Data were collected as part of the Expect study I, a prospective cohort study performed in the south-eastern part of the Netherlands with the purpose of validating a number of first-trimester obstetric prediction models. Pregnant women were recruited in 36 midwifery practices and 6 hospitals between July 2013 and January 2015, with follow-up until December 2015. Eligibility criteria for the Expect study were: less than 16 weeks of gestation and a minimum age of 18 years. The Medical Ethical Committee of the Maastricht University Medical Centre evaluated the study protocol and declared that no ethical approval was necessary (MEC 13-4-053). All participating women gave online informed consent.
Figure 1 shows the flowchart of the study population. For this study, we included only women that filled out the questionnaire at 8 weeks of gestation or later, allowing most women to have adapted their diet or started using supplements. On the basis of this restriction, 136 women had to be excluded. We also excluded one woman with the missing values in all dietary questions. Finally, 2477 women were available for analysis.
Women were asked to complete an online questionnaire before 16 weeks of gestation (or a paper version if requested), containing questions about a range of variables including socio-demographic characteristics, lifestyle, obstetric history, medical conditions, and family history. Supplement use before and during pregnancy was asked for, focusing on folic acid, vitamin D, prenatal vitamins, general multivitamins, and calcium preparations, as well as dietary intake of calcium and vitamin D.
The Dutch FFQ-TOOL™ (FFQ = food-frequency questionnaire) was used for the selection of food products contributing to calcium and vitamin D intake . For the present study, we only focused on calcium intake. Based on food and nutrient intake data for 20–45 year old (non-pregnant) women who participated in the Dutch National Food Consumption Survey 2007–2010 (DNFCS 2007–2010) and food composition data from the Dutch Food Composition (NEVO) Table 2010, which were embedded in the Dutch FFQ-TOOL™, we made the FFQ-TOOL select those food products that cumulatively covered > 80% of the variance in calcium intake . The selection procedure resulted in 18 food items, for which both the frequency of use (reference period: last month) and the average daily amount of use were asked: milk and buttermilk; yoghurt and fromage frais (with or without fruit); yoghurt drinks and other dairy beverages; chocolate milk; custard and pudding; Dutch cheese; non-Dutch cheese and cream cheese; cheese spread; bread spread (sub types: margarine; low-fat margarine; and butter); cooking fat (bake and fry products); and fish (subtypes: fat fish such as salmon, mackerel, eel, and white harring; lean fish such as codfish, tilapia, panga fish and trout; white fish filet; smoked or steamed fish; herring; and fish fingers. Milk, milk products, and cheese were included in the questionnaire as major sources of calcium, bread spread, bake and fry products, and fish as major sources of vitamin D.
We chose to include questions on food products that cumulatively cover > 80% of the variance in calcium intake and eliminate food products with small calcium contents. This consideration was made to encourage participants to complete the full questionnaire of the Expect study I. Covering the complete dietary calcium intake in this study was not feasible, as the FFQ was part of an intensive questionnaire containing several pregnancy-related topics, with the purpose of validating a number of first-trimester obstetric prediction models. The food items in the questionnaire covered an estimated 62% of total absolute dietary calcium intake. In case dietary calcium intakes were either 0 mg/day or over 1750 mg/day, women were contacted to check whether any unintended errors were made. In the group of women with dietary intakes above the tolerable upper level of 2500 mg per day, four women reported correct intakes and the rest made an unintended error, which was corrected after contact with the participant. Since intakes < 200 mg calcium in the mostly Caucasian population of this study are relatively low, we checked whether exclusion of these outliers (dietary calcium intake < 200 mg, n = 106) affected median dietary calcium intake and the percentage of total inadequate intake in an extra-sensitivity analysis.
Questions on potential calcium-containing supplements such as prenatal vitamins, general multivitamins, and calcium supplements, were included for this study. We requested time and period of use (start of usage before and during pregnancy, when potentially stopped, current use), brand and any subtype, frequency of use per week, and amount of tablets per day.
Calcium was standardized to the elemental form in milligrams, based on the labels. We contacted the manufacturers for the clarification when the exact elementary amount of calcium in the supplement was unclear.
Baseline characteristics were analyzed and presented as percentages. Missing values in the baseline characteristics of the Expect I cohort regarding education level (n = 3) and body mass index (BMI) (n = 5), were imputed using stochastic regression imputation based on predictive mean matching .
We calculated individual daily dietary calcium intake by multiplying frequency of consumption by consumed amounts of all the assessed food products and combining product intake (grams per day) with calcium content of each product according to the Dutch Food Composition Table of 2010 (NEVO-online 2010)  and DNFCS2007-2010 . Missing frequency and amount values were imputed with the modal value of all the valid values for the specific variable. To account for the incomplete coverage of the food-frequency questionnaire, we adjusted the estimated calcium intake values (estimated intake*100/61.65). In this way, adjusted total calcium intakes were used in the analyses and presented in the results.
We calculated percentages of women using the different calcium-containing supplements for each week of gestation. We calculated daily calcium intake from supplement use by combining frequency, amount of supplements, and content of specific supplements among current users at 8 weeks of gestation. In case a participant used a supplement but did not know the exact (subtype) brand, the modal value was imputed. Median values of calcium supplement intake at 8 weeks of gestation were calculated.
Median values of total calcium intake, dietary calcium intake, and calcium intake from supplement use were calculated and presented in milligrams per day, with interquartile range (IQR). Total calcium intake was compared to the Recommended Dietary Allowance (RDA) of 1000 mg/day and the Estimated Average Requirement (EAR) of 800 mg/day. An intake level of 800 mg calcium is expected to satisfy the needs of 50% of all pregnant women [26, 27]. The EAR cut point method proposed by the Institute of Medicine (IOM) was used to assess the level of inadequacy in calcium intake in our population [20, 28].
Analyses were performed using IBM SPSS statistics version 23.