Study design and setting
This cross-sectional study was conducted from October 2015 through February 2017 at the Department of Gynaecology and Obstetrics Hospital Serdang, Selangor, Malaysia. Hospital Serdang is a public hospital located in Sepang district of Selangor, which caters for a population of about 2 million residents from three districts: Petaling, Sepang and Hulu Langat. This study adhered to the STROBE guidelines for observational studies.
Participants
Pregnant women were conveniently recruited while admitted to the labour suite’s Patient Assessment Centre of Hospital Serdang for delivery. Inclusion criteria were Malaysian, aged 19 to 40 years, singleton pregnancy and week of pregnancy ≥ 37 weeks. Pregnant women who were diagnosed with pre-existing systemic disease, pregnancy complications, and had a history of bone and renal disorders, were excluded from the study.
Using an estimated prevalence of vitamin D deficiency (<25 nmol/L) in pregnant Malaysian women of 37% [23]and precision of 7% with a 95% confidence interval, the sample size was calculated to be 192 using the formula [37]: . Additio of 20% for non-response gave a minimum sample size of 245 pregnant women.
Data collection and blood sampling
Information on maternal ethnicity, education level, employment status and household income were self-reported using an interviewer-administered questionnaire. Maternal age, gestational age, last menstrual period (LMP), first booking (date, weeks, and ultrasound scan), gravidity, pre-pregnancy weight and heights were obtained from hospital electronic medical record and antenatal record. Gestational age was determined by LMP and confirmed by the first dating scan. Body Mass Index (BMI) was calculated as body weight divided by squared of body height (kg/m2). Venous blood was collected from pregnant women on the day of labour. All collected blood samples were processed within the collection day. After centrifugation, plasma was aliquoted and buffy coat was collected for subsequent deoxyribonucleic acid (DNA) extraction.
The duration of exposure to sunlight per week was estimated using a questionnaire adapted from a previous study [36]. Pregnant women were asked about their outdoor activities from 7 am to 7 pm over the past one month. Information on the type of activity, duration (in minutes), usual outdoor attire, frequency (per week), use of glove, umbrella, and sunscreen were recorded. Based on the attire worn, the percent of body surface area (BSA) exposed to sunlight was estimated using the "Rule of Nine".
In general, the climate in Selangor, Malaysia, is warm to hot with an average daily sunshine of 6 hours throughout the year. The region is typically affected by north-east monsoon (November-February; rainy season) and south-west monsoon (May to September, relatively dry season). Time spent outdoors and average available sunlight may vary between rainy and dry season [37]. As 25OHD has 3 weeks of half-life in blood circulation, exposure to sun for about 4 weeks before drawing the blood sample may better correlate with the circulating 25OHD. Therefore, in the present study month of blood sampling was dichotomised into December to March and April to November.
To estimate the skin colour of the study participants, the Fitzpatrick scale [38] was used. Based on the women skin colour and skin tanning evaluation, study participants were classified into six different skin phototypes. In this study, as the sample size of each skin type subset was small, particularly skin type I, II, V and VI, skin colour types were dichotomised into light skin colour (Fitzpatrick scale I to III) and dark skin colour (Fitzpatrick scale IV and V) [39].
Daily vitamin D intake from dietary and supplemental sources, was assessed using a vitamin D-specific semi-quantitative Food Frequency Questionnaire (FFQ), which was adapted from a previous study [40]. Pregnant women were asked to recall the brand (for commercial food), frequency and serving size of the listed food they had consumed over the past one month. At the same time, the pregnant women also provided the information regarding their supplemental intakes over the past one month: supplements (e.g. brand name, type of supplements, and specific nutrient), frequency and dosage of intake. To minimise the bias on depth and nature of probing, sun exposure and FFQ was administered by only one trained interviewer. The complete questionnaires used in this study is shown in Supplementary File 1.
Measurement of plasma 25OHD
Plasma concentrations of 25OHD3 and 25OHD2 were determined using ultra-high-performance chromatography (UHPLC) and summed to calculate total plasma 25OHD. The inter-assay coefficient of variation at 50nmol/L were 6% and 7% for 25OHD3 and 25OHD2, respectively. Multiple cut-offs (<25, <30 and <50 nmol/L) were used to describe the prevalence of maternal VDD. The cut-off of National Academy of Medicine (formerly known as Institute of Medicine [IOM]) (<30nmol/L) which is associated with an increased risk of VDD [41] was used in the analysis of factors associated with maternal VDD.
Genotyping
DNA was extracted from the buffy coat using the QIAamp DNA blood kit (QIAGEN, Germany) following to the manufacturer's protocol. DNA yields and quality were determined using the NanoVue Plus UV spectrophotometer (GE Healthcare, USA).
Genotyping of GC SNPs rs4588 and rs7041 were carried out by restriction fragment length polymorphism (RFLP) technique. PCR was performed using a thermocycler (Thermo Fisher Scientific, USA). The PCR reaction mixture was prepared in a total volume of 20 µL: 10 μL of 2X GoTaq® G2 Green Master Mix (Promega, USA), 1 µL each of 10nmol forward primer (5'-AAATAATGAGCAAATGAAAGAAGAC-3’) and reverse primer (5'- CAATAACAGCAAAGAAATGAGTAGA-3'), 3 µL of nuclease-free water and 5 µL of DNA (15 ng/µL). The PCR conditions for amplification included an initial step of denaturation at 95°C for 10 minutes followed by 35 cycles of denaturation at 95°C for 45 s, annealing at 51°C for 45s and elongation at 72°C for 45s, and finally a step of final extension step at 72°C for 7 min. The PCR product, a 483-bp fragment, was then digested separately using restriction enzyme, HaeIII (for rs7041) and StyI (for rs 4588) (New England Biolabs Inc., USA) in a total reaction volume of 20 µL. The digested products were then evaluated by electrophoresis on 1.5% agarose gel stained with ethidium bromide.
Statistical analysis
Statistical analysis was performed using SPSS version 21.0 (SPSS Inc., Chicago, USA). All the variables have less than 5% missing data and single imputation technique was applied [43]. All the continuous variables were assessed for normality using skewness and kurtosis test. Mean and standard deviation (SD) were presented for normally distributed variables, whereas median and interquartile range (IQR) were presented for skewed variables. All the categorical variables were reported as numbers and proportions. Differences in the proportion of GC SNPs and diplotypes between ethnicity and the Hardy-Weinberg Equilibrium (HWE) for each SNPs were examined using Chi-square test.
Individual associations between factors and VDD (25OHD <30nmol/L) were determined using univariate binary logistic regression. To further evaluate the joint associations of demographic, lifestyle and genetic factors with the risk of maternal VDD, multivariate logistic regression was performed. Variables that had p-value <0.25 in univariate analyses and biologically important were included in multivariate analyses. Cramer's V and Pearson's correlation coefficient were used to test the associations between variable. Variables with r2> 0.6 were considered highly correlated and excluded from the multivariate regression. Two separate multivariate models were constructed to determine the factors associated with VDD. Model 1 included selected demographic characteristics, lifestyle factors and the two GC SNPs (rs7041 and rs4588); Model 2 consisted of the demographics, lifestyle factors and GC diplotypes. All the statistical significance was specified at p-value <0.05.