Impact of vitamin D supplementation on preterm births and premature rupture of membranes: a study protocol for a double-blind randomised controlled trial

Abstract

Introduction

Preterm birth (PTB) is a major adverse outcome in developing countries, including India, with serious adverse consequences. Although the pathophysiology is not fully understood, maternal biomarkers influencing placental function may contribute to premature rupture of membranes and PTB. Vitamin D₃ supplementation during pregnancy may lower the risk of PTB, particularly in populations with a high prevalence of vitamin D deficiency (~90% in India). However, available biomarkers for PTB prediction are non-confirmatory, costly and not part of routine care. The aim of the study is to evaluate whether 2000 IU/day vitamin D₃ supplementation during pregnancy reduces PTB compared with 500 IU/day (standard care).

Methods and analysis

Singleton pregnant women ≤18 weeks gestation with deficiency of 25-hydroxyvitamin D (<20 ng/mL) attending antenatal clinics will be randomised (n=902) in a double-blind trial to receive either 500 IU or 2000 IU/day of vitamin D₃ with 1000 mg elemental calcium until delivery. Women with chronic illness, infections (HIV, positive Venereal Disease Research Laboratory test and positive hepatitis B surface antigen test), assisted conception or planned relocation before study completion will be excluded. Baseline details such as socio-demographic data, diet, anthropometry, ultrasound findings and blood pressure will be collected. Monthly antenatal visits will include clinical monitoring, supplement distribution and compliance checks. Blood samples at baseline and second and third trimesters and cord blood will be collected to assess vitamin D-related markers, pregnancy-associated plasma protein A and haemoglobin. Urinary calcium-to-creatinine ratio will be measured mid-study. Birth outcomes and infant anthropometry will be recorded. Baseline and endline characteristics will be compared between study arms using the χ2 test or Fisher’s exact test for categorical variables (depending on the expected frequencies) and the independent samples t-test or Mann–Whitney U test for continuous variables (following normality assessment).

Ethics and dissemination

The study has been funded by the Department of Biotechnology, India (Grant Ref: BT/PR41941/MED/97/548/2021). Ethical clearance has been obtained from Institutional Ethics Committee (Ref No. IEC/344/2021), St. John’s Medical College & Research Institute and Ethics Committee of Bangalore Medical College & Research Institute (BMCRI/EC/25/2024). Study data will be disseminated through peer-reviewed publications and conference presentations.

Trial registration

Trial has been prospectively registered with Clinical Trials Registry - India (CTRI/2021/10/037658).

STRENGTHS AND LIMITATIONS OF THE STUDY.

• The trial employs double blinding (participants and study investigators) to minimise performance and detection bias.

• Serial blood sampling at baseline, each trimester and delivery will enable longitudinal assessment of vitamin D–related biomarkers across both study arms.

• Gestational age at birth will be confirmed using the Best Obstetric Estimate approach, combining the last menstrual period with first-trimester ultrasound dating, minimising misclassification of the primary outcome.

• The sample size calculation was based on an assumed effect size, as direct evidence on the effect of 2000 IU/day vitamin D₃ supplementation on preterm birth was unavailable at the time of study design.

• Restricting enrolment to vitamin D–deficient pregnant women attending tertiary care centres in Bengaluru may limit the generalisability of findings, and expansion to additional recruitment sites may pose challenges in maintaining consistency of clinical practices and data collection procedures.

Introduction

Preterm births (PTBs), defined as live births before 37 weeks of gestation, are a leading global cause of infant mortality, accounting for over one-third of all neonatal fatalities.¹ They can be further categorised into spontaneous and medically indicated types, with spontaneous cases including idiopathic preterm labour and those preceded by premature rupture of membranes (PROM),² wherein membrane rupture prior to labour onset represents a distinct pathway that can initiate preterm delivery. Their prevalence in India ranges from 10% to 18%, as against the global prevalence of 10%, bringing along adverse consequences for the infant, including respiratory, immunological and cognitive impairments, underscoring the need for addressing their impact on both the mother and the child, while developing prevention strategies.³,⁶ There is a growing consensus that PTBs have multifactorial origins, including socio-demographic, environmental, psychological, biological, genetic and nutritional—many of which exist in tandem, especially among the low-income populations.⁴

