Efficacy of maternal B12 supplementation in vegetarian women for improving infant neurodevelopment: protocol for the MATCOBIND multicentre, double-blind, randomised controlled trial

Introduction Vitamin B12 deficiency is widely prevalent across many low- and middle-income countries, especially where the diet is low in animal sources. While many observational studies show associations between B12 deficiency in pregnancy and infant cognitive function (including memory, language and motor skills), evidence from clinical trials is sparse and inconclusive. Methods and analysis This double-blind, multicentre, randomised controlled trial will enrol 720 vegetarian pregnant women in their first trimester from antenatal clinics at two hospitals (one in India and one in Nepal). Eligible mothers who give written consent will be randomised to receive either 250 mcg methylcobalamin or 50 mcg (quasi control), from enrolment to 6 months post-partum, given as an oral daily capsule. All mothers and their infants will continue to receive standard clinical care. The primary trial outcome is the offspring’s neurodevelopment status at 9 months of age, assessed using the Development Assessment Scale of Indian Infants. Secondary outcomes include the infant’s biochemical B12 status at age 9 months and maternal biochemical B12 status in the first and third trimesters. Maternal biochemical B12 status will also be assessed in the first trimester. Modification of association by a priori identified factors will also be explored. Ethical considerations and dissemination The study protocol has been approved by ethical committees at each study site (India and Nepal) and at University College London, UK. The study results will be disseminated to healthcare professionals and academics globally via conferences, presentations and publications. Researchers at each study site will share results with participants during their follow-up visits. Trial registration number CTRI/2018/07/015048 (Clinical Trial Registry of India); NCT04083560 (ClinicalTrials.gov)

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Other than as permitted in any relevant BMJ Author's Self Archiving Policies, I confirm this Work has not been accepted for publication elsewhere, is not being considered for publication elsewhere and does not duplicate material already published. I confirm all authors consent to publication of this Work and authorise the granting of this licence.  Study design benefits from preceding qualitative patient and public involvement (PPI), and nested qualitative sub-study will ensure participant feedback and process evaluation to optimise trial conduct.
 The study is being conducted across two diverse socio-demographic populations, to increase generalisability of study findings.

Study limitations
 Different regulatory structures in two different countries.
 Normal/Routine care delivered in the two study sites differs. This could lead to differences in maternal and new-born care and monitoring between study sites.

INTRODUCTION
Malnutrition during pregnancy is a global health problem. Micronutrient deficiencies are associated with adverse developmental outcomes in children, including poorer neurocognitive functioning potentially leading to poorer educational achievement and economic prospects in affected children [1,2].
Vegetarian diets and those low in animal source foods are risk factors for B-12 deficiency [7,8]. During pregnancy sufficient B-12 intake is vital for foetal brain development and deficiency in pregnancy is linked to infant birth defects, intrauterine growth restriction, preterm delivery and neural tube defects [9][10][11][12][13][14]. Many observational studies document that B-12 deficiency during pregnancy and early infancy adversely affects infant cognitive function [15,16] including memory [17], language and motor skills [18].
The evidence supporting a causal link between maternal vitamin B-12 levels and infant cognitive outcomes from Randomised Controlled Trials (RCTs) is inconclusive [11]. Maternal B-12 supplementation given as 50mcg a day from <14 weeks' gestation through to 6 weeks postpartum did not improve infant neurodevelopmental outcomes at 9 months in a previous trial [11]. However, hyperhomocysteinemia (a marker of B-12 insufficiency) during pregnancy was negatively associated with infant cognitive outcomes, suggesting that the dose or duration of B-12 supplementation may have been inadequate. Additionally, a high rate of participant attrition (51.4%), poor monitoring of supplement compliance, and lack of biochemical evaluation of B-12 status in the supplemented group further limit the conclusiveness of the findings from this trial.
Other RCTs that assessed a 250mcg dose of B-12 per day throughout pregnancy until 3 months' postpartum in Bangladesh [19], and >2000mcg from 17 to 34 weeks' gestation in India [20] did not evaluate neurodevelopment outcomes in infancy. Hence, there is insufficient evidence concerning timing, dose, duration and efficacy of B-12 supplementation. To address this evidence gap, we are conducting a double-blind RCTs in one LMIC (India) and one low income country (Nepal), which will evaluate the efficacy of 250mcg/day vitamin B12 from the first trimester to 6 months' post-partum on infant neurodevelopment outcomes measured at age 9 months.

Primary objective
To determine the effect of 250mcg daily oral maternal vitamin B-12 supplementation on infant neurodevelopment as compared to 50mcg.

Secondary objectives
To determine the effect of 250mcg versus 50mcg of oral maternal vitamin B-12 supplementation on biochemical parameters of B-12 status including: a. Maternal -at the end of the third trimester; b. Infant -at 9 months post-partum.

Tertiary objectives
To determine the:

Setting
This study will be conducted across two sites, in Delhi, India and Kathmandu, Nepal. Both countries have a documented high prevalence of B-12 deficiency. [5,6] Participants Approximately 700 women deliver annually at Sitaram Bhartia Institute of Science and Research (SBISR), New Delhi, India. SBISR is a privately funded, tertiary hospital catering to the higher and middle-income populace. Approximately 22, 000 women deliver annually at Paropakar Maternity and Women's Hospital (PMWH), Kathmandu, Nepal. PMWH is a government hospital facility and caters to people from poorer socio-economic strata. Data from SBISR from our earlier work (2011)(2012)(2013)(2014) documented that most of the mothers delivering at SBISR were well-educated (96.8% college education), working ladies (63.2%) living in well-earning (Mean Gross Family Income 16000 USD) nuclear families (57.9%) [21]. A directly comparable dataset was not available from PMWH.

Feasibility Data and Preliminary Work
Feasibility of data and preliminary qualitative work was conducted for the trial in 2017-18. Feasibility of recruitment within the given timelines was studied on the basis of previous data available from the study sites. The lead author (JN) examined neurodevelopmental outcome of 413 babies born at SBISR, in a cohort study between 2011 and 2014. The project had a similar antenatal recruitment and postdelivery infant neurodevelopmental follow-up design as proposed in this trial [22]. Approximately 2/3 (67%) of subjects completed the cohort study. The loss to follow-up (33%) and speed of recruitment have been taken into account in the sample size calculations and the recruitment timeline for this trial.
No similar recruitment and retention data were available from PMWH. Kathmandu. Homemade recipes which include egg and/or dairy products as a main ingredient were incorporated in the FFQ. Locally available household utensils were standardised to measure the amount of food consumed to maintain consistence across the sites.
All research staff from India and Nepal were jointly trained in study design, research methodology, good clinical practice, study protocol and study tools. Training lectures were video recorded and made available to the teams at both study sites as a reference material for continuous training.

Eligibility
Potentially eligible participants are screened at first presentation to the antenatal clinic at <12 weeks of gestation in accordance with the following selection criteria.

Inclusion Criteria:
 Vegetarian women defined as a self-reported dietary pattern that includes veganism and/or people who do eat egg and/or do consume milk and/or a portion meat/chicken/fish < once a month;  Living within an a-priori defined geographical area; o Delhi -national capital region; o Nepal -10-km radius of PMWH;  Able to understand at least one of English, Hindi or Nepalese. Anthropometric measurements and signs of micronutrient deficiency (anaemia and rickets) are recorded during each visit as part of routine care. Metabolic disorders (congenital hypothyroidism, congenital adrenal hyperplasia, cystic fibrosis, biotinidase deficiency, phenylketonuria and galactosemia) are screened for and maternal support for breastfeeding by a paediatrician or lactation counsellor is provided at 7-14 days.
Study specific: Supplements are provided to mothers at each visit, with instructions for usage until 6 months after childbirth. Any morbidities and deficiencies are recorded and treated as per institutional guidelines. Maternal and infant tolerance for the supplementation including any gastrointestinal symptoms are recorded at each visit, as is maternal compliance. Provision of supplements to the mother in both groups is be stopped at 6 months post-partum.
The hospital evaluation at 9 months (±14 days) includes neurodevelopmental assessment by a developmental therapist using the Developmental Assessment Scales for Indian Infants (DASII) -an adapted and validated version of the Bayley's Scale for Infant Development Third Edition (BSID III) and blood sampling for estimation of Hb, vitamin B-12, homocysteine and holo-transcobalamin concentrations [25]. The DASII motor scale assesses control of gross and fine motor muscle group whilst cognitive, personal and social skills development is assessed through the mental scale. All neurodevelopmental assessments is conducted by a trained developmental therapist at the institute in a soundproof room. In premature babies corrected age is used to calculate developmental quotients. Additional assessments include a complementary feeding assessment by a nutritionist using a 72-hour dietary recall and home environment assessment using the adapted Bradley HOME inventory [24] by a trained field worker.
Compliance: Research staff dispense blister packaging of ten B-12 capsules and study specific bags for participants to store, carry and keep supplements in one place. In case of loss of supplement or inability to come for follow-up, participants are able to contact the researcher at any time to request more supplements. The supplement packaging is numbered with a serial number. To maximise compliance, weekly reminders are sent through the preferred mode of communication as specified by the trial participants. Each participant is provided with a bag to save empty packaging strips and instructed to carry the bag with her at the next visit to monitor compliance.

Biochemical analysis
All samples are processed as one batch at study completion. Maternal samples are assessed for blood levels for B-12, homocysteine, holo-transcobalamin, 25(OH)D, folate, CBC and RBC indices in the first
Exploratory Outcome(s):  Haemoglobin (Hb) of infants at 9 months (+14 days) post-partum;  Infant anthropometry including weight, height, length and head circumference at 9 months after birth in all subjects.

