Supplemental choline to prevent and treat learning and memory deficits of early-life iron deficiency (The SupCHO Study): study protocol for a randomized, placebo-controlled trial in Ugandan infants with iron deficiency anemia

Supplemental choline to prevent and treat learning and memory deficits of early-life iron deficiency (The SupCHO Study): study protocol for a randomized, placebo-controlled trial in Ugandan infants with iron deficiency anemia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Supplemental choline to prevent and treat learning and memory deficits of early-life iron deficiency (The SupCHO Study): study protocol for a randomized, placebo-controlled trial in Ugandan infants with iron deficiency anemia Sarah Cusick, Ezekiel Mupere, Paul Bangirana, Reagan Baluku, Maria Kroupina, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7041039/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Nov, 2025 Read the published version in Trials → Version 1 posted 5 You are reading this latest preprint version Abstract Background: Iron deficiency (ID) limits the neurodevelopmental potential of more than 200 million children each year. Iron therapy started when IDA is first diagnosed—typically by screening for anemia at or detection of clinical symptoms of IDA at 12 months of age—does not fully correct earlier ID-mediated brain dysfunction, underscoring the need for low-cost, easily implementable adjunct therapies to iron to treat or prevent this dysfunction in high-risk populations. Supplementation with the essential nutrient choline lessens damage done to the developing hippocampus when given with iron in pre-clinical rodent models, and choline supplementation improves hippocampus-mediated memory and learning in 2-3-year-old children with Fetal Alcohol Spectrum Disorders, a condition associated with hippocampal damage and one for which ID is a component of the neuropathology. Choline has not been tested in children with IDA. Our overall aim is to conduct a randomized, placebo-controlled clinical trial to test whether nine months of daily choline supplementation along with standard iron therapy improves hippocampus-dependent neurobehavioral outcomes in Ugandan infants with IDA. Methods: Three hundred 6-month-old infants with IDA who present to immunization clinics at Mulago and Kawempe National Referral Hospitals in Kampala, Uganda, will be randomized to iron plus choline or iron plus placebo. Iron (oral ferrous sulfate 2 mg/kg/day) will be given for the first three months of follow-up, and a dispersible tablet of choline (200 mg as choline bitartrate) or identical placebo will be given daily for all nine months of follow-up. We will conduct neurobehavioral tests assessing hippocampus-specific memory and attention and global cognition at enrollment (when each infant is 6 months of age) and after nine months of follow-up (when each infant is 15 months of age). Discussion: If we find a neurobehavioral benefit when choline is given along with iron, choline could be added immediately to standard of care treatment for IDA. This low-cost intervention could safely mitigate the brain dysfunction of early-life ID that is often not diagnosed until the hippocampal critical window is closing, providing life-long benefit for both the individual and the economic and social prosperity of entire regions. Trial registration: Clinical trials.gov# NCT06527391; Registered 24 July 2024 Choline iron deficiency brain development nutrients and brain hippocampus Figures Figure 1 Figure 2 Administrative information Note: the numbers in curly brackets in this protocol refer to SPIRIT checklist item numbers. The order of the items has been modified to group similar items (see http://www.equator-network.org/reporting-guidelines/spirit-2013-statement-defining-standard-protocol-items-for-clinical-trials/). Title {1} Supplemental choline to prevent and treat learning and memory deficits of early-life iron deficiency (The SupCHO Study): study protocol for a randomized, placebo-controlled trial in Ugandan infants with iron deficiency anemia Trial registration {2a and 2b}. Clinical trials.gov# NCT06527391; Registered 24 July 2024 Protocol version {3} 2 nd May 2025; Protocol version 1.1 Funding {4} E.W. “AL” Thrasher Award, Thrasher Research Fund (Primary funder) University of Minnesota Masonic Institute for the Developing Brain Interdisciplinary Faculty Research Award (Supplemental funder) Author details {5a} Sarah E. Cusick, PhD, Department of Pediatrics, University of Minnesota Ezekiel Mupere MBChB, MMED, MS, PhD, Department of Paediatrics and Child Health, Makerere University Paul Bangirana, PhD, Department of Psychiatry, Makerere University Reagan I. Baluku, Global Health Uganda Maria Kroupina PhD, Department of Pediatrics, University of Minnesota Carol Cheatham, PhD, Department of Psychology and Neuroscience, University of North Carolina at Chapel Hill Jeffrey Wozniak, PhD, Department of Psychiatry and Behavioral Science, University of Minnesota Michael K. Georgieff MD, Department of Pediatrics, University of Minnesota Name and contact information for the trial sponsor {5b} Thrasher Research