Vitamin D is a fat-soluble secosteroid, primarily known for regulating calcium and phosphate metabolism. The widespread prevalence of vitamin D deficiency (VDD) among pregnant women in a tropical country such as India, ranging from 34.3% to 96.3%, is alarming.⁷ Low levels of circulating vitamin D are proven to be associated with co-morbidities during pregnancy such as gestational diabetes, preeclampsia and PTB, as well as an increased risk of infections to the mother and baby, rickets and low birth weight.⁸ Additionally, vitamin D supports placental function, hormone secretion, immune modulation and antimicrobial defence, all essential for maintaining feto-maternal tolerance.⁹,¹² Evidence suggests increased respiratory symptoms in the child at 3–5 years born to mothers with low circulating vitamin D levels during pregnancy.¹³,¹⁵ Numerous studies have consistently demonstrated associations between low serum vitamin D levels and the incidence of PTB among various geographical locations and ethnicities. This is mediated through the vital immunomodulatory role of 1,25-dihydroxyvitamin D on diverse populations of vitamin D receptor (VDR)-expressing cells within the decidua, where any insult during this period may lead to PTB,⁹ revealing a compelling link between maternal vitamin D status and the risk of delivery before term.¹⁶,¹⁹ The relationship between vitamin D and infections is of particular interest in the context of PTB, since approximately 40% of PTB are associated with infections.²⁰

However, current literature presents conflicting findings regarding vitamin D supplementation during pregnancy, with discrepancies stemming from differences in dosage, frequency, type (ergocalciferol/cholecalciferol), method of administration (oral/intravenous) and duration of supplementation. Given these factors, it is worth investigating whether increasing the current antenatal dose of 500 IU of vitamin D for pregnant women could be more effective. This has been backed by recommendations from reputed organisations acknowledging the need for a minimum dose ranging from 1500 to 2000 IU to maintain 25-hydroxyvitamin D (25(OH)D) levels >30 ng/mL during pregnancy.^{21 22} This assertion has further been supported by international studies examining the efficacy and safety of supplementing 2000 IU of vitamin D during pregnancy.^{23 24} While literature on this topic in India is sparse, a study in Maharashtra (n=100) evaluating the impact of supplementing 2000 IU of vitamin D3 on the incidence of PTB concluded a greater incidence in the control group (38%) when compared with the experimental group (8%).²⁵

Our study primarily aims to evaluate whether daily supplementation of 2000 IU of vitamin D3, initiated at randomisation (14–18 weeks of gestation) until delivery reduces PTB incidence in pregnant women with VDD (25(OH)D<20 ng/mL), with PROM examined as a secondary outcome given its role as a mechanistic precursor to spontaneous PTB.

Methods

This trial protocol has been prepared and reported in accordance with the Standard Protocol Items: Recommendations for Intervention Trials (SPIRIT) 2025 guidelines. The completed SPIRIT checklist is provided as online supplemental file 1.

This manuscript describes protocol version 2, dated 6 August 2025. The protocol version corresponds to the version registered at the Clinical Trials Registry – India (CTRI/2021/10/037658). The study has been funded by the Department of Biotechnology, Government of India (Grant Ref: BT/PR41941/MED/97/548/2021). The funding agency was not involved in the design of the trial and will not be involved in the conduct, analysis or reporting.

Study design

A double-blind randomised controlled trial comparing 500 IU of vitamin D3/day and 1000 mg of elemental calcium (current standard of care in antenatal care (ANC)) with 2000 IU of vitamin D3/day and 1000 mg of elemental calcium during pregnancy starting from randomisation (14–18 weeks of gestation) to delivery.

Study setting

The study will be conducted at the outpatient department of the obstetrics and gynaecology department of tertiary care hospitals in Bengaluru, Karnataka. The first participant was enrolled in August 2024, and subsequent follow-ups are expected to continue until September 2026. The study protocol is presented in figure 1.