Sample size considerations
A planned sample of 720 pregnant women, 360 per study site, will be randomly assigned to experimental (n=360; n=180 in per study site) or control (n=360; n=180 in per study site) groups. This is based on the number of infants needed to complete the neurodevelopmental screen in each of the two randomised groups to detect a difference in Development Quotient (DQ) at nine months (primary outcome) of 2 points (22). The difference of 2 DQ points was chosen because it is the smallest effect size thought to be clinically significant and hence a null trial result is likely to exclude a clinically relevant effect of maternal B12 supplementation on infant cognitive function. The sample size has been inflated by 35% to account for attrition and assumes 90% statistical power and a 5% two-sided significance level. variables. These models include interaction terms to explore conditional differences between the trial's arms. If necessary, transformations are applied to the response variables to fulfil the model's assumptions. Covariates include trial site, infant feeding, gender, feeding by gender, weight z-score at birth, weight gain between birth and 3 months, and maternal body mass index (BMI). Z-scores for height and weight are calculated according to the World Health Organisation [26] growth standards using the R language for statistical computing [27].
We will examine the impact of B-12 supplementation on randomised groups in two subgroups -those that have any of the following conditions vs. all others (high risk vs. low risk):  Maternal morbidity during pregnancy will be classified as high-risk (including gestational diabetes, pregnancy-induced hypertension/pre-eclampsia/eclampsia and absent or reversed end diastolic flow); and low-risk;  Birth weight z-score categorized as high-risk (small for gestational age [SGA]: <10th centile for birth weight); and low-risk (appropriate for weight [>10th centile for birthweight]);  Gestation categorized as preterm (low-risk; 33-37 weeks gestation); very preterm (high-risk; 28-32 weeks gestation); and term (low-risk: >37 weeks gestation). Extremely preterm (<28 weeks) will be exited;  Higher risk neonatal course categorized as high risk (if neonate has: APGAR score <7 at 1 minute, hypoglycaemia, hyperbilirubinemia or prolonged neonatal intensive care stay >7 days); and low risk.
 Infants with documented neurological disease/anomaly/illness will be sub-grouped separately (high risk) and compared to those with no disease/anomaly illness (low risk).

Data management and dissemination
Data will be collected under the regulation of the data protection and management guidelines given by Governments of India and Nepal, and as per the UK Data Protection Act 2018. Data will be securely stored in an E-database with password protection. The E-database was designed using Access. Live data entry will be conducted at the study sites and tested for accuracy using duplicate data entry at an offsite location at periodic intervals.
The study results will be disseminated globally with academic and health professionals via conferences, presentations and publications. Researchers at each study site will share emerging results with participants during their follow-up visits. Regular site meetings with press offices, short films, radio spots, periodic blog posts, Twitter and Facebook messages, and online articles are also

Qualitative evaluation proposed:
The qualitative sub-study aims to explore knowledge, attitudes and perceptions of pregnant women towards B-12 supplementation and child development, and potential barriers to implementation.
Better understanding these domains across the two study sites may inform the perceived need for the intervention, help define patient-centric outcomes and allow the evaluation of any differences between the two arms.
We will use convenience sampling and estimate interviewing 30 participants across both trial sites.
We will develop and pilot a semi-structured topic guide in collaboration with PPI representatives serving on the Trial Steering Committee (TSC). The topic guide will be designed in English and translated into either Hindi or Nepali for administration across the study sites. The topic guide will then be back translated to English to ensure consistency and accuracy of content. It is intended to help direct the interview whilst remaining sensitive to unsolicited themes. An interviewer with prior training and experience in qualitative methods will facilitate the semi-structured interviews.
Data will be collected in quiet rooms in either hospital, away from other patients and staff. Interviews will be facilitated in English, Hindi or Nepalese. All interviews will be audio recorded, transcribed, translated into English (where necessary) and then crosschecked against the original recording. The translated transcripts will then be coded using the Atlas Ti 7.2 software for qualitative analysis. [28] A research team member will code and categorise data, and prepare an initial narrative. A final report will then be prepared with input from the interviewers and wider research team. documents and electronic databases. Every effort is made to arrange interviews and examination at a time convenient to the participants without causing much disruption in their daily routine. Study data is stored securely and is only accessible to trial staff and authorised personnel. The Principal Investigator at each study site will submit an annual progress report, end of trial notification and a final report to the Institutional Ethics Committee, host organisations and the funders.
We used the SPIRIT checklist for writing our protocol [29].

DISCUSSION
This study will have considerable impact on current and future policy initiatives focusing on micronutrient supplementation in India and Nepal by underpinning micronutrient supplementation policy in India as envisaged in the recently released "National Health Policy", and the micronutrient initiative in Nepal. The project will provide key insights to strengthen existing national nutrition programmes like "National Iron plus Initiative", "Janini Suraksha Yojna" and "Janini Shishu Suraksha Kariyakram" initiated by the Government of India and similar initiatives by the Government of Nepal.
Because of the high worldwide prevalence of maternal B-12 deficiency (74%), the study findings will be highly relevant for maternal and neonatal health in nutritionally vulnerable and at-risk populations globally. The work will also provide evidence on the utility, dose, duration and time of initiation of Vitamin B12 supplementation in pregnant women/infants.

DECLARATIONS
Based on the SPIRIT guidelines.

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Ethical Considerations and
 The study is being conducted across two diverse socio-demographic populations, to increase generalisability of study findings.

Study limitations
 Different regulatory structures in two different countries.
 Normal/Routine care delivered in the two study sites differs. This could lead to differences in maternal and new-born care and monitoring between study sites.

INTRODUCTION
Malnutrition during pregnancy is a global health problem. Micronutrient deficiencies are associated with adverse developmental outcomes in children, including poorer neurocognitive functioning potentially leading to poorer educational achievement and economic prospects in affected children [1,2].
Vegetarian diets and those low in animal food source are risk factors for B-12 deficiency [7,8].
Sufficient B-12 intake during pregnancy is vital for foetal brain development. Deficiency of B-12 in pregnancy is linked to infant birth defects, intrauterine growth restriction, preterm delivery and neural tube defects [9][10][11][12][13]. Many observational studies detail the association between B-12 deficiency during pregnancy or early infancy with subsequent poorer infant neurodevelopment [14] and cognitive function [15,16] including memory [17], language and motor skills [18].
There is limited evidence from randomised controlled trials (RCTs) regarding effect of vitamin B-12 supplementation and infant cognitive outcomes [19,20,21]. An infant supplementation trial reported neurodevelopmental benefit of infant B-12 supplementation with folate [21]. Except for vitamin B12 in relation to gross motor functioning, there was no significant benefit of giving vitamin B12 or folic acid alone on early child development. In another trial, maternal vitamin B-12 supplementation given as 50mcg a day from <14 weeks' gestation through to 6 weeks postpartum did not improve infant neurodevelopmental outcomes at 9 months [19]. In this trial hyperhomocysteinemia (a marker of B-12 insufficiency) during pregnancy was negatively associated with infant cognitive outcomes, suggesting that the dose or duration of B-12 supplementation may not have been adequate.
Additionally, a high rate of participant attrition (51.4%), poor monitoring of supplement compliance, a lack of biochemical improvement in B-12 status in the supplemented group mothers and lack of infant biochemical evaluation for B-12 status in later infancy further limit the inferences that can be drawn from this trial. Other RCTs that assessed a 250mcg dose of B-12 per day throughout pregnancy until 3 months' postpartum in Bangladesh [22], and >2000mcg from 17 to 34 weeks' gestation in India [23] did not evaluate neurodevelopment outcomes in infancy. Hence, there is insufficient evidence concerning timing, dose, duration and efficacy of B-12 supplementation. To address this evidence gap, we are conducting a double-blind multicentric RCT in one LMIC (India) and one low income country (Nepal), which will evaluate the efficacy of 250mcg/day vitamin B12 supplementation in vegetarian mothers from the first trimester to 6 months' post-partum on infant neurodevelopment outcomes measured at age 9 months. It is noteworthy that several published protocols report ongoing work on the subject from India and Nepal [24,25]. The proposed work differs significantly in several aspects from them.
We specifically chose to supplement vegetarian women as a high-risk population in view of the lower intake of B-12 and higher prevalence of deficiency. We chose the dose of 50 mcg as a quasi-control group as it has been specifically documented earlier to prevent deterioration in deficiency in Indian pregnant women. In addition, given the progressive decline in B-12 levels through pregnancy documented in the placebo group of an earlier trial and the potentially serious neonatal consequences of severe B-12 deficiency highlighted above, a pure placebo control was thought to be unethical. We chose an interventional dose of 250 mcg as it has been shown in an earlier trial to improve B-12 levels [22] although neurodevelopment was not assessed in this trial.

Primary objective
To determine the effect of 250mcg daily oral vitamin B-12 supplementation of vegetarian pregnant and post-partum women on infant neurodevelopment as compared to 50mcg.

Secondary objectives
To determine the effect of 250mcg versus 50mcg of oral vitamin B-12 supplementation of vegetarian pregnant and post-partum women on biochemical parameters of B-12 status including: a. Maternal -at the end of the third trimester; b. Infant -at 9 months post-partum.