Fund, Allison Martinez, tel +1 (801) 240-4753, email [email protected] University of Minnesota Masonic Institute for the Developing Brain, Hannah Shryer, tel +1 (612) 365-8400, email: [email protected] Role of sponsor {5c} Study sponsors/funders have no role in study design; collection, management, analysis, and interpretation of data; writing of the report; or submission for publication and will have no authority over any of these activities. Introduction Background and rationale {6a} Iron deficiency (ID) remains the most common nutritional deficiency in the world despite prevention and food fortification efforts (1). In regions of the world such as Uganda, iron deficiency anemia (IDA)--the most severe stage of ID--affects one in every four children under five years of age (2). Early-life ID is a primary cause of long-term brain impairment because of cognitive deficits that persist for decades, despite diagnosis and iron treatment (3–6). Thus, early-life ID leads to unmet educational and job potential, thereby perpetuating an entrenched, transgenerational cycle of poverty and low societal productivity (7). The neurobehavioral pathology frequently occurs before a child is diagnosed as iron-deficient by hemoglobin because in all developing mammals including humans, the brain becomes iron-deficient before the red cells (8). In many low-income countries, including Uganda, there is no screening program for anemia, and ID is not diagnosed until clinical symptoms of severe ID anemia develop--well past the time that therapeutic iron can fully protect the brain (9) . Because the global prevalence of IDA among children 6-59 months of age is approximately 30% (10), the need to treat the brain dysfunction caused by this early-life ID that is not fully remediable with iron therapy is urgent. Preclinical models show that a major factor contributing to long-term deficits is the timing of iron repletion. When repletion does not precede or coincide with the developing brain’s peak need for iron, the brain is impacted long term, despite subsequent iron repletion. The hippocampus is particularly vulnerable to early life ID because of its high metabolic demand (11–14). It is the brain’s major processor for recognition learning and memory. In humans, it grows and differentiates particularly rapidly in the last trimester of pregnancy through the first two years of life, encompassing two peak risk periods for ID--the neonatal period and toddlerhood (9,15,16). Iron is essential to support the very high energy (ATP) neuronal energy demands of structural development of the hippocampus through its critical role in mitochondrial cytochrome function and ATP generation (11–14). Iron also regulates JARID histone demethylase activity of Brain Derived Neurotrophic Factor IV (BDNF-IV), a critical growth factor involved in neuronal growth and differentiation and synaptic plasticity (17–19). Without adequate iron to support this rapid growth during its critical growth spurt, permanent structural damage, characterized by simplified and abnormal dendritic arborization, occurs to not only the neurons in the hippocampus, but also to subsequent developing higher-order structures in the brain that depend on proper connections from the hippocampus during development (20,21). These include frontal lobe structures supporting attention and behavior. Because this damage frequently occurs before a child is diagnosed as iron-deficient by hemoglobin, iron therapy is often started too late to fully protect the brain (9). Developmentally informed, timed supplementation with the essential nutrient choline as an adjunct therapy to iron in children with IDA may be part of the solution to protect the brain. We have shown that compared to iron alone, choline given with iron to iron-deficient rat pups during critical periods of hippocampal development improves hippocampus-dependent learning and memory function across the lifespan(22,23). Mechanistically, choline counteracts early-life ID’s long-term harmful effects on the hippocampus by reversing negative epigenetic marks in synaptic plasticity genes, improving energy efficiency in neurons, and stabilizing neuronal structure (11,24,25). Timed choline is effective in models of other conditions characterized by hippocampal damage (26–28), including fetal alcohol spectrum disorders (FASD), where brain ID is part of the neuropathology (29–32). Choline’s positive effects on hippocampal function seen in preclinical models of FASD has been successfully translated to young children. We recently led the first clinical trial of choline supplementation in young children with FASD(33,34)and found that nine months of daily choline supplementation as 500 mg choline bitartrate in a fruit-flavored drink was feasible for parents to administer and was well-tolerated with no serious adverse events. Choline improved memory performance in 2-3-year-old children more so than in 4-5-year-old children, as measured by the Elicited Imitation (EI) task (a hippocampus-dependent recognition memory task), suggesting a critical window early in postnatal development (34). Follow-up studies after four years