Figure 1. Study protocol flow diagram. ANC, antenatal care; D3, cholecalciferol; IU, International Unit; PTH, parathyroid hormone; USG, ultrasonography; VDBP, vitamin D–binding protein; VDR, vitamin D receptor.

Eligibility criteria and recruitment

Apparently healthy pregnant women of reproductive age (18–40 years) attending the routine antenatal clinic between 14 and 18 weeks of gestation (as assessed by the last menstrual period (LMP)), willing to consent and comply with the study protocol regimen, blood collection and supplementation regimen will be included in the study. Trained research staff will assess the eligibility of all participants presenting to the ANC clinic and obtain a written informed consent from each for the use of their data and biological specimens (if applicable) in ancillary studies. Pregnant women with multiple (eg, twin) pregnancies, chronic morbidities, ongoing medications/treatment, infections (such as HIV, positive Venereal Disease Research Laboratory test and positive hepatitis B surface antigen test), assisted pregnancies, current vitamin D supplementation, or plans to discontinue or relocate from the study area before study completion will be excluded. On successful screening, the vitamin D status of all consented participants will be ascertained as an eligibility criterion for randomisation, and only those with a total serum 25(OH)D<20 ng/mL will be randomised. Women who do not meet this threshold will be referred for routine ANC. This restriction means that findings from this trial will be specifically applicable to vitamin D–deficient singleton pregnancies and may not be generalisable to women with vitamin D insufficiency or sufficiency.

Several strategies will be employed to promote participant retention and ensure complete follow-up throughout the trial. Participants will be contacted monthly via telephone calls to remind them of upcoming antenatal visits and supplement collection. In cases of missed visits, home visits will be conducted by trained research staff to re-engage participants and collect data where possible. The enrolment will be closely monitored to ensure that monthly targets are met. Should recruitment lag necessitate the addition of new sites, all new site staff will undergo the same training and certification procedures as the primary sites before enrolment commences, including standardisation of anthropometric measurements, biological sample handling and data entry protocols. New sites will be incorporated into the randomisation schedule. As recruitment numbers may vary across sites, the study site will be included as a covariate in all primary outcome analyses to account for any site-level imbalances.

Intervention

The intervention will be provided into two groups: one group will receive the experimental high dose of vitamin D 2000 IU/day), and the other group will receive the standard vitamin D dose (500 IU/day). Both groups will receive a similar dose of elemental calcium, which will be consumed as two tablets daily (one in the morning and the other at night) to attain the requisite trial dose. Participants will be given a regimen box containing tablets within blister packs adequate for 35 days (each blister pack arranged as 7×2 per pack). A diagram of the regimen blister pack is given in online supplemental file 2. Both groups will receive tablets randomly, between 14 and 18 weeks of gestation, and will continue until delivery. Counselling according to the ANC guidelines will also be provided. The tablets are manufactured by Excellamed Laboratories Pvt. (Maharashtra, India) and approved and certified by the Food Safety and Standards Authority of India for Good Manufacturing Practices.

Assignment of intervention: allocation and blinding

Participants will be individually randomised in a 1:1 ratio to either of the two intervention arms: (1) 2000 IU vitamin D/day or (2) 500 IU vitamin D/day, using permuted block randomisation, based on an electronically generated sequence produced by an independent statistician, stratified by the study site. The randomisation codes will be held by a statistician independent of the study team and will remain sealed until trial completion. Allocation concealment will be ensured using sealed, opaque supplement boxes, pre-labelled by an independent pharmacist in accordance with the randomisation schedule. Eligible participants at each site will be assigned the next available sequentially numbered box for that site by the research staff, who will have no knowledge of the allocation at the time of enrolment. The tablets for both arms are identical in physical characteristics (appearance, colour, size, shape, weight and taste) to maintain blinding.