Tertiary objectives
To determine the: a. Socio-economic mediators (income, education, profession) of the relationship between maternal B12 status, supplementation and infant neurodevelopment;

Patient and Public Involvement:
To represent the participants' perspective a Patient and Public Involvement (PPI) panel was established consisting of four parent pairs who had delivered a baby within the previous 2 years. One parent pair is also included in continued project development and the Trial Oversight Committee (TOC). We further conducted a qualitative study with pregnant mothers at both study sites to better understand the factors likely to influence the uptake and implementation of the proposed intervention, and proposed barriers to implementation. This qualitative study established that most respondents were unaware of B-12 dietary sources and the importance of B-12. All the participants understood the burden of the intervention and were eager to know about symptoms, treatment and prognosis of B-12 deficiency. Respondents' willingness to accept the intervention and drawing extra blood from the baby as a part of routine sampling for checking B-12 levels guided the study design.
Perceived barriers including concerns around potential side effects of the B-12 supplement, relocation  All participants presenting at the two study sites will be informed of the role and importance of vitamin B-12 in human health and its sources. This will be done through educational banners and posters and through community workshops.
 The initial information about the trial will be given by the doctor providing antenatal care to the mother. Subsequent detailed consent and recruitment will be conducted by study research staff.
 In order to improve compliance and study retention, reminder text messages are sent weekly to participating mothers.
 To incentivise women (sponsoring various biochemical tests at the Indian trial site and covering transportation cost at the Nepal site) at both sites to avoid the risk of financial stress resulting from study participation.
 To add Bradleys' Home Observation for Measurement of the Environment (HOME) inventory to the assessment given the divergent environmental profile of the two study sites [28] in order to assess the infant's environment and possible confounders of neurodevelopment.
The study results will be disseminated globally with academic and health professionals via conferences, presentations and publications. Researchers at each study site will share emerging results with participants during their follow-up visits.
We adapted a pre-validated Food Frequency Questionnaire (FFQ) for Vitamin B-12 intake developed by Gael et al. [29] to the Indian and Nepalese settings. For our purpose, the original Vitamin B12 FFQ by Gael et al. containing 30 questions on consumption of food and beverages containing B12 was reviewed independently by two nutritionists. A market survey was done to obtain information on egg and milk products in the diet as well as B-12 fortified items available in local markets of Delhi and Kathmandu. Homemade recipes which included egg and/or dairy products as a main ingredient were incorporated in the FFQ. Locally available household utensils were standardised to measure the amount of food consumed to maintain consistence across the sites.
All research staff from India and Nepal were jointly trained in study design, research methodology, good clinical practice, study protocol and study tools. Training lectures were video recorded and made available to the teams at both study sites as a reference material for continuous training.

Eligibility
Potentially eligible participants are screened at first presentation to the antenatal clinic at <12 weeks of gestation in accordance with the following selection criteria.

Inclusion Criteria:
 Vegetarian women defined as a self-reported dietary pattern that includes veganism and/or people who do eat egg and/or do consume milk and/or a portion meat/chicken/fish < once a month;  Living within an a-priori defined geographical area; o Delhi -national capital region; o Nepal -10-km radius of PMWH;  Able to understand at least one of English, Hindi or Nepalese.

Exclusion Criteria:
 Wish for medical termination of pregnancy;  Aged <18 or >35 years;  Anticipate moving out of the a-priori defined geographical area before or after delivery;  Treated for infertility;  Having a known pre-diagnosed mental health disorder including depression, drug or alcohol abuse;  Currently participating or having participated in another study within four weeks prior to trial start;  Having a known allergy to vitamin B-12 or another supplement constituent.

Randomisation
As presented in Figure 1, all eligible and consenting participants are randomised with an allocation ratio of 1:1 to either the intervention (250mcg), or control (50mcg) group. The randomisation sequence is generated at a single third-party location (non-study site) using a computer-generated sequence stratified for location. The sequence is allocated to serial numbers by a neutral person in each country using sequentially numbered, sealed, opaque envelopes. Codes within envelopes will identify boxes labelled with 5-digit codes. Each box contains enough dosages to cater to the study period of one participant. The strips containing intervention and control doses of B-12 are identical in taste, smell and appearance. All participants, recruiters, developmental therapists, laboratory staff and the data analyst are blinded to the B-12 allocation until final analysis.

Procedure
At enrolment participants are invited to provide demographic details including age, height, weight, ethnicity, education and socio-economic status. Their preferred channel of subsequent communication (phone, text message, email) is also noted. Participants are allocated a study identification number and accompanied for blood sampling. The timing of blood sampling is synchronised with that of any other samples being drawn in the first trimester to minimise unnecessary venepunctures. Twelve ml of blood sample is drawn into 2 plain vials (5 ml each) and 2 ml is drawn into an EDTA vial. The EDTA sample is processed immediately for complete blood counts by coulter counter.The plain (serum separator tubes) vial samples are centrifuged at 3500 rpm for 10 minutes at the study site. The separated serum is transported on ice and stored at -70° C at round the clock temperature and custody monitored at both the study sites . Participants are given B-12 supplement doses to last five days beyond the expected date of their next visit after being fully informed as to the supplement and its administration. Within 48 hours, participants are contacted to confirm initialisation of supplementation and re-emphasise compliance. Subsequent maternal visits take place monthly until 36 weeks and then weekly. At these visits, all participants visit their obstetrician as part of routine care. Participants also meet with the research staff at a pre-specified location at the study site. To maximise compliance, weekly supplement and pre-appointment reminders are sent via the preferred mode of communication. Acquired morbidity (gestational diabetes, hypertension, and hypothyroidism) and medication history are noted in case record forms (CRFs). Empty supplement packets are collected at each appointment as an indicator of adherence. In the third trimester, mothers' dietary B-12 intake is assessed using a pre-standardised and adapted FFQ and blood sampling will be repeated using the same procedures as the first trimester.
Neonatal assessment: During childbirth, a medical officer and/or paediatrician monitors the birth and post-delivery course of neonates for any morbidity potentially influencing neurodevelopment.
Gestational age, growth retardation, congenital abnormalities and APGAR (appearance, pulse, grimace, activity, respiration) score is also routinely documented for neonates. Cord pH is measured for all new-borns with delayed cry and who require any intervention beyond the basic steps of resuscitation (ambu bagging, intubation, or pharmacological treatment). Any seizures, neurological problems, hypoglycaemia, hypothermia, hyperbilirubinemia are screened, diagnosed, evaluated, and managed as per institutional guidelines. As per the standard practice, all new-borns are screened for hearing deficits, vision, and critical congenital heart disease as per institutional protocols. Preterm neonates transferred to the neonatal unit are followed as per their customized follow-up and early intervention plans as recommended by the primary care provider. Neurodevelopment and biochemical assessment of preterm neonates is carried out as per their corrected ages.
Infant follow-up is conducted at 7-14 days, 4, 8, 12 and 16 weeks and at 6 and 9 months of age. At these visits, the infants undergo routine care as well as study specific evaluations (Visit Schedule: Supplementary Table 1). Routine care: Each mother-infant dyad sees a paediatrician for age-appropriate routine care, which involves clinical evaluation, preventative care and vaccination as per standard procedure.
Anthropometric measurements and signs of micronutrient deficiency (anaemia and rickets) are recorded during each visit as part of routine care. Metabolic disorders (congenital hypothyroidism, congenital adrenal hyperplasia, cystic fibrosis, biotinidase deficiency, phenylketonuria and galactosemia) are screened for and maternal support for breastfeeding by a paediatrician or lactation counsellor is provided at 7-14 days. Study specific: Supplements are provided to mothers at each visit, with instructions for usage until 6 months after childbirth. Any morbidities and deficiencies are recorded and treated as per institutional guidelines. Maternal and infant tolerance for the supplementation including any gastrointestinal symptoms are recorded at each visit, as is maternal compliance. Provision of supplements to the mother in both groups will be stopped at 6 months post-partum.
The hospital evaluation at 9 months (±14 days) includes neurodevelopmental assessment by a developmental therapist using the Developmental Assessment Scales for Indian Infants (DASII) -an adapted and validated version of the Bayley's Scale for Infant Development Third Edition (BSID III) [30] and blood sampling for estimation of Hb, vitamin B-12, homocysteine and holo-transcobalamin concentrations. The DASII motor scale assesses control of gross and fine motor muscle group whilst cognitive, personal and social skills development is assessed through the mental scale. All neurodevelopmental assessments will be conducted by a trained developmental therapist at the institute in a soundproof room. In premature babies corrected age is used to calculate developmental quotients. Additional assessments include a complementary feeding assessment by a nutritionist using a 72-hour dietary recall and home environment assessment using the adapted Bradley HOME inventory [28] by a trained field worker.
Compliance: Research staff dispense blister packaging of ten B-12 capsules and study specific bags for participants to store, carry and keep supplements in one place. In case of loss of supplement or inability to come for follow-up, participants are able to contact the researcher at any time to request more supplements. The supplement packaging is numbered with a serial number. To maximise compliance, weekly reminders are sent through the preferred mode of communication as specified by the trial participants. Each participant is provided with a bag to save empty packaging strips and instructed to carry the bag with her at the next visit to monitor compliance.

Exploratory Outcome(s):
 Haemoglobin (Hb) of infants at 9 months (+14 days) post-partum;  Infant anthropometry including weight, height, length and head circumference at 9 months after birth in all subjects.