showed that choline improved memory function, non-verbal intelligence, behavioral symptoms of Attention Deficit Hyperactivity Disorder, and visual-spatial skills (35). After seven years, choline-supplemented children had better of executive functioning and speed of processing, with better white matter organization on MRI scan (36). These circuit- and structure-specific improvements observed in this study and subsequent similar studies guide the selection of the neurobehavioral (NB) tests used in this protocol, which include those that assess hippocampal function (working memory), behavior, and attention. Choline supplementation has not been tested in children with IDA, yet it may be a low-cost adjunct therapy to iron that could be used immediately. To fill this gap, we propose a randomized, placebo-controlled trial of choline in children with IDA living in Uganda. We will shift enrollment age earlier than in the FASD study to age 6 months to maximize NB impact by capturing more of the epoch of rapid hippocampal growth and differentiation. This trial is significant because of the high prevalence of early-life ID globally, its long-term individual and intergenerational consequences, and the possibility that an inexpensive and immediately available nutritional intervention may lessen those consequences. Objectives {7} Our primary objective is to conduct a randomized, placebo-controlled clinical trial to test whether nine months of daily supplementation with the essential nutrient choline along with standard iron therapy improves hippocampus-dependent neurobehavioral outcomes in 6-month-old infants with iron deficiency anemia. We hypothesize that infants with iron deficiency anemia who receive iron and choline will have better scores on specific neurobehavioral tests of recognition memory than infants who receive iron and placebo. Trial design {8} The Supplemental Choline to Prevent and Treat Learning and Memory Deficits of Early Iron Deficiency (SupCHO) study is a randomized, double-blinded, placebo-controlled, superiority clinical trial testing if 6-month-old infants with IDA who receive iron with choline have better scores on NB tests of hippocampal and global function at 15 months of age compared to infants who receive iron with placebo. The study will be conducted using a 1:1 allocation ratio, two arms, with parallel groups. We will recruit and enroll from immunization clinics at Mulago National Referral Hospital and Kawempe National Referral Hospital, both in Kampala, Uganda. Study randomization and baseline and follow-up assessments will be based at Mulago Hospital. The SupCHO protocol adheres to the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines and was registered at www.clinicaltrials.gov (NCT06527391). Methods: Participants, interventions and outcomes Study setting {9} Children will be enrolled from the immunization clinics at Mulago National Referral Hospital and Kawempe National Referral Hospital in Kampala, Uganda. All follow-up visits will take place at Mulago National Referral Hospital. These hospitals are approximately 3.5 km apart and are both staffed by study medical officers and co-investigators. Uganda is the only country where data will be collected. Eligibility criteria {10} Study-specific, protocol-trained medical officers and nurses will conduct the assessments required to determine eligibility according to the inclusion and exclusion criteria: Inclusion criteria: 1) Age 6 months +/- 28 days; 2) Hemoglobin (Hb) 7 - = 80; 4) T<37.5°C; 5) Malaria-negative based on Rapid Diagnostic Test (RDT); 6) Mother is HIV-negative by HIV RDT. Exclusion criteria: 1) Developmental disorder; 2) Severe malnutrition (severe wasting or bipedal edema); 3) Known sickle cell disease; 4) Neurologic disorder, brain injury, or other condition affecting brain development; 5) Not currently breastfeeding; 6) Birthweight < 2000 g. Who will take informed consent? {26a} The screening consent process will take place in a private, partitioned-off space near the immunization clinic at Kawempe National Referral Hospital or Mulago National Referral Hospital, depending on where the child presents. All study nurses and medical officers conducting the screening are trained in Good Clinic Practice. Children found to be eligible for the full study after screening will be enrolled in the study office in either Kawempe National Referral Hospital or Mulago National Referral Hospital in a study room that is separated by the rest of the unit by a closed door. Once the medical officer/nurse determines that a potential participant meets all the inclusion criteria and has none of the exclusion criteria, he or she will go through the consent process for the full study with the caregiver. Both the screening and full-study consent forms will be available in English and Luganda, the local language. The medical officer or nurse will read through the entire consent forms with the caregiver of the potential participant child. All questions will be answered completely. At the end of the full-study consent discussion, the parent/guardian will demonstrate understanding by answering 10 comprehension