In the event of a medical emergency where knowledge of the allocated intervention is essential for clinical management, emergency unblinding may be initiated by the treating physician or principal investigator by contacting the independent statistician holding the randomisation codes. The Data Safety Monitoring Board (DSMB) and institutional review board will be notified of any unblinding event within 24 h. Participants who are unblinded will be withdrawn from the intervention but will continue to be followed up and included in the intention-to-treat (ITT) analysis unless they withdraw consent.

Sample size

Vitamin D supplementation during pregnancy is associated with improvement in maternal vitamin D status and reduction in the risk of PTB; however, evidence regarding the effect of different supplementation doses on gestational age at birth remains limited.²⁶ In the study by Naghshineh and Sheikhaliyan,²⁷ conducted primarily to evaluate pre-eclampsia outcomes, PTB was reported as a secondary outcome, identified in 24% in the no intervention group and 6% in the 600 IU/day supplementation group, corresponding to a relative difference of approximately 75% between groups.

For the present study, a PTB rate of 6% was assumed for the 500 IU group based on the comparable supplementation dose. Although no published trial has specifically evaluated PTB rates with 2000 IU of vitamin D₃ supplementation, it was considered reasonable to assume that a higher supplementation dose may provide additional benefit compared with 500–600 IU/day supplementation. Therefore, for the purpose of sample size estimation, a conservative relative difference of approximately 68% was assumed for the 2000 IU group compared with the baseline rate of 6% in the 500 IU group, resulting in an expected PTB rate of approximately 2% in the 2000 IU arm. This corresponds to an absolute difference of 4% between the two study groups.

Assuming a two-sided significance level of 5% and power of 80% to detect an absolute difference of 4% in PTB rates between the groups (6% vs 2%), the minimum required sample size was estimated to be 411 participants per group. After accounting for an anticipated 10% attrition rate, the final required sample size was 902 pregnant women, with approximately 451 participants in each group.

Statistical methods

Data will be analysed using Stata V.16. Categorical variables will be summarised as frequencies and percentages. Continuous variables will be presented as mean±SD or median with IQR, based on distributional assessment using the Shapiro–Wilk test.

Analysis population

The primary analysis will follow the ITT principle, including all randomised participants analysed in the groups to which they were allocated, regardless of adherence or protocol deviations. A per-protocol (PP) analysis, restricted to participants who completed the intervention with adequate adherence (defined as consumption of ≥75% of assigned supplements) and had no major protocol violations, will be conducted as a pre-specified sensitivity analysis to assess the robustness of ITT findings.

Missing data

The extent and pattern of missing data will be examined prior to analysis. If missingness is minimal (<10%), complete case analysis will be performed. Where substantial missing data are observed, multiple imputation by chained equations will be considered under a missing-at-random assumption. Sensitivity analyses will be conducted where appropriate to assess the robustness of the findings. For the primary outcome, participants lost to follow-up or with missing outcome data will be retained in the ITT analysis under a missing-at-random assumption.

Baseline and general analysis

Baseline sociodemographic and pregnancy-related characteristics will be compared between the 500 IU and 2000 IU groups. Categorical variables will be compared using the Chi-square test or Fisher’s exact test depending on expected cell frequencies. Continuous variables will be compared using the independent samples t-test or Mann–Whitney U test, as appropriate. Standardised mean differences will also be reported to evaluate baseline balance independent of sample size.

Site considerations

Randomisation will be stratified by recruitment site; however, as all participating sites are government hospitals of broadly similar clinical setting and patient profile, a meaningful cluster effect is not anticipated, and participants across sites will be treated as independent observations. Study site will nevertheless be included as a covariate in all primary regression models to account for any residual between-site variability.

Primary outcome: preterm birth (500 IU vs 2000 IU)

The incidence of PTB will be compared between the two groups in the ITT population. Variables demonstrating baseline imbalance between arms at p<0.10, along with the study site, will be adjusted for in the multivariable model. Log-binomial regression will be used to estimate crude and adjusted risk ratios (RR) with 95% CIs. Should convergence issues arise, Poisson regression with log link and robust variance estimation will be applied as a pre-specified alternative. The PP sensitivity analysis will be conducted using the same modelling approach.