Sample size considerations
A planned sample of 720 pregnant women, 360 per study site, will be randomly assigned to experimental (n=360; n=180 in per study site) or control (n=360; n=180 in per study site) groups. This is based on the number of infants needed to complete the neurodevelopmental screen in each of the two randomised groups to detect a difference in Development Quotient (DQ) at nine months (primary outcome) of 2 points (26). The difference of 2 DQ points was chosen because it is the smallest effect size thought to be clinically significant and hence a null trial result is likely to exclude a clinically relevant effect of maternal B12 supplementation on infant cognitive function. The sample size has been inflated by 35% to account for attrition and assumes 90% statistical power and a 5% two-sided significance level.

Statistical analysis
The primary and secondary outcomes are analysed using an intention-to-treat analysis by a blinded statistician. Comparability of participants assigned to study groups at enrolment is assessed using tests of association for categorical variables and by fitting generalised linear models for continuous  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y   14 variables. These models include interaction terms to explore conditional differences between the trial's arms. If necessary, transformations are applied to the response variables to fulfil the model's assumptions. Covariates include trial site, infant feeding, gender, feeding by gender, weight z-score at birth, weight gain between birth and 3 months, and maternal body mass index (BMI). Z-scores for height and weight are calculated according to the World Health Organisation [31] growth standards using the R language for statistical computing [32].
We will examine the impact of B-12 supplementation in two subgroups -those that have any of the following conditions vs. all others (high risk vs. low risk):  Maternal morbidity during pregnancy will be classified as high-risk (including gestational diabetes, pregnancy-induced hypertension/pre-eclampsia/eclampsia and absent or reversed end diastolic flow); and low-risk;  Birth weight z-score categorized as high-risk (small for gestational age [SGA]: <10th centile for birth weight); and low-risk (appropriate for weight [>10th centile for birthweight]);  Gestation categorized as preterm (low-risk; 33-37 weeks gestation); very preterm (highrisk; 28-32 weeks gestation); and term (low-risk: >37 weeks gestation). Extremely preterm (<28 weeks) will be exited;  Higher risk neonatal course categorized as high risk (if neonate has: APGAR score <7 at 1 minute, hypoglycaemia, hyperbilirubinemia or prolonged neonatal intensive care stay >7 days); and low risk.
 Infants with documented neurological disease/anomaly/illness (Developmental Quotient (by DASII scale of the infant at 9 months) will be sub-grouped separately (high risk) and compared to those with no disease/anomaly illness (low risk).

Data management and dissemination
Data will be collected under the regulation of the data protection and management guidelines given by Governments of India and Nepal, and as per the UK Data Protection Act 2018. Data will be securely stored in an E-database with password protection. The E-database was designed using Access. Live data entry will be conducted at the study sites and tested for accuracy using duplicate data entry at an offsite location at periodic intervals.
The study results will be disseminated globally with academic and health professionals via conferences, presentations and publications. Researchers at each study site will share emerging results with participants during their follow-up visits. Regular site meetings with press offices, short  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y   15 films, radio spots, periodic blog posts, Twitter and Facebook messages, and online articles are also planned to strengthen the trial's online presence and reach young and middle-aged internet users for whom the information may be of interest.

Qualitative evaluation proposed:
The qualitative sub-study aims to explore knowledge, attitudes and perceptions of pregnant women towards B-12 supplementation and child development, and potential barriers to implementation.
Better understanding these domains across the two study sites may inform the perceived need for the intervention, help define patient-centric outcomes and allow the evaluation of any differences between the two arms.
We will use convenience sampling and estimate interviewing 30 participants across both trial sites.
We will develop and pilot a semi-structured topic guide in collaboration with our PPI representatives serving on the Trial Steering Committee (TSC). The topic guide will be designed in English and translated into either Hindi or Nepali for administration across the study sites. The topic guide will then be back translated to English to ensure consistency and accuracy of content. It is intended to help direct the interview whilst remaining sensitive to unsolicited themes. An interviewer with prior training and experience in qualitative methods will facilitate the semi-structured interviews.
Data will be collected in quiet rooms in either hospital, away from other patients and staff. Interviews will be facilitated in English, Hindi or Nepalese. All interviews will be audio recorded, transcribed, translated into English (where necessary) and then crosschecked against the original recording. The translated transcripts will then be coded using the Atlas Ti 7.2 software for qualitative analysis. [33] A research team member will code and categorise data, and prepare an initial narrative. A final report will then be prepared with input from the interviewers and wider research team.

Ethical considerations:
The The trial is being conducted in accordance to the principles and rules of the Declaration of Helsinki.
Informed and written consent is obtained from all participants by the research teams at each study  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  anonymity. The participants are identified only by a participant identification number on all trial documents and electronic databases. Every effort is made to arrange interviews and examination at a time convenient to the participants without causing much disruption in their daily routine. Study data is stored securely and is only accessible to trial staff and authorised personnel. The Principal Investigator at each study site will submit an annual progress report, end of trial notification and a final report to the Institutional Ethics Committee, host organisations and the funders.
We used the SPIRIT checklist for writing our protocol [34].

DISCUSSION
This study will have considerable impact on current and future policy initiatives focusing on micronutrient supplementation in India and Nepal by underpinning micronutrient supplementation policy in India as envisaged in the recently released "National Health Policy", and the micronutrient initiative in Nepal. The project will provide key insights to strengthen existing national nutrition programmes like "National Iron Plus Initiative", "Janini Suraksha Yojna" and "Janini Shishu Suraksha Kariyakram" initiated by the Government of India and similar initiatives by the Government of Nepal.
Because of the high worldwide prevalence of maternal B-12 deficiency (74%), the study findings will be highly relevant for maternal and neonatal health in nutritionally vulnerable and at-risk populations globally.
As one of the few large-scale trials of B-12 supplementation in pregnancy, the results of the study would be generalisable to vegetarian expecting mothers; in whom deficiency of Vitamin B12 is most prevalent in South Asia. a key aim of the project is to reset significant elements of the research agenda in maternal and child health. We aim to contribute to the evidence base on improving neurodevelopmental outcomes in vulnerable infants. While a positive outcome for the trial would help develop an intervention program to reduce developmental delay using Vitamin B-12 as a supplementation in pregnant women, a negative or null result would also add to existing knowledge and lay the groundwork for future work on the subject especially around utility, dose, duration and time of initiation of Vitamin B12 supplementation in pregnant women/infants.    14. Azad C, Jat KR, Kaur J, Guglani V, Palta A, Tiwari A, Bansal D. Vitamin B12 status and neurodevelopmental delay in Indian infants: a hospital-based cross-sectional study.

Instructions to authors
Complete this checklist by entering the page numbers from your manuscript where readers will find each of the items listed below.
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In your methods section, say that you used the SPIRITreporting guidelines, and cite them as:  Allocation: implementation #16c Who will generate the allocation sequence, who will enrol participants, and who will assign participants to interventions 8  trimesters. Maternal biochemical B-12 status will also be assessed in the first trimester. Modification of association by apriori identified factors will also be explored.

Ethical Considerations and Dissemination:
The Study Protocol has been approved by ethical committees at each site and at University College London. The study results will be disseminated to healthcare professional and academics globally via conferences, presentations and publications.
Researchers at each study site will share results with participants during their follow-up visits.

ARTICLE SUMMARY Study strengths
 Large-scale, multi-centre randomised controlled, double-blind trial across a low-middle income country and low-income country to determine efficacy of maternal vitamin B-12 supplementation during pregnancy on neurodevelopmental outcomes six-month postpartum.
 Study design benefits from preceding qualitative patient and public involvement (PPI), and nested qualitative sub-study that will ensure participant feedback and process evaluation to optimise trial conduct.
 The study is being conducted across two diverse socio-demographic populations, to increase generalisability of study findings.

Study limitations
 Normal/Routine care differs at the two sites, which could lead to differences in the conduct and monitoring of the trial.
 Health system regulatory structures differ in the two countries. can potentially lead to poorer educational achievement and economic prospects [1,2].
Partaking in a vegetarian diet, or one low in animal food sources, is a key risk factor in developing B-12 deficiency [7,8]. Sufficient B-12 intake during pregnancy is vital for foetal brain development [9].
There is limited evidence from randomised controlled trials (RCTs) regarding effect of vitamin B-12 supplementation and infant cognitive outcomes [20,21,22]. An infant supplementation trial observed neurodevelopmental benefit of infant B-12 supplementation with folate and documented 0.45 (95% CI 0.19, 0.73) and 0.28 (95% CI 0.02, 0.54) higher SD-units in the domains of gross motor and problemsolving functioning, respectively [22]. However, in this study there was no significant benefit of giving vitamin B-12 or folic acid alone on early child development (with the exception of vitamin B-12 in relation to gross motor functioning [22]). In another trial, maternal vitamin B-12 supplementation given as 50mcg a day from <14 weeks' gestation through to 6 weeks postpartum did not improve infant neurodevelopmental outcomes at 9 months [21]. In this trial hyperhomocysteinemia (a marker of B-12 insufficiency) during pregnancy was negatively associated with infant cognitive outcomes. The study documented a deterioration in biochemical B-12 status in the placebo group while the rise in B-12 status in the supplemented group (50 mcg) was inconsistent (no significant difference between first and third trimester levels). Additionally, a high rate of participant attrition (51.4%), poor monitoring of supplement compliance, a lack of biochemical improvement in B-12 status in the supplemented group mothers and lack of infant biochemical evaluation for B-12 status in later infancy further limit the inferences that can be drawn from this trial. Other studies that assessed a 250mcg dose of B-12 per day throughout pregnancy until 3 months' post-partum in Bangladesh [23], and >2000mcg from 17 to 34 weeks' gestation in India [24] did not evaluate neurodevelopment outcomes in infancy. Hence, there is insufficient evidence concerning timing, dose, duration and efficacy of B-12 supplementation with reference to infant neurodevelopment. Although, several published protocols report ongoing work on B-12 supplementation from India and Nepal [25,26], the proposed work differs significantly in several aspects.
First, we specifically chose to supplement vegetarian women, as they are a high-risk population in view of their low B-12 intake and high prevalence of B-12 deficiency. Second, an interventional dose of 250 mcg was chosen because it has been shown in an earlier trial to improve B-12 levels in pregnancy [23] although infant neurodevelopment was not assessed in this trial. Third, we chose 50 mcg B-12 /day as a quasi-control group as this dose has been shown to prevent deterioration in B-12 status during pregnancy in India [21,22]. Furthermore, given the progressive decline in B-12 levels through pregnancy (documented in the placebo group of earlier trials [21,22]), and the potentially serious neonatal consequences of severe B-12 deficiency highlighted above, a pure placebo control was thought to be unethical.