questions. If the caregiver answers fewer than eight questions correctly, the informed consent process will be repeated until at least eight questions can be answered correctly. If the caregiver successfully completes the questionnaire and agrees for the child to participate in the study, he/she will sign the consent form or provide a thumb print if he or she cannot sign his or her name. An impartial witness will also sign the form. The impartial witness will be present during the entire consent discussion if the participant’s caregiver is unable to read. Additional consent provisions for collection and use of participant data and biological specimens {26b} In addition to the consent forms for screening and enrollment in the full study, we have separate consent forms for storage and future use of biological specimens and a data-use agreement. All consent forms are available in English and Luganda, and the informed consent process will be conducted in whichever of the two languages the caregiver prefers. Interventions Explanation for the choice of comparators {6b} All children will receive three months of liquid oral ferrous sulfate (2 mg/kg/day) as treatment for iron-deficiency anemia. Additionally, each child will be randomized to 200 mg of choline bitartrate or identical placebo tablet as a liquid-dispersible tablet. The benefit of choline for brain development in young children has not been established, nor has a potential role for choline as an adjunct therapy with iron to treat iron deficiency anemia. Thus, the comparator for choline is a placebo tablet. Intervention description {11a} Randomization and study supplement: On Day 0, children will be randomized to nine months of daily choline bitartrate or identical placebo tablet as a 200 mg dispersible tablet all manufactured under GMP by Toronto Institute of Pharmaceutical Technology. Treatment assignment (choline or placebo) will be made in a 1:1 ratio by permuted randomization in random block lengths. Investigators, field staff, and participants will be blinded to treatment assignment. Supplement instruction: For the choline/placebo tablets, caregivers will be instructed to put the tablet in a small amount (60 mL or ¼ C) of liquid, e.g., breastmilk, water, or juice, to be dispersed. An initial one-month (28 days) supply of the study tablets will be sent home with each mother at enrollment along with a dosage calendar where days can be ticked off as well as written instructions for use. The supplements and calendar will be replenished each month at a home visit. Home visitors will check the preceding month’s calendar and verify adherence by pill counts. Iron syrup will be given the first three months of follow-up, while choline and placebo tablets will be given for all nine months of follow-up. Criteria for discontinuing or modifying allocated interventions {11b} Choline is an essential nutrient and occurs naturally in foods (37). Choline is found in abundance in an animal-based diet, but many Ugandans, especially if iron-deficient, may not consume sufficient choline. Choline can cause vomiting, heavy sweating and salivation, low blood pressure, and liver damage when given in high doses (>1000 mg) (37–39), but there are no known adverse reactions to the nutrient supplement at the dose given in this trial. Children 2-3 y with FASD who consumed 500 mg of choline bitartrate daily for nine months had a greater incidence of fishy body odor, but this odor did not lead any of the children to discontinue the supplement (33, 34). We will actively monitor all physical symptoms at home visits and phone calls, tallying the symptoms weekly. If at any time the study pediatrician deems it medically necessary for a child, the supplement will be stopped. The supplement may also be stopped at any time by the caregiver. We will track adherence with pill counts during home visits and provide counsel as needed, but a decision to stop the supplement by the caregiver will not preclude the child from participation in study follow-up. Tablet doses and allocations will not be modified over the course of the study. Strategies to improve adherence to interventions {11c} All caretakers will be given a calendar with nine 28-day months on which to tick off each daily dose of the study supplement. Starting with the Day 0, continuing monthly at the home visits, and ending at the Month 9 clinic visit, we will take a printable color photo of the child at each age that can be pasted into the calendar, with the nine months of the study collectively showing the child’s monthly development from age 6 – 15 months. The calendar also has the World Health Organization (WHO) height and weight charts, and these growth measures will be tracked at each home visit, so that there is also a visual depiction of the child’s growth. In addition to talking to the caretaker and examining the dosing calendar, we will count pills remaining in the supplement bottle and compare it to the number of pills expected at each home visit. Percent adherence for the month will be calculated as (doses taken/doses prescribed) * 100. Counsel will be given as needed to address any concerns