Secondary outcomes (500 IU vs 2000 IU)

The incidence of all secondary outcomes (including PROM, gestational hypertension, gestational diabetes, severe anaemia, stillbirth, fetal death, low birth weight and small-for-gestational age (SGA) birth) will be analysed using the same regression framework as the primary outcome. Crude and adjusted RRs with 95% CIs will be reported using log-binomial or Poisson regression with robust variance estimation as appropriate.

Repeated biomarker measurements

Serum 25(OH)D and other biomarkers measured longitudinally across pregnancy (eg, at enrolment, mid-pregnancy and near term) will be analysed using linear mixed-effects models, with time, treatment arm and their interaction as fixed effects, and a random intercept for each participant to account for within-subject correlation. This approach accommodates unbalanced data and participants with partially missing timepoints under the missing-at-random assumption. Results will be reported as estimated marginal means with 95% CIs at each timepoint and as the overall treatment-by-time interaction effect.

Study procedures

Pregnant women ≤18 weeks of gestation as assessed by the LMP and confirmed by ultrasound dating will be recruited for the study. The schedule for participant recruitment, intervention and assessments is described in table 1.

Table 1. Enrolment and intervention schedule.

	Screening	Randomisation	Post-randomisation
Time points		Monthly pregnancy visit (randomisation) (14–18 weeks)	Monthly pregnancy visit (22–26 weeks)	Monthly pregnancy visit (30–34 weeks)	Delivery
Enrolment
Eligibility screening	x
Informed consent	x
Randomisation		x
Interventions
High-dose vitamin D (2000 IU/day)
Standard of care (500 IU/day)
Assessments
Socio-demographics		x
Maternal anthropometry		x	x	x	x
Maternal blood pressure		x	x	x	x
Maternal morbidity		x	x	x
Maternal blood sample collection for biomarkers (vitamin D, PTH, PAPP-A, VDR and VDBP)		x	x	x	x
Sun exposure levels		x	x	x
Physical activity levels		x	x	x
Diet recalls		x	x	x
Urinary calcium and creatinine			x
Birth outcome					x
Birth weight and length					x
Infant mortality					x
Infant morbidity					x
Infant anthropometry					x

IU, International Unit; PAPP-A, pregnancy-associated plasma protein A; PTH, parathyroid hormone; VDBP, vitamin D-binding protein; VDR, vitamin D receptor.

At recruitment, standardised and validated interview-based questionnaires will be administered to obtain information on socio-economic status,²⁸ obstetric history and morbidity, anthropometry, physical activity, habitual dietary intake, clinical details, morbidity, blood pressure monitoring and venous blood sample for the preterm biomarker analysis. The pregnant women will be followed up until delivery during their routine antenatal follow-up visits. The detailed assessments will include the following;

Anthropometry

Weight (kg) (at each trimester) using a digital calibrated and validated weighing scale (SECA 874, Hamburg, Germany) to the nearest 10 g, height (cm) (only at recruitment) using the wall mounted/portable stadiometer (calibrated to the nearest 0.1 cm; SECA 213, Hamburg, Germany), mid-upper arm circumference (MUAC, cm), at each monthly follow-up visit using the non-elastic measuring tape (SECA 201, Hamburg, Germany) (calibrated to the nearest 0.1 cm), skinfold thicknesses (biceps, triceps and subscapular) measured by the calibrated Holtain callipers to the nearest 0.1 mm (Holtain, Crymych, UK). Body composition parameters such as fat percentage, fat mass and fat free mass will be computed using standard formulae.²⁹

Blood pressure monitoring

At recruitment and every monthly follow-up visit, blood pressure will be monitored using the digital calibrated and validated instrument (Omron HEM-7130, Kyoto, Japan) against the standard mercury sphygmomanometer. Appropriate cuff sizes will be procured.