Primary objective
To determine the effect of 250mcg versus 50mcg daily oral vitamin B-12 supplementation of pregnant women from the first trimester to 6 months post-partum on infant neurodevelopment at age 9 months (specifically the impact on the Motor and Mental developmental quotient (DQ) by Development Assessment Scale for Indian Infants (DASII scale).

Secondary objectives
To determine the effect of 250mcg versus 50mcg of oral vitamin B-12 supplementation in pregnancy and 6 months post-partum on biochemical parameters of B-12 status (Vitamin B-12 levels, Homocysteine and Holo-transcobalamin) in mothers at the end of the third trimester and in infants at age 9 months.

1.
To evaluate the influence of the following effect modifiers: a. Socio-economic indicators (income, education, profession) on the relationship between maternal B12 status, supplementation and infant neurodevelopment; e. Maternal and infant B-12 status and neurodevelopment at 9 months after birth.

2.
To determine the interaction of maternal iron and vitamin D status with the relationship between infant vitamin B-12 status and infant neurodevelopment at 9 months.

METHODOLOGY Trial Registration:
The study is registered as a primary clinical trial at the WHO International Clinical Trials Registry   supplement, relocation and financial difficulties. All of these preceding factors were considered in the study design, development of patient information, consultation and follow-up procedures as follows:

Feasibility Data and Preliminary Work
 All participants at the two study sites will be informed of the role, sources and importance of vitamin B-12 in human health using educational banners and posters and through community workshops.
 The initial information about the trial will be given by the doctor providing antenatal care to the mother. Subsequent detailed consent and recruitment will be conducted by study research staff.
 In order to improve compliance and study retention, reminder text messages will be sent weekly to participating mothers.
 To avoid the risk of financial loss to participants, various biochemical tests at the Indian trial site will be sponsored, and transportation costs at the Nepal site will be covered.
 Bradleys' Home Observation for Measurement of the Environment (HOME) inventory was added to the assessment given the divergent environmental profile of the two study sites [28], in order to assess the infant's environment and possible environmental confounders of neurodevelopment.
The study results will be disseminated globally to academics and health professionals via conferences, presentations and publications. Researchers at each study site will share results with participants during their follow-up visits.
Also, in preparation for this trial we adapted a pre-validated Food Frequency Questionnaire (FFQ) for All research staff from India and Nepal will be jointly trained in study design, research methodology, good clinical practice, the study protocol and study tools. Training lectures will be video-recorded and made available to the teams at both study sites as a reference material for continuous training.

Eligibility
Potentially eligible participants will be screened at first presentation to the antenatal clinic at <12 weeks of gestation in accordance with the following criteria: Inclusion Criteria:  Consuming a vegetarian diet -including veganism and/or people who do eat egg and/or do consume milk and/or a portion meat/chicken/fish < once a month;  Living within an a-priori defined geographical area; o Delhi -national capital region; o Nepal -10-km radius of PMWH;  Able to understand at least one of English, Hindi or Nepalese.  Anticipating moving out of the a-priori defined geographical area before or after delivery;  Undergoing treatment for infertility;  Having a known pre-diagnosed mental health disorder, including depression, drug or alcohol abuse;  Currently participating or having participated in another study within four weeks prior to the trial commencing;  Having a known allergy to vitamin B-12 or another supplement constituent.

Consent
Eligible participants meeting the selection criteria will be given time to think over continuation of pregnancy, site of delivery, participation in the trial and any other considerations. They will be contacted within 48 hours by the research team regarding their decision to participate or not and will 10 be invited to provide written or video-recorded consent if relevant. Participants will be able to opt into the trial any time before 12 weeks of gestation.

Randomisation
As presented in Figure 1, all eligible and consenting participants will be randomised with an allocation ratio of 1:1 to either the intervention (250mcg), or control (50mcg) group. The randomisation sequence will be generated at a single, third-party location (non-study site) using a computergenerated sequence stratified for location. The sequence will be allocated to serial numbers by a neutral person in each country using sequentially numbered, sealed, opaque envelopes. Codes within envelopes will identify boxes labelled with 5-digit codes. Each box will contain enough dosages to cater to the study period of one participant. The strips containing intervention and control doses of B-12 will be identical in taste, smell and appearance. All participants, recruiters, developmental therapists, laboratory staff and the data analyst will be blinded to the B-12 allocation until results of final analysis are made available.

Procedure
Upon enrolment, demographic and clinical details, including age, height, weight, ethnicity, education and socio-economic status, will be collected. The participants' preferred channel of subsequent communication (phone, text message, email) will be noted. Participants will be allocated a study identification number and accompanied by the researcher for blood sampling. The timing of blood sampling will be synchronised with that of any other samples being drawn in the first trimester to minimise unnecessary venepunctures. Twelve ml of blood sample will be drawn into two plain vials (five ml each) and two ml will be drawn into an EDTA vial. The EDTA sample will be processed immediately for complete blood counts by coulter counter. The plain (serum separator tubes) vial samples will be centrifuged at 3500 rpm (Rotor radius 15 cm) for 10 minutes at the study site. The separated serum will be transported on ice and consistently stored at -70° C and will be monitored at both the study sites. Participants will be given enough B-12 supplement doses to last five days beyond the expected date of their next visit, after being fully informed about the supplement and its administration. Participants will be contacted 48 hours after receiving the B-12 doses to confirm initialisation of supplementation and re-emphasise compliance.
Subsequent maternal visits will take place monthly until 36 weeks, and then weekly thereafter. At these visits, all participants will visit their obstetrician as part of routine care. Participants will also meet with the research staff at a pre-specified location at the study site. To maximise compliance, weekly supplement and pre-appointment reminders will be sent via the pre-specified preferred mode of communication. Acquired morbidity (gestational diabetes, hypertension, and hypothyroidism) and medication history will be noted in case record forms (CRFs). Empty supplement packets will be collected at each appointment as an indicator of adherence. In the third trimester, mothers' dietary B-12 intake will be assessed using a pre-standardised and adapted FFQ, and blood sampling will be repeated using the same procedures as in the first trimester.
Neonatal assessment: During childbirth, a medical officer and/or paediatrician will monitor the birth and post-delivery course of neonates for any morbidity that might potentially influence neurodevelopment. Gestational age, growth retardation, congenital abnormalities and APGAR (appearance, pulse, grimace, activity, respiration) score will be routinely documented for neonates.
Cord pH will be measured for all new-borns with a delayed cry and who require any intervention beyond the basic steps of resuscitation (ambu bagging, intubation, or pharmacological treatment).
Any seizures, neurological problems, hypoglycaemia, hypothermia, or hyperbilirubinemia will be assessed, diagnosed, evaluated, and managed as per institutional guidelines. As per standard practice, all new-borns will be screened for hearing deficits, vision, and critical congenital heart disease according to institutional protocols. Preterm neonates transferred to the neonatal unit will be monitored as per their customized follow-up and early intervention plans, implemented as recommended by the paediatrics and/or primary care provider. Neurodevelopment and biochemical assessment of preterm neonates will be carried out as per their corrected gestational ages.
Infant follow-up will be conducted at 7-14 days, 4, 8, 12 and 16 weeks and at 6 and 9 months of age.
At these visits, the infants will undergo routine care as well as study specific evaluations (see Visit Schedule: Supplementary Table 1). Routine care: Each mother-infant dyad will see a paediatrician for age-appropriate routine care, which will involve clinical evaluation, preventative care and vaccination as per standard procedures in each site. Anthropometric measurements and signs of micronutrient deficiency (anaemia and rickets) will congenital adrenal hyperplasia, cystic fibrosis, biotinidase deficiency, phenylketonuria and galactosemia) will be screened for and maternal support for breastfeeding by a paediatrician or lactation counsellor will be provided at 7-14 days.
Study specific: Supplements will be provided to mothers at each visit, with instructions to take these until 6 months after childbirth. Any morbidities and deficiencies will be recorded and treated as per institutional guidelines. Maternal and infant tolerance for the supplementation including any gastrointestinal symptoms will be recorded at each visit, as will maternal compliance. Provision of supplements to the mother in both groups will be stopped at 6 months post-partum.
The hospital evaluation at 9 months (±14 days) will include neurodevelopmental assessment by a developmental therapist using the Developmental Assessment Scales for Indian Infants (DASII) -an adapted and validated version of the Bayley's Scale for Infant Development Third Edition (BSID II) [30] and blood sampling for estimation of Hb, vitamin B-12, homocysteine and Holo-transcobalamin concentrations. The DASII motor scale assesses gross and fine motor skills, whilst cognitive, personal and social skills development are assessed through the DASII mental scale. All neurodevelopmental assessments will be conducted by a trained developmental therapist at the institute in a soundproof room. In premature babies, corrected gestational age will be used to calculate developmental quotients. Additional assessments will include a complementary feeding assessment by a nutritionist using a 72-hour dietary recall and home environment assessment using the adapted Bradley HOME inventory [28] by a trained field worker.
Compliance: Research staff will dispense blister packaging of ten B-12 capsules and study specific bags for participants to store, carry and keep supplements in one place. In case of loss of supplement or inability to come for follow-up, participants will be able to contact the researcher at any time to request more supplements. The supplement packaging will be numbered with a serial number. To maximise compliance, weekly reminders will be sent through the preferred mode of communication as specified by the trial participants. Each participant will be provided with a bag to save empty packaging strips and instructed to carry the bag with her at the next visit to monitor compliance.

