Study design and participants
The present study was embedded in the Longitudinal Twin Study (LoTiS), an ongoing twin pregnancies and birth cohort study conducted in Chongqing. Chongqing is situated in southwestern China at latitude of 29.35° N and characterized by a subtropical monsoon humid climate. This city has insufficient sunshine (1000-1400 h/year), especially in winter and spring. The LoTiS twin cohort study was established in January 2016 and aims to unravel the complex interplay between genes and the environment in specifying early life determinants of illness in infancy (Chinese Clinical Trial Registration Number:ChiCTR-OOC-16008203) and was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (No. 201530). Written informed consent was obtained from all participants. This subgroup study included dichorionic twin pregnant women with a prepregnancy BMI in the normal range (18.5~23.9) as well as daily multivitamin (vitamin D: 500IU) supplementation from the first trimester to delivery. Mothers with chronic metabolic diseases and those using immunosuppressants were excluded. Twin pairs with a birth weight <1500 g, significant malformations, or genetic disorders were also excluded. All twin offspring received daily usage of vitamin D supplements (400 IU/day) from birth, and had a pediatric follow-up visit thereafter at a corrected age of 6 months.
Data collection
Maternal sociodemographic data (age, height, weight, education, occupation, parity, mode of conception), lifestyle behaviors before pregnancy (smoking and alcohol use) and pre-existing conditions were collected by standardized questionnaire in the first follow-up during 11-16 gestational weeks. The standardized questionnaire was self-designed for LoTiS cohort study, and detailed information was presented in Supplementary File 1. Information about vitamin D supplementation and other nutrients was collected using a structured questionnaire during the third trimester. The detailed information of this questionnaire is presented in Supplementary File 2. Pregnancy complications, maternal and neonatal outcomes including gestational age, preterm premature rupture of membranes (PPROM, <37 gestational weeks), neonatal gender, birthweight and small-for-gestational age (SGA, defined as a weight below the 10th percentile for GA and sex [29]) were collected from medical records.
Measurement of serum 25[OH]D and classification criteria
Peripheral blood samples were collected from the mothers in the third trimester, and cord blood samples were immediately collected from newborns after placenta delivery by using a coagulation-promoting blood collection tube. The specimen was transported to the Maternal and Fetal Medical Laboratory under refrigeration, where they were centrifuged to obtain serum. There the serum 25[OH]D level was measured by high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-MS-MS, Waters, USA), which is the gold standard method [30]. The concentrations of 25[OH]D3 and 25[OH]D2 were measured separately, and the total level of 25[OH]D was the sum of 25[OH]D3 and 25[OH]D2.
Serum 25[OH]D is the best estimator to assess the body vitamin D levels in the body [31]. For analysis, we divided mothers into 3 groups based on 25[OH]D levels: 25[OH]D levels <20 ng/mL indicated a deficiency, 25[OH]D levels in the range of 20-30 ng/mL indicated an insufficiency, and 25[OH]D levels >30 ng/mL were considered sufficiently high [32]. The same cut-offs were used for neonates according to the Chinese standard [33].
Infant growth assessment and skin prick test
Infants growth was monitored for weight and length at the corrected age of 6 months. The corrected age was defined as the infant’s chronological age minus the difference between term birth (40 weeks) and chronological gestational weeks of delivery. Weight and length were simultaneously measured unclothed by a digital measuring bed (Beideneng, Shanghai, China) operated by trained nurses at the Department of Child Health Care of Chongqing Health Center for Women and Children. The indexes of weight, height and BMI were converted to Z-scores for sex and age according to the WHO Child Growth Standards software (https://www. who. int/childgrowth/software/en/).
On the same day, a skin prick test (SPT) was performed to preliminarily diagnose food allergies. The SPT was performed on the volar surface of the forearm with a lancet through the use of a drop of an allergen extract and test results were produced in 15 to 20 min. The routine test screened for food types including milk, egg white, egg yolk, peanut, fish, wheat, soybean, citrus, and apple.
Statistical methods
All statistical analyses were performed with SPSS version 25.0 (IBM, Armonk, NY, USA). Categorical variables are presented as the count and percentage, and were analyzed by the Chi-squared or Fisher's exact test. Continuous variables are presented as means and standard deviation, and were analyzed by Student’s t-test, LSD student’s t test, one-way analysis of variance or the nonparametric test. Linear correlation analysis was used to explore the correlation between 25[OH]D levels in mothers and newborns and the correlation of 25[OH]D levels between co-twins. Multivariable linear regression analysis was used to detect the associations between neonatal 25[OH]D levels and related clinical characteristics. All tests were two-tailed and p< 0.05 was considered statistically significant.