Diet intake

Food frequency will be recorded at recruitment and once in each trimester by a validated food frequency questionnaire.³⁰ During each monthly follow-up visit, a short 24-hour open-ended diet recall questionnaire will be administered to record the habitual dietary intake. The calculations for macro-nutrient and micro-nutrient intake will be computed using standard food composition tables (Indian Council of Medical Research, National Institute of Nutrition or United States Department of Agriculture database).

Physical activity levels

A validated physical activity questionnaire will be administered once at each trimester to understand the physical activity level of the study participants during pregnancy.³¹

Clinical examination

Routine clinical examination will be performed by an obstetrician at the outpatient department. If a participant acquires any kind of morbidity (pregnancy-induced hypertension, gestational diabetes mellitus or infections), they will continue to be part of the study. However, if the morbidity is found related to the supplement, the participant will be withdrawn from the study and will seek routine care from the obstetrician/gynaecologist.

Biological sample collection

Samples of whole venous blood (8 mL) drawn at baseline and during each trimester will be used to estimate the concentration of vitamin D, VDR, vitamin D binding protein (VDBP) and parathyroid hormone (PTH). The blood sample will be collected in EDTA and plain vacutainers (4 mL each). Haemoglobin concentration will be estimated using an automated haematology analyser (Horiba Medical, Japan). The remaining blood from EDTA and plain vacutainers will be centrifuged at 3000 rpm for 10 min to separate the plasma and serum. Both serum and plasma will be aliquoted into pre-labelled cryovials and stored at −80°C until analysis by trained laboratory staff. The total 25(OH)D and PTH levels in serum samples will be measured using electrochemiluminescence immunoassay (Cobas 6000, Roche Diagnostics, Switzerland). Instrument-specific quality controls (QC) will be employed to ensure the accuracy of the measurements, with an intra-day and inter-day coefficient of variation (CV) <5% across all levels. The VDBP, VDR and pregnancy-associated plasma protein A levels will be assessed by quantitative sandwich enzyme immunoassay technique using ELISA kits following manufacturer’s instructions. For quality check, in-house serum pooled and spiked samples will be employed with CVs <8% for both inter-day and intra-day measurements with a recovery rate between 95% and 105% for the spiked samples. Urine tests will be conducted once in the middle of the intervention to assess urinary calcium and creatinine.

Supplement consumption

Participants, as per the randomisation, will receive the assigned supplement along with instructions for its consumption. Additionally, they will be provided with iron and folic acid supplementation, consisting of a daily dose of 5 mg of folic acid until the 14th week of gestation, followed by 0.4 mg of folic acid and 60 mg of elemental iron for the remainder of the pregnancy, as well as two doses of tetanus toxoid in accordance with ANC guidelines.

Adherence record

Adherence will be monitored using tablet counts, including the count of the unused tablets, return of the empty blister packs and record of the tablets consumed based on the total blister packs provided to the participants each month. Further monitoring will be done via telephone calls, spot checks and home visits. It will be calculated as the number of tablets consumed divided by the total number of tablets dispensed over the study period, expressed as a percentage. Participants with >75% adherence will be classified as adherent for the purpose of the PP analysis. This threshold is consistent with adherence definitions used in comparable supplementation trials.³² Participants falling below this threshold will be retained in the ITT analysis.

Safety monitoring

All participants will be screened for urinary calcium-to-creatinine ratio (a priori caution limit of non-fasting urinary calcium-to-creatinine ratio of 0.8 mg/mg or ≥2.27 mmol/mmol) in the second trimester to rule out hypervitaminosis D. Supplementation will be stopped if the urinary calcium-to-creatinine ratio exceeds 1.0 (mg/dL/mg/dL). If excessive levels are observed, the participant will be withdrawn from supplementation but will continue to be monitored and followed up until the study period. Any morbidity or symptoms of allergy diagnosed during the study period will be recorded, and if found to have a causal effect on the study outcome, supplementation will be withheld, and appropriate medical assistance will be given.