Biochemical analysis
All saved samples will be processed as one batch after the completion of the study. The maternal samples will be used to determine the blood levels for Vitamin B-12, Homocysteine, Holo-

Outcomes:
The following study outcomes will be investigated:

Sample size considerations
A planned sample of 720 pregnant women, 360 per study site, will be randomly assigned to experimental (n=360; n=180 in per study site) or control (n=360; n=180 in per study site) groups. This is based on the number of infants needed to complete the neurodevelopmental screen in each of the two randomised groups to detect a difference in Development Quotient (DQ) at nine months (primary outcome) of 2 points (presumed mean DQ in this population of infants at 9 months of 96.9, SD 7.07 on basis of earlier work)( [27]. The difference of 2 DQ points was chosen because it is the smallest effect size thought to be clinically significant and hence a null trial result is likely to exclude a clinically relevant effect of maternal B12 supplementation on infant cognitive function. The sample size has been inflated by 35% to account for attrition, and assumes 90% statistical power and a 5% two-sided significance level.

Statistical analysis
The primary and secondary outcomes will be analysed using an intention-to-treat analysis by a blinded statistician. Comparability of participants assigned to study groups at enrolment will be assessed using tests of association for categorical variables and by fitting generalised linear models for continuous variables. These models will include interaction terms to explore conditional differences between the trial's arms. If necessary, transformations will be applied to the response variables to fulfil the model's assumptions. Covariates will include trial site, infant feeding, gender, feeding by gender, weight zscore at birth, weight gain between birth and 3 months, and maternal body mass index (BMI). Z-scores for height and weight will be calculated according to the World Health Organisation [31] growth standards using the R language for statistical computing [32].
Subgroup analysis will be conducted to examine the differential impact of B-12 supplementation in high versus low risk mothers and infants. Mothers identified as having pregnancy related or other morbidity will be classified as high-risk (e.g. including gestational diabetes, pregnancy-induced hypertension/pre-eclampsia/eclampsia and absent or reversed end diastolic flow); and low-risk.
Babies will be defined as high risk if they are any of a combination of the following:  Small for gestational age [SGA], defined as <10th centile for birth weight;  Very preterm (28-32 weeks gestation); Extremely preterm babies (<28 weeks) will be excluded from the analysis;  Experience of a higher risk neonatal course defined as experience of APGAR score <7 at 1 minute, hypoglycaemia, hyperbilirubinemia or prolonged neonatal intensive care stay >7 days);  Infants with documented neurological disease/anomaly/illness will be sub-grouped separately (high risk) and compared to those with no disease/anomaly illness (low risk).

Data management and dissemination
Data will be collected under the regulation of the data protection and management guidelines given by Governments of India and Nepal, and as per the UK Data Protection Act 2018. Data will be securely 15 stored in an E-database with password protection. The E-database will be designed using Access. Live data entry will be conducted at the study sites and tested for accuracy using duplicate data entry at an offsite location at periodic intervals.
The study results will be disseminated globally with academic and health professionals via conferences, presentations and publications. Researchers at each study site will share emerging results with participants during their follow-up visits. Regular site meetings with press offices, short films, radio spots, periodic blog posts, Twitter and Facebook messages, and online articles are also planned to strengthen the trial's online presence and reach young and middle-aged internet users for whom the information may be of interest.

Qualitative evaluation proposed
A qualitative sub-study aims to explore knowledge, attitudes and perceptions of pregnant women towards B-12 supplementation and child development, and potential barriers to implementation.
Better understanding these domains across the two study sites may inform the perceived need for the intervention, help define patient-centric outcomes and allow the evaluation of any differences between the two arms.
We will use convenience sampling, and aim to interview 30 participants across both trial sites. We will develop and pilot a semi-structured topic guide in collaboration with our PPI representatives serving on the Trial Steering Committee (TSC). The topic guide will be designed in English and translated into either Hindi or Nepali. The topic guide will then be back-translated to English to ensure consistency and accuracy of content. It is intended to help direct the interview whilst remaining sensitive to unsolicited themes. An interviewer with prior training and experience in qualitative methods will facilitate the semi-structured interviews.
Data will be collected in quiet rooms in each hospital, away from other patients and staff. Interviews will be facilitated in English, Hindi or Nepalese, depending on the preference of the participant. All interviews will be audio recorded, transcribed, translated into English (where necessary) and then crosschecked against the original recording. The translated transcripts will then be coded using the Atlas Ti 7.2 software for qualitative analysis [33]. A research team member will code and categorise data, and prepare an initial narrative. A final report will then be prepared with input from the interviewers and wider research team.

Ethical considerations:
The  submitted and, where necessary, obtained approval from the above parties for all substantial amendments to the original approved documents.
The trial will be conducted in accordance to the principles and rules of the Declaration of Helsinki.
Informed and written consent will be obtained from all participants by the research teams at each study site. The trial will comply with the Data Protection Act (UK), 2018, ensuring the participants' anonymity. The participants will be identified only by a participant identification number on all trial documents and electronic databases. Every effort will be made to arrange interviews and examination at a time convenient to the participants, without causing much disruption in their daily routine. Study data will be stored securely and will only be accessible to trial staff and authorised personnel. The Principal Investigator at each study site will submit an annual progress report, end of trial notification and a final report to the Institutional Ethics Committee, host organisations and the funders.
We used the SPIRIT checklist for writing our protocol [34].

DISCUSSION
A positive result from this study will have considerable impact on current and future policy initiatives focusing on micronutrient supplementation in India and Nepal, by underpinning micronutrient supplementation policy in India as envisaged in the recently released "National Health Policy", and the micronutrient initiative in Nepal. The project will provide key insights to strengthen existing national nutrition programmes like "National Iron Plus Initiative", "Janini Suraksha Yojna" and "Janini Shishu Suraksha Kariyakram" initiated by the Government of India and similar initiatives by the Government of Nepal. Because of the high worldwide prevalence of maternal B-12 deficiency (74%), the study findings will also be highly relevant for maternal and neonatal health in nutritionally vulnerable and at-risk populations globally.
Using a 50 mcg quasi-control group limits the inferences that can be drawn from a negative result (neurodevelopment comparable between the two groups). However, the following interpretations might be possible: 1. Findings: Improvement in B-12 parameters from first trimester to third trimester is statistically significant in both groups and the change is statistically comparable between two groups for mother and baby. Substantial proportion of mother/infants in both groups improve from B-12 deficient to replete state. 2. Findings: Improvement in B-12 parameters from first trimester to third trimester is significant in either 250 mcg alone or both groups, but 250 mcg causes higher rise (statistically significant) in B-12 biochemical parameters than 50 mcg for mother and baby.
Interpretation: 250 mcg is more efficacious than 50 mcg in improving biochemical B-12 status.
Since neurodevelopment is equivalent between two groups a higher dose of supplementation does not offer any neurodevelopmental advantage despite better biochemical B-12 status. Our study specifically seeks to address the question of optimum dose for vegetarian pregnant women.
As one of the few large-scale trials of B-12 supplementation in pregnancy, the results of this study would be generalisable to vegetarian expecting mothers. We aim to contribute to the evidence base on improving neurodevelopmental outcomes in vulnerable infants. While a positive outcome for the trial would help to develop an intervention program to reduce developmental delay using Vitamin B-12 as a supplementation in pregnant women, a negative or null result would also add to existing knowledge and lay the groundwork for future work on the subject especially around utility, dose,
Based on the SPIRIT guidelines.

Instructions to authors
Complete this checklist by entering the page numbers from your manuscript where readers will find each of the items listed below.
Your article may not currently address all the items on the checklist. Please modify your text to include the missing information. If you are certain that an item does not apply, please write "n/a" and provide a short explanation.
Upload your completed checklist as an extra file when you submit to a journal.

Methods and Analysis:
This double blind, multi-centre randomised controlled trial will enrol 720 vegetarian pregnant women in their first trimester from ante-natal clinics at two hospitals (one in India and one in Nepal). Eligible mothers who give written consent will be randomised to receive either 250 mcg methylcobalamin or 50 mcg (quasi control), from enrolment to 6 months post-partum, given as an oral daily capsule. All mothers and their infants will continue to receive standard clinical care.
The primary trial outcome is the offspring's neurodevelopment status at 9 months of age, assessed using the Development Assessment Scale of Indian Infants. Secondary outcomes include the infant's biochemical B-12 status at age 9 months and maternal biochemical B-12 status in the first and third trimesters. Maternal biochemical B-12 status will also be assessed in the first trimester. Modification of association by apriori identified factors will also be explored.