with the supplement. Relevant concomitant care permitted or prohibited during the trial {11d} All children will receive a three-month course of oral iron therapy. Any child found to be anemic during the finger-prick hemoglobin check at the month 6 (age 12 months) follow-up home visit will be given an additional three-month course of iron. Any medical care needed by the child while enrolled in the study will be permitted, and no medically prescribed care will be prohibited. Provisions for post-trial care {30} There are no provisions for post-trial care. Each participant is covered by Sanlam Insurance while enrolled in the trial, with provision for up to $10,000 for any research-related injury in accordance with applicable Ugandan laws. Outcomes {12} Primary outcome : Elicited Imitation outcomes (number of steps correctly recalled and number of steps recalled in the correct order for both immediate and delayed recall) at age 15 months, i.e., after nine months of daily supplementation with choline or placebo. For each of these outcomes, we will compare groups using a one-way analysis of covariance (ANCOVA). Secondary outcomes. Mean composite Z-score on Mullen, scores on each scale of the BRS, and proportion of time the child views an animation viewing across a computer screen on the Early Childhood Vigilance Test at age 15 months in the choline vs. placebo groups. Participant timeline {13} All 300 participants will be enrolled at age 6 months, will be given a 3-month course of oral ferrous sulfate (2 mg/kg/day), and will be randomized to receive a dispersible tablet of choline (200 mg as choline bitartrate) or placebo daily for nine months of follow-up, i.e., until 15 months of age ( Figure 1 ). We will conduct biweekly surveillance via home visits and phone calls for adherence and illness throughout the nine-month-follow-up period and will compare neurobehavioral test scores between the choline and placebo groups at age 15 months (Figure 2). Pre-Screening We will identify potentially eligible participants during their routine visits at age 14 weeks and 6 months to the immunization clinics at Kawempe National Referral Hospital or Mulago National Referral Hospital. These hospitals are approximately 3.5 km apart and are both staffed by study medical officers and co-investigators. 14-week visit (most children will likely be identified at this visit): Study staff will identify age-eligible children who attend the clinic for their 14-week routine immunizations. The caregiver of the child will be told about the study and invited to bring the child for study screening during their 6-month routine visit. The caregiver will be reminded that the biological mother must be present at screening at 6 months. 6-month visit: Age-eligible children who were not identified at their 14-week visit can be screened directly at their 6-month routine immunization visit if they are with their biological mother. In this case, study staff will ask the mother if she is interested in participating, and if yes, she will be invited for screening that very day. The biological mother must be present at screening because she must be screened for HIV by RDT as part of the inclusion criteria. Study screening (at 6-month visit): All official screening will be at the child’s 6-month visit and will take place at Mulago National Referral Hospital or Kawempe National Referral Hospital, depending on where they were first identified. At this visit, mothers of age-eligible, afebrile infants will provide written screening consent for a finger-prick blood sample from their child and themself. The child’s blood sample will be used to measure zinc protoporphyrin (ZPP), hemoglobin, and for a malaria Rapid Diagnostic Test (RDT). The maternal sample will be used for an HIV RDT test. If the child meets the inclusion criteria below and has none of the exclusion criteria, he or she is eligible for enrollment. Diagnosis of IDA by two point-of-care tests: We will measure hemoglobin (Hb) by HemoCue and ZPP by hematofluorometer to diagnose IDA. Per WHO recommendations, anemia will be defined as Hb < 11.0 g/dL in children 6 months of age (40). ZPP is a hemoglobin precursor that accumulates in the developing red blood cells when iron is not available to synthesize hemoglobin (41,42). Thus, a high ZPP (≥ 80µmol/mol heme) reflects iron-deficient erythropoiesis. We will have front-face hematofluorometers onsite at both Kawempe National Referral Hospital and Mulago National Referral Hospital (Protofluor-Z, Helena Biosciences) to measure ZPP in real time. If a child has a hemoglobin concentration of < 7.0 g/dL, h fetal alcohol spectrum disorders he/she is not eligible for the current study and will be referred for treatment at the pediatric clinic at Mulago National Referral Hospital or Kawempe National Referral Hospital. Enrollment: Once a child meets all inclusion criteria, he/she can be enrolled. Informed consent for the full study will take place on the day of screening and thus can be at either Mulago National Referral Hospital or Kawempe National Referral Hospital. After consent and enrollment, the child will be scheduled for a