Birth outcomes

At delivery, details such as the delivery type, gestational age at birth, any maternal or neonatal morbidity and labour complications will be recorded. Similarly, information about the neonate such as sex, appearance–pulse–grimace–activity–respiration score, birth outcome classification (normal birth outcomes, ie, babies born beyond 37 weeks of gestation; PTBs; babies born <37 weeks of gestation), birth weight (SECA 354) (low birth weight <2500 g), SGA (birth weight <10th percentile of their gestational age), PROM and baby anthropometric measurements including baby length (SECA 417), circumferences (MUAC, head, chest and abdomen; SECA 212) and skinfold thickness (biceps, triceps and subscapular) (Holtain, Crymych, UK) will be recorded within 72 h of birth. Gestational age will be determined using the Best Obstetric Estimate (BOE) approach, prioritising first-trimester ultrasound dating wherever available. LMP-based gestational age will be used only when ultrasound records are unavailable. In case of discordance between LMP and ultrasound dating of more than 7 days, the ultrasound estimate will take precedence. Birth outcomes will be recorded by trained research staff from hospital delivery records and verified against antenatal ultrasound documentation. PROM will be ascertained from clinical notes recorded by the attending obstetrician at delivery. Cord blood (as a surrogate measure of the neonate) will be collected by a staff nurse attending the delivery to determine the total vitamin D level to assess the blood biomarkers status. Quality assurance and control protocols will be followed during blood handling.

Data will be collected by trained research staff, who will receive initial training at the start of the study and periodically undergo validation procedures to ensure that inter- and intra-observer variations are minimised, in accordance with the study requirements.

Patient and public involvement

The patients or the public were not involved in the design or conduct of the study. However, the findings of the study will be disseminated to all the relevant public stakeholders including pregnant women, obstetricians, gynaecologists, participants and their family members.

Trial status

The participant enrolment for the trial started in August 2024 and is expected to be completed by March 2026. Given that participants will be followed up until delivery, we expect results by the end of 2026.

Data management

Data will be collected by trained research staff, who will receive training at the start of the study and periodically undergo validation procedures to ensure that inter- and intra-observer variations are minimised, in accordance with the study requirements. Quantitative data will be collected following the study’s standard operating procedures (SOP) by trained researchers. The data will be coded with a subject number and entered (real-time, wherever possible) in a database using REDCAP database (a public domain for epidemiologic data capture) and secured in the data server at the host institute via the cellular telephone network or wireless internet. Periodic manual and statistical random checks will be conducted to confirm the completeness of the data. The data collected will be validated, organised and preserved as per the norms of the Host Institute. Details on biochemical parameters assessed and the QA/QC values will be recorded into the laboratory books and discussed during the weekly meetings. All instruments used will be regularly calibrated and standardised as per user instrument instructions. The data from REDCAP will be transposed to Excel/Stata V.16 for statistical analysis.

Outcomes

A summary table for the primary and secondary outcomes and definitions is given in table 2. All outcome analyses are restricted to women with confirmed vitamin D deficiency (25(OH)D<20 ng/mL) at baseline, and findings should be interpreted within this specific population. The primary outcome for this study is PTB, defined as a live birth before 37 weeks of gestation, determined using the Best Obstetric Estimate (BOE) approach, considering both the LMP and data from the ultrasound dating scan during the first trimester of pregnancy. PROM, defined as rupture of the fetal membranes prior to labour onset, is included as a secondary outcome, given its mechanistic relevance to PTB.³³ In case ultrasound scans are not available for a participant, LMP-based gestational age will be used. The secondary outcomes for this study will also include gestational hypertension, gestational diabetes and severe anaemia. Other infant outcomes such as stillbirths, fetal deaths, low birth weight, SGA, delivery type and infant anthropometry will also be examined.

Table 2. Definitions of primary and secondary outcomes for the study.

Outcomes	Definitions
Primary outcomes
Preterm birth	Live birth before 37 completed weeks of gestation, or <259 days since the first day of the woman’s last menstrual period1 34
Secondary outcome
Premature rupture of membranes	Rupture of the fetal membranes before the onset of labour contractions33 35
Gestational hypertension	Blood pressure >140 mmHg systolic or >90 mmHg diastolic on two separate readings, at least 4 h apart after 20 weeks of gestation in a woman with previously normal blood pressure. In cases in which the systolic pressure is >160 mmHg or the diastolic pressure is >110 mmHg, the diagnosis may be confirmed with a repeat measurement over a shorter interval.36
Gestational diabetes	Impaired glucose tolerance with onset or first recognition during pregnancy37
Severe anaemia	Haemoglobin concentration <7.0 g/L38
Stillbirth	Fetal death >28 weeks of gestation39
Fetal death	A product of human conception, regardless of gestational age which does not show any signs of life after expulsion or extraction, whether or not the umbilical cord has been cut or placenta remains attached.40
Low birth weight	Live birth weighing <2500 g41
Small-for-gestational-age birth	Live birth with size-for-gestational age <10th percentile on the INTERGROWTH-21st standard42

Oversight and monitoring

To ensure the study upholds the highest standards of safety and ethical compliance, the trial is being overseen by the DSMB, which convenes on a quarterly basis to discuss the progress of the study. The members include Dr. Christopher Robert Sudfeld (Associate Professor of Global Health and Nutrition, Harvard T.H. Chan School of Public Health, Boston, USA) as the Committee Chairperson, Dr. Bharati Kulkarni (Director, National Institute of Nutrition, Hyderabad, India), Dr. Nita Bhandari (Senior Scientist and Director, Society for Applied Studies, New Delhi, India), Dr. Shalini Hegde (Associate Professor, Department of Paediatric Surgery, St. John’s Medical College Hospital, India) and Dr. Tinku Thomas (Professor & Head, Department of Biostatistics, St. John’s Medical College, Bangalore, India). The DSMB is also monitoring all severe adverse events (SAEs) on a monthly basis and may request unmasking the data for safety concerns.

The DSMB will have the authority to recommend termination of the trial, with the final decision to terminate resting with the principal investigator in consultation with the institutional ethics committee. Two categories of stopping guidelines have been pre-specified. For safety, supplementation will be stopped for individual participants if the urinary calcium-to-creatinine ratio exceeds 1.0 mg/dL/mg/dL or if circulating 25(OH)D levels exceed 50 ng/mL. Should these safety thresholds be exceeded in >10% of recruited participants, the DSMB will evaluate whether trial-wide stopping is warranted. Additionally, if the rate of SAEs differs significantly between arms at p≤0.05, the DSMB will consider unblinding and stopping the trial for safety concerns. For efficacy, a two-sided Haybittle–Peto stopping rule of p<0.001 will be applied to the primary outcome of PTB, assessed using the χ2 or Fisher’s exact test, to determine whether the trial should be stopped early due to overwhelming evidence of effect.

Protocol amendments

Any modifications to this protocol will be initiated by the principal investigator and must receive approval from the institutional ethics committee prior to implementation. Substantial amendments, those that may affect the safety of the participants, the scientific validity of the study or the conduct of the trial, will additionally be submitted to the DSMB for review and updated on the Clinical Trials Registry - India (CTRI) portal. All approved amendments will be documented with a revised version number and date and communicated to the site investigators and research staff before implementation.

Ethics and dissemination

Ethical clearance for this trial has been obtained from the Institutional Ethics Committee (Ref No. IEC/344/2021), St. John’s Medical College & Research Institute and the Ethics Committee of Bangalore Medical College & Research Institute (BMCRI/EC/25/2024) and has been prospectively registered with the Clinical Trials Registry - India (CTRI/2021/10/037658). Written informed consent will be obtained from all participants prior to enrolment, covering their participation in the trial and the use of their data and biological specimens in ancillary studies. The informed consent process will be conducted by trained research staff in the participant’s preferred language, ensuring adequate time for questions and voluntary decision-making. A copy of the participant informed consent form is provided as online supplemental file 3. The findings from this trial will be disseminated through peer-reviewed publications and conference presentations. Results will also be shared with government and non-government stakeholders at state and national levels.