Ethical Considerations and Dissemination:
The Study Protocol has been approved by ethical committees at each study site (India and Nepal) and at UCL, UK. The study results will be disseminated to healthcare professionals and academics globally via conferences, presentations and publications.
Researchers at each study site will share results with participants during their follow-up visits.

ARTICLE SUMMARY Study strengths
 Large-scale, multi-centre, randomised controlled, double-blind trial across a low-middle income country (LMIC) and low-income country (LIC) to determine the efficacy of maternal vitamin B-12 supplementation during pregnancy on neurodevelopmental outcomes sixmonth post-partum.
 Study design benefits from the preceding qualitative patient and public involvement (PPI) and nested qualitative sub-study, ensuring participant feedback and process evaluation to optimise trial conduct.
 The study is being conducted across two diverse socio-demographic populations to increase generalisability of study findings.

Study limitations
 Normal/Routine care differs at the two sites, which could lead to differences in the conduct and monitoring of the trial.
 Health system regulatory structures differ in the two countries. can potentially lead to poorer educational achievement and economic prospects [1,2].
Partaking in a vegetarian diet, or one low in animal food sources, is a key risk factor in developing B-12 deficiency [7,8]. Sufficient B-12 intake during pregnancy is vital for foetal brain development [9].
There is limited evidence from randomised controlled trials (RCTs) regarding the effect of vitamin B-12 supplementation and infant cognitive outcomes [20,21,22]. An infant supplementation trial observed neurodevelopmental benefits of infant B-12 supplementation with folate, and documented 0.45 (95% CI 0.19, 0.73) and 0.28 (95% CI 0.02, 0.54) higher SD-units in the domains of gross motor and problem-solving functioning, respectively [22]. However, in this study there was no significant benefit of giving vitamin B-12 or folic acid alone on early child development (with the exception of vitamin B-12 in relation to gross motor functioning [22]). In another trial, maternal vitamin B-12 supplementation given as 50mcg a day from <14 weeks' gestation through to 6 weeks postpartum did not improve infant neurodevelopmental outcomes at 9 months [21]. In this trial hyperhomocysteinemia (a marker of B-12 insufficiency) during pregnancy was negatively associated with infant cognitive outcomes. The study documented a deterioration in biochemical B-12 status in the placebo group, while the rise in B-12 status in the supplemented group (50 mcg) was inconsistent (no significant difference between first and third trimester levels Although, several published protocols report ongoing work on B-12 supplementation in India and Nepal [25,26], the proposed work differs significantly in several ways. First, we specifically chose to supplement vegetarian women, as they are a high-risk population due to their low B-12 intake, and as such have a high prevalence of B-12 deficiency. Second, an interventional dose of 250 mcg was chosen because it has been shown in an earlier trial to improve B-12 levels in pregnancy [23] (although infant neurodevelopment was not assessed in this trial). Third, we chose 50 mcg B-12 /day as a quasi-control group as this dose has been shown to prevent deterioration in B-12 status during pregnancy in India [21]. Furthermore, given the progressive decline in B-12 levels throughout pregnancy (documented in the placebo group of earlier trials [21,22]), and the potentially serious neonatal consequences of severe B-12 deficiency highlighted above, a pure placebo control was thought to be unethical.

Primary objective
To determine the effect of 250mcg versus 50mcg daily oral vitamin B-12 supplementation of pregnant women from the first trimester to 6 months post-partum on infant neurodevelopment at age 9 months.

Secondary objectives
To determine the effect of 250mcg versus 50mcg of oral vitamin B-12 supplementation in pregnancy and 6 months post-partum on biochemical parameters of B-12 status (Vitamin B-12 levels, Homocysteine and Holo-transcobalamin) in mothers at the end of the third trimester and in infants at age 9 months.
To evaluate the influence of the following effect modifiers: a. Socio-economic indicators (income, education, profession) on the relationship between maternal B12 status, supplementation and infant neurodevelopment; e. Maternal and infant B-12 status and neurodevelopment at 9 months after birth.

2.
To determine the interaction of maternal iron and vitamin D status with the relationship between infant vitamin B-12 status and infant neurodevelopment at 9 months.

METHODOLOGY Trial Registration:
The study is registered as a primary clinical trial at the WHO International Clinical Trials Registry  Respondents were also willing to accept the intervention and the need for drawing extra blood from the baby as a part of routine sampling for B-12 levels. Perceived barriers to the study included concerns around potential side effects of the B-12 supplement, relocation mid-trial, and financial difficulties. All of these factors were considered in the study design, development of patient information, consultation, and follow-up procedures as follows:

Feasibility Data and Preliminary Work
 All participants at the two study sites will be informed of the role, sources and importance of vitamin B-12 in human health using educational banners and posters and through community workshops;  The initial information about the trial will be given by the doctor providing antenatal care to the mother. Subsequent detailed consent and recruitment will be conducted by study research staff;  In order to improve compliance and study retention, reminder text messages will be sent weekly to participating mothers;  To avoid the risk of financial loss to participants, various biochemical tests at the Indian trial site will be sponsored, and transportation costs at the Nepal site will be covered;  Bradleys' Home Observation for Measurement of the Environment (HOME) inventory was added to the assessment given the divergent environmental profile of the two study sites [28], in order to assess the infant's environment and possible environmental confounders on neurodevelopment; The study results will be disseminated globally to academics and health professionals via conferences, presentations and publications. Researchers at each study site will share results with participants during their follow-up visits.
Also, in preparation for this trial we adapted a pre-validated Food Frequency Questionnaire (FFQ) for Vitamin B-12 intake [29] to the Indian and Nepalese settings. In brief, the original Vitamin B-12 FFQ by All research staff from India and Nepal will be jointly trained in study design, research methodology, good clinical practice, the study protocol, and study tools. Training lectures will be video-recorded and made available to the teams at both study sites as a reference material for continuous training.

Eligibility
Potentially eligible participants will be screened at first presentation to the antenatal clinic at <12 weeks of gestation in accordance with the following criteria: Inclusion Criteria:  Consuming a vegetarian diet -including veganism and/or people who do eat egg and/or do consume milk and/or a portion meat/chicken/fish < once a month;  Living within an a-priori defined geographical area; o Delhi -national capital region; o Nepal -10-km radius of PMWH;  Able to understand at least one of the following languages-English, Hindi or Nepalese.  Anticipating moving out of the a-priori defined geographical area before or after delivery;  Undergoing treatment for infertility;  Having a known pre-diagnosed mental health disorder, including depression, drug or alcohol abuse;  Currently participating or having participated in another study within four weeks prior to the trial commencing;  Having a known allergy to vitamin B-12 or another supplement constituent.

Consent
Eligible participants meeting the selection criteria will be given time to think over continuation of pregnancy, site of delivery, participation in the trial and any other considerations. They will be contacted within 48 hours by the research team regarding their decision to participate, and will be 10 invited to provide written or video-recorded consent. Participants will be able to opt into the trial any time before 12 weeks of gestation.

Randomisation
As presented in Figure 1, all eligible and consenting participants will be randomised with an allocation ratio of 1:1 to either the intervention (250mcg), or control (50mcg) group. The randomisation sequence will be generated at a single, third-party location (non-study site) using a computergenerated sequence stratified for location. The sequence will be allocated to serial numbers by a neutral person in each country using sequentially numbered, sealed, opaque envelopes. Codes within envelopes will identify boxes labelled with 5-digit codes. Each box will contain enough dosages to cater to the study period of one participant. The strips containing intervention and control doses of B-12 will be identical in taste, smell and appearance. All participants, recruiters, developmental therapists, laboratory staff, and the data analyst will be blinded to the B-12 allocation until results of final analysis are made available.