neuropsychological testing/Randomization Day (Day 0) at Mulago National Referral Hospital within 10 days of screening/enrollment. Participants are informed on the full consent form that if for any reason they are not able to come for the Day 0 visit within 10 days, they cannot remain in the study. All children will be started on iron on the day of enrollment. For the iron syrup, the study pharmacist will instruct each mother how to draw up the correct amount of iron syrup (ferrous sulfate, 2/mg/kg-day) in a medication syringe. Any child found to be ill at screening will be referred immediately to the Mulago Paediatric Assessment Center or to the Kawempe Outpatient Department, depending on the screening site, for diagnosis and treatment. Emergencies will be referred to the Acute Care Unit at each hospital. Day 0 Testing/Randomization Day (all at Mulago National Referral Hospital): Each enrolled child’s official Day 0 will take place at Mulago National Referral Hospital. On this day, they will have baseline neuropsychological testing (see below), a physical exam, dietary assessment, and a venous blood draw for measurement of iron status biomarkers. At the end of this visit, the child will be given a blinded randomized treatment assignment for choline or placebo (Figure 1). Dietary Assessment: We will assess dietary consumption of choline on Day 0 and at the end of the follow-up period to verify that this consumption does not differ between treatment groups. Trained study staff will administer a 24-hour food recall developed specifically for the Ugandan diet (43) to each mother to answer for their child at enrollment and at the follow-up visit when the child is 15 months of age. Estimates of portion size will be made using standard Ugandan plates, cups, and spoons. We will calculate the choline content of the foods using the USDA database (44). Neurobehavioral testing: Enrolled children will be evaluated by hippocampus-specific and general behavioral tests at Day 0 (6 months of age) and after nine months of daily supplementation, at 15 months of age (45–48). Measurements at enrollment will be used to characterize overall function of the groups pre-intervention, while the study outcome measures will be NB test scores at 15 months of age. Task descriptions 1) Elicited Imitation (EI) : This test provides a comprehensive understanding of hippocampal function by interleaving components of immediate and delayed recall to assess working memory, a more advanced hippocampus-dependent task (49–52). At age 15 months, children will be presented with multi-piece props and are able to manipulate them to provide a baseline measure of spontaneous occurrences of the target behaviors. After this period, a tester will model a multi-step sequence using the props. The sequence will be modeled twice with narration. Infants will be allowed to model the sequence immediately (immediate recall, a measure of encoding) and after 15 minutes (delayed recall, a measure of memory). All sessions will be recorded and coded by trained observers. To ensure reliability, 20% of videos will be coded by a second trained observer. Performance will be scored as the number of target actions recalled and number of actions produced in the correct order for both immediate and delayed conditions. We have extensively used and published on this paradigm to examine hippocampal function in infants with prenatal metabolic and nutritional deficiencies (34,53). Ongoing quality control for EI scoring will occur via biweekly Zoom calls for the duration of the trial, with the EI trainer independently scoring 20% of the videos. EI is not a reliable measure of hippocampal function at age 6 months, but we will capture baseline hippocampal function in the global cognition tests that we will perform(50,54). 2) The Mullen Scales of Early Learning (MSEL): MSEL is a global cognition test, measuring the domains of gross motor, fine motor, visual reception, receptive language, and expressive language (55). We have used the Mullen multiple times in our studies in Uganda (45,46). 3) The Behavior Rating Scales developed by Lozoff (56) for use in studying ID will be coded from videotaped Mullen testing sessions. These scales have three composite scores that assess behaviors (fearfulness, impulsivity, and low positive affect) secondary to the monoaminergic effects of ID. Two testers will observe each testing session. One will administer the Mullen, while the other will observe behavior. At the end of the session, both testers will complete a BRS scoresheet and compare scores for quality control. Approximately one-fifth of videos from all sessions will be coded independently by a Ugandan master coder to ensure reliability. 