The Effect of Vitamin D3 Therapy on 25(OH)D, 1,25(OH)2D, VDBP, and 24,25(OH)2D Maternal Serum Levels in Pregnant Women With Vitamin D Deficient and Insufficient
Phase: PHASE2
Conditions: Vitamin D Deficiency
Sponsor: Indonesia University

Study Protocol and Sta.s.cal Analysis Plan 
June 30th 2023 
 
The Eﬀect of Vitamin D3 Therapy on 25(OH)D, 1,25(OH)2D, VDBP, and 
24,25(OH)2D Maternal Serum Levels in Pregnant Women With Vitamin D 
Deﬁcient and Insuﬃcient   
 
 
STUDY PROTOCOL 
This is a randomized controlled trial of vitamin D therapy of 5,000 IU daily or 50,000 IU weekly 
during pregnancy. The study will be conducted at Cipto Mangunkusumo NaDonal Center General 
Hospital and Koja DisDct Hospital in Jakarta, Indonesia from April 2021 – December 2023. All the 
pregnant women will be screened for eligibility when they present to the clinic for antenatal care 
visits and will be oﬀered enrolment if they meet the following inclusion criteria: gestaDonal age 
of ≤ 14 weeks, vitamin D deﬁcient or insuﬃcient (25(OH)D <30 ng/ml], and positive fetal heart rate 
from ultrasound examination. Individuals are not eligible if they have any of the following 
exclusion criteria: mulDple pregnancy, pregnancy with congenital anomaly, hyperemesis 
gravidarum, diarrhea, complicated medical history (hypertension, diabetes mellitus, heart, 
kidney, or liver disease), or use of any dietary supplement containing vitamin D prior to 
enrolment. Informed consent will be obtained from all parDcipants. The study was approved by 
ethical commi\ee at Cipto Mangunkusumo NaDonal Center General Hospital (KET – 
785/UN2.F1/ETIK/ PPM.00.02/ 2019). 
Participants are randomly assigned to one of two parallel intervention groups, with allocation 
concealment: vitamin D3 (cholecalciferol) 5,000 IU/week (Imedco) or 50,000 IU/week (Imedco). 
Assignment is based on a computer-generated randomization list, with 1:1 allocation, using 
permuted blocks of size 6. All participants are given a standard prenatal multivitamin (Emineton) 
which contains fumarate Fe 90 mg; folic acid  0.4 mg; vitamin B6 3 mg; vitamin B12 5 mcg; 
sulphate cupric 0.35 mg, sulphate cobalt 0.15 mg, sulphate mangan 5 mcg, vitamin C 60 mg, 

vitamin E 5 mg, and phosphate calcium 60 mg. Interventions in both groups are given for four 
weeks. 
Baseline blood tests, including serum 25(OH)D, 1,25(OH)2D, VDBP, and 24,25(OH)2D are 
performed at recruitment. A medical history, physical, and ultrasound examinaDon are 
performed. ParDcipants complete a quesDonnaire about sunlight exposure. Thereaier, 
parDcipants are contacted weekly at their homes to evaluate the adherence and the symptoms 
related to pregnancy complicaDons and hypo-/hypercalcemia. Adherence to the supplementaDon 
regimen is assessed by maternal self-report and capsule counts at the end of the study visit. Aier 
four weeks of intervenDons, the maternal venous blood is collected to assess serum 25(OH)D, 
1,25(OH)2D, VDBP, and 24,25(OH)2D. Safety thresholds for the study include hypervitaminosis D 
(25(OH)D D ≥100 ng/mL). 
Serum 25(OH)D is quanDﬁed by direct compeDDve Chemilumiscence Immunoassay (CLIA) using 
LIAISON®. Serum 1,25(OH)2D is quanDﬁed by Liquid Chromatography-tandem Mass Spectrometry 
(LC-MS/MS) using ImmuTube®. Serum VDBP is quanDﬁed by Enzyme-Linked Immunosorbent 
Assay (ELISA) using QuanDkine®. Serum 24,25(OH)2D is quanDﬁed by Acquity I Class Binary 
Solvent Manager FTN , Xevo TQXS Tandem Mass Spectrometry (Waters CorporaDon). All 
laboratory test will be performed at the Prodia Laboratory, Jakarta.  
 
STATISTICAL ANALYSIS PLAN 
Data will be processed using IBM SPSS version 25 program. Each dependent variable is tested for 
homogeneity variance using the Kolmogorov Smirnov normality test. Baseline data are described 
and summarized either by means and standard deviaDons for the normally distributed variables, 
or medians and interquarDle ranges for the non-normally distributed variables. Bivariate analysis 
of qualitaDve data is tested using Chi-Square for the normally distributed variables or Fisher test 
for the non-normally distributed variables. Bivariate analysis of quanDtaDve data is tested using 
T-test for the normally distributed variables or Mann Whitney U-test test for the non-normally 
distributed variables. The data is supplemented with 95% conﬁdence interval with a signiﬁcance 
limit of p ≤ 0.05.