Procedure
Upon enrolment, demographic and clinical details, including age, height, weight, ethnicity, education and socio-economic status, will be collected. The participants' preferred channel of subsequent communication (phone, text message, email) will be noted. Participants will be allocated a study identification number and accompanied by the researcher for blood sampling. These samples will be taken at the same time as other samples being drawn in the first trimester to minimise unnecessary venepunctures. Twelveml of blood sample will be drawn into two plain vials (5ml each) and 2ml will be drawn into an EDTA vial. The EDTA sample will be processed immediately for complete blood counts by coulter counter. The plain (serum separator tubes) vial samples will be centrifuged at 3500 rpm (Rotor radius 15 cm) for 10 minutes at the study site. The separated serum will be transported on ice and consistently stored at -70° C, and will be monitored at both the study sites. Participants will be given enough B-12 supplement doses to last five days beyond the expected date of their next visit, after being fully informed about the supplement and its administration. Participants will be contacted  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y   11 48 hours after receiving the B-12 doses to confirm initialisation of supplementation and re-emphasise compliance.
Subsequent maternal visits will take place monthly until 36 weeks, and then weekly thereafter. At these visits, all participants will visit their obstetrician as part of routine care. Participants will also meet with the research staff at a pre-specified location at the study site. To maximise compliance, weekly adherence and pre-appointment reminders will be sent via the pre-specified preferred mode of communication. Acquired morbidity (gestational diabetes, hypertension, and hypothyroidism) and medication history will be noted in case record forms (CRFs). Empty supplement packets will be collected at each appointment as an indicator of adherence. In the third trimester, mothers' dietary B-12 intake will be assessed using a pre-standardised and adapted FFQ, and blood sampling will be repeated using the same procedures as in the first trimester.
Neonatal assessment: During childbirth, a medical officer and/or paediatrician will monitor the birth and post-delivery health of neonates for any morbidity that might potentially influence neurodevelopment. Gestational age, growth retardation, congenital abnormalities and APGAR (appearance, pulse, grimace, activity, respiration) score will be routinely documented for neonates.
Cord pH will be measured for all new-borns with a delayed cry and who require any intervention beyond the basic steps of resuscitation (ambu bagging, intubation, or pharmacological treatment).
Any seizures, neurological problems, hypoglycaemia, hypothermia, or hyperbilirubinemia will be assessed, diagnosed, evaluated, and managed as per institutional guidelines. As per standard practice, all new-borns will be screened for hearing deficits, vision, and critical congenital heart disease according to institutional protocols. Preterm neonates transferred to the neonatal unit will be monitored as per their customized follow-up and early intervention plans, implemented as recommended by the paediatrics and/or primary care provider. Neurodevelopment and biochemical assessment of preterm neonates will be carried out as per their corrected gestational ages.
Infant follow-up will be conducted at 7-14 days, 4, 8, 12 and 16 weeks and at 6 and 9 months of age.
At these visits, the infants will undergo routine care as well as study specific evaluations (see Visit Schedule: Supplementary Table 1). Routine care: Each mother-infant dyad will see a paediatrician for age-appropriate routine care, which will involve clinical evaluation, preventative care and vaccination as per standard procedures in each site. Anthropometric measurements and signs of micronutrient deficiency (anaemia and rickets) will  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y   12 be recorded during each visit as part of routine care. Metabolic disorders (congenital hypothyroidism, congenital adrenal hyperplasia, cystic fibrosis, biotinidase deficiency, phenylketonuria and galactosemia) will be screened for and maternal support for breastfeeding by a paediatrician or lactation counsellor will be provided at 7-14 days.
Study specific: Supplements will be provided to mothers at each visit, with instructions to take until 6 months after childbirth. Any morbidities and deficiencies will be recorded and treated as per institutional guidelines. Maternal and infant tolerance for the supplementation, including any gastrointestinal symptoms, will be recorded at each visit, as will maternal compliance. Provision of supplements to the mother in both groups will be stopped at 6 months post-partum.
The hospital evaluation at 9 months (±14 days) will include neurodevelopmental assessment by a developmental therapist using the Developmental Assessment Scales for Indian Infants (DASII) -an adapted and validated version of the Bayley's Scale for Infant Development Third Edition (BSID II) [30] -and blood sampling for estimation of Haemoglobin, Vitamin B-12, Homocysteine and Holotranscobalamin concentrations. The DASII motor scale assesses gross and fine motor skills, whilst cognitive, personal and social skills development are assessed through the DASII mental scale. All neurodevelopmental assessments will be conducted by a trained developmental therapist at the institute in a soundproof room. In premature babies, corrected gestational age will be used to calculate developmental quotients. Additional assessments will include a complementary feeding assessment by a nutritionist using a 72-hour dietary recall, and a home environment assessment using the adapted Bradley HOME inventory [28] by a trained field worker.
Compliance: Research staff will dispense blister packaging of 10 B-12 capsules and study specific bags for participants to store, carry, and keep supplements in one place. In case of loss of supplement or inability to come for follow-up, participants will be able to contact the researcher at any time to request more supplements. The supplement packaging will be numbered with a serial number. To maximise compliance, weekly reminders will be sent through the preferred mode of communication as specified by the trial participants. Each participant will be provided with a bag to save empty packaging strips and instructed to carry the bag with her at the next visit to monitor compliance.

Outcomes:
The following study outcomes will be investigated:

Sample size considerations
A planned sample of 720 pregnant women, 360 per study site, will be randomly assigned to experimental (n=360; n=180 in per study site) or control (n=360; n=180 in per study site) groups. This is based on the number of infants needed to complete the neurodevelopmental screen in each of the two randomised groups to detect a difference in Development Quotient (DQ) at nine months (primary outcome) of 2 points (presumed mean DQ in this population of infants at 9 months of 96.9, SD 7.07  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y   14 on basis of earlier work)( [27]. The difference of 2 DQ points was chosen because it is the smallest effect size thought to be clinically significant, and hence a null trial result is likely to exclude a clinically relevant effect of maternal B12 supplementation on infant cognitive function. The sample size has been inflated by 35% to account for attrition, and assumes 90% statistical power and a 5% two-sided significance level.

Statistical analysis
The primary and secondary outcomes will be analysed using an intention-to-treat analysis by a blinded statistician. Comparability of participants assigned to study groups at enrolment will be assessed using tests of association for categorical variables and by fitting generalised linear models for continuous variables. These models will include interaction terms to explore conditional differences between the trial's arms. If necessary, transformations will be applied to the response variables to fulfil the model's assumptions. Covariates will include trial site, infant feeding, gender, feeding by gender, weight zscore at birth, weight gain between birth and 3 months, and maternal body mass index (BMI). Z-scores for height and weight will be calculated according to the World Health Organisation [31] growth standards using the R language for statistical computing [32].
Subgroup analysis will be conducted to examine the differential impact of B-12 supplementation in high versus low risk mothers and infants. Mothers identified as having pregnancy-related or other morbidity will be classified as high-risk (e.g. including gestational diabetes, pregnancy-induced hypertension/pre-eclampsia/eclampsia and absent or reversed end diastolic flow); and low-risk.

Data management and dissemination
Data will be collected under the regulation of the data protection and management guidelines provided by the Governments of India and Nepal, and as per the UK Data Protection Act 2018. Data will be securely stored in an E-database with password protection. The E-database will be designed using Access. Live data entry will be conducted at the study sites and tested for accuracy using duplicate data entry at an offsite location at periodic intervals.
The study results will be disseminated globally with academic and health professionals via conferences, presentations and publications. Researchers at each study site will share emerging results with participants during their follow-up visits. Regular site meetings with press offices, short films, radio spots, periodic blog posts, and Twitter and Facebook messages are also planned to strengthen the trial's online presence and reach young and middle-aged internet users for whom the information may be of interest.

Qualitative evaluation
A qualitative analysis of the information learned during preliminary PPI work aimed to explore knowledge, attitudes and perceptions of pregnant women towards B-12 supplementation and child development, and the potential barriers to implementation. Better understanding these domains across the two study sites informed the perceived need for the intervention, helped define patientcentric outcomes and allowed the evaluation of any differences between the two arms.
We used convenience sampling to recruit and interview 25 participants across both trial sites (SBISR 12; PMWH 13). We developed and piloted a semi-structured topic guide in collaboration with our PPI representatives serving on the Trial Steering Committee (TSC). The topic guide was designed in English and translated into either Hindi or Nepali. The topic guide was then back-translated into English to ensure consistency and accuracy of content. It was created with the intention to help direct the interview whilst remaining sensitive to unsolicited themes. We ensured that an interviewer with prior training and experience in qualitative methods facilitated the semi-structured interviews.
Data was collected in quiet rooms in each hospital, away from other patients and staff. Interviews were facilitated in English, Hindi or Nepalese, depending on the preference of the participant. All interviews were audio recorded, transcribed, translated into English (where necessary) and then crosschecked against the original recording. The translated transcripts were then coded using the Atlas Ti 7.2 software for qualitative analysis [33]. A research team member coded and categorised the data, and prepared an initial narrative. A report detailing the findings is in the process of being finalised, with input from the interviewers and wider research team.  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  The trial will be conducted in accordance with the principles and rules of the Declaration of Helsinki.
Informed and written consent will be obtained from all participants by the research teams at each study site. The trial will comply with the Data Protection Act (UK), 2018, ensuring the participants' anonymity. The participants will be identified only by a participant identification number on all trial documents and electronic databases. Every effort will be made to arrange interviews and examinations at a time convenient to the participants, without causing much disruption in their daily routine. Study data will be stored securely and will only be accessible to trial staff and authorised personnel. The Principal Investigator at each study site will submit an annual progress report, end of trial notification and a final report to the Institutional Ethics Committee, host organisations and the funders.
We used the SPIRIT checklist for writing our protocol [34].

DISCUSSION
A positive result from this study will have considerable impact on current and future policy initiatives, by underpinning current micronutrient supplementation policy in India and Nepal. The project will provide key insights to strengthen existing Indian governmental nutrition programmes such as "National Iron Plus Initiative", "Janini Suraksha Yojna" and "Janini Shishu Suraksha Kariyakram".
Because of the high worldwide prevalence of maternal B-12 deficiency (74%), the study findings will also be highly relevant to maternal and neonatal health in nutritionally vulnerable and at-risk populations globally.
Using a 50 mcg quasi-control group limits the inferences that can be drawn from a negative result (neurodevelopment comparable between the two groups). The study outcome could produce several results, which could be interpreted as follows: 1. Findings: Improvement in B-12 parameters from first trimester to third trimester is statistically significant in both groups, and the change is statistically comparable between two groups for  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y mother and baby. Substantial proportion of mother/infants in both groups improve from B-12 deficient to replete state.
Interpretation: 250 mcg dose is as effective as 50 mcg in improving biochemical parameters and both doses result in comparable neurodevelopment. 250 mcg dosing offers no additional advantage over 50 mcg.
2. Findings: Improvement in B-12 parameters from first trimester to third trimester is significant in either 250 mcg alone or both groups, but 250 mcg causes higher rise (statistically significant) in B-12 biochemical parameters than 50 mcg for mother and baby.
Interpretation: 250 mcg is more efficacious than 50 mcg in improving biochemical B-12 status.
Since neurodevelopment is equivalent between two groups a higher dose of supplementation does not offer any neurodevelopmental advantage despite better biochemical B-12 status.

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