

Thyroid cancer (C73) Other endocrine glands² (C37, C74–75)

III. Central nervous system tumors and miscellaneous intracranial and intraspinal neoplasms IV. Neuroblastoma and other

peripheral nervous cell tumors

VI. Renal tumors IX. Soft-tissue and other

extraosseous sarcomas Folic acid (before and/or during

pregnancy) No use

Use in one pregnancy Use in two or more pregnancies Multivitamins (before and/or during pregnancy)

No use

Use in one pregnancy Use in two or more pregnancies Total amount of folic acid from multivitamin and folic acid

Total amount of folic acid from multivitamin supplements and folic acid supplements (before and/or during pregnancy)

No use (0 mg)

Multivitamins only (~ 0.2 mg) Folic acid only (0.4 mg) Folic acid and multivitamins (~ 0.6 mg)

Paternal country of origin⁶

Maternal age at the very first childbirth (for total and breast cancer only) Legislators, senior officials, and managers

Professionals

Technicians and associate professionals

Clerks

Service workers and shop and market sales workers

Paternal country of origin≥40 ⁶ Norway For paternal country of origin

Year of childbirth Maternal country of origin⁶

Norway

High-income countries Low/middle-income countries

Agricultural, forestry, and fishery workers

Craft and related trades workers Plant and machine operators and assemblers

Elementary occupations Maternal smoking

Never Sometimes

≤10 cigarettes daily

>10 cigarettes daily

Daily smoking, unknown amount

Design

Population-based cohort study Population-based cohort study Population-based cross-sectional study

Statistical models Time-dependent Cox proportional

hazard regression models Cox proportional hazard

regression models Log-binomial regression

Statistical software SPSS version 22 and STATA

version 13 SPSS version 22 and STATA

version 13 STATA version 14

1 International Classification of Diseases, 10^th version

2 Malignant neoplasm of thymus, adrenal gland, and other endocrine glands and related structures excluding endocrine pancreas, ovary, and thyroid.

3 International Classification of Childhood Cancer, third edition (Steliarova-Foucher et al. 2005)

4 We have used approximately (~) because multivitamins used in Norway during 1999–2010 initially contained 0 mg of folic acid and later contained 0.2 mg of folic acid. Folic acid supplements contained 0.4 mg of folic acid throughout the study period (Norwegian Scientific Committee for Food Safety 2015).

5 According to the class scheme of Erikson, Goldthorpe and Portocarero (EGP) (Erikson and Goldthorpe 1992)

6 According to the classification of the World Health Organization’s Health Statistics and Information Systems’ estimates for 2000–2012, available from

(http://www.who.int/healthinfo/global_burden_disease/estimates/en/index1.html)

7 Standard Classification of Occupation (STYRK-08 2011) (10 major groups)