4) The Early Childhood Vigilance Test (ECVT) is a computerized test to evaluate attention that was modeled after Ruff’s puppet vigilance paradigm (57). The total time required for all tests is approximately 1.5-2 hours. Rest and snack breaks will be given as needed. Venipuncture: After neurobehavioral testing, we will collect 3-5 mL of whole blood from each child. We will use whole blood for CBC. The remaining blood will be centrifuged, and plasma/RBC pellet will be collected, aliquoted, and frozen onsite at -80°C for analysis of plasma iron and inflammatory biomarkers (onsite by ELISA assay). Follow-up Clinic Visit at age 15 months (+/- 2 weeks) : All children will have a scheduled follow-up clinic visit after nine months of follow up, i.e., at 15 months of age. This follow-up visit will include neurobehavioral tests, a venous blood draw of 3-5 mL, and a 24-hour food recall as at the enrollment visit. Surveillance and Follow-up: Home visits: All enrolled children will receive an insecticide-treated bednet. For the next nine months of follow-up, we will have alternating biweekly (every two weeks +/- 3 days) home visits and phone calls for each enrolled study child to assess adherence to the study tablets (tablet counts for entire nine-month follow-up period) and iron syrup (volume check, for first three months of follow-up) and to inquire about any illnesses or side effects. Any ill child with history of fever, persistent diarrhea or vomiting, lethargy, or anorexia, will be transported to the clinic for diagnosis and treatment. Mothers will also be instructed to call the study clinic any time their child is ill and will be given transportation refund to attend the clinic for treatment. The home visitor will assess the home environment using the Infant-toddler version of the HOME Inventory (The Home Observation for the Measurement of the Environment Inventory) form at the first visit, and at each home visit, dietary strategies to prevent future ID through increased consumption of locally available food that is high in bioavailable iron will be presented using local materials and methods. Hemoglobin will be measured by finger-prick blood sample using HemoCue at the home visit at 6 months follow-up (12 months of age). Any child with hemoglobin < 11.0 g/dL will receive an additional 3-month course of iron therapy. Receipt of a second course of iron therapy will be controlled for in final analyses. Sample size {14} This study is powered for the primary outcome, Elicited imitation scores (continuous) at 15 months of age in the choline vs. placebo group. Our sample size gives a detectable difference (80% power) in a t-test of Cohen's d = 0.3, i.e., roughly one-third a SD unit, where the SD describes variation of the outcome within group. Allowing 15% lost to follow-up gives 150 children per group (choline and placebo), 300 children total. Recruitment {15} We will identify eligible children approximately 2.5 months prior to enrollment at their 14-week visit to the immunization clinic when they receive their polio and pneumococcal conjugate immunizations (58). The Immunization Clinics at Mulago and Kawempe National Referral Hospitals are among the largest in the country. Approximately 140 children attend the clinics each month for their 14-week visit (E. Mupere, personal communication). Since roughly one in four Ugandan children has IDA, we anticipate enrolling the 300 children with IDA comfortably in 12-18 months, with a target of 4-5 children enrolled each week. Assignment of interventions: allocation Sequence generation {16a} The tablet manufacturer, Toronto Institute of Pharmaceutical Technology (TIPT), will generate the allocation sequence (1:1; choline:placebo) by permuted randomization in random block lengths using 3-digit numbers, ranging from 101 - 400. These 3-digit numbers will correspond to participants’ study ids. Each study id (linked to a treatment assignment) will be printed directly onto nine supplement (tablet) bottles, representing one bottle for each of the nine months of supplementation. The link between treatment assignment and study id will be kept at TIPT only. Study staff, investigators, and caregivers will all be blinded to treatment assignment. Concealment mechanism {16b} Study id numbers that are linked to the randomly assigned treatment assignment will be printed directly onto supplement bottles. Each id will be printed onto nine separate bottles of study tablet, along with the bottle number, e.g., Study id 101, bottle 1; Study id 101, bottle 2, up to Study id bottle 9. Study staff on the ground will only give out the regimens according to sequential study id number and will not know or have access to the link between the id number and treatment assignment. Implementation {16c} The allocation sequence will be generated by TIPT, the tablet manufacturer. Participants will be enrolled by study nurses and pediatricians at Mulago and Kawempe National Referral Hospitals. Assignment of study id’s will be done by the study dispenser/pharmacist, thus assigning each participant a blinded treatment assignment. Assignment of interventions: Blinding Who will be blinded {17a} Trial participants, care providers, outcome assessors, data analysts, and investigators will all be blinded to treatment assignment until all outcomes for the trial, including plasma biomarkers, have been collected, all trial data have been entered into the study database and verified, and the final analytical data set is generated and locked. Procedure for unblinding if needed {17b} Because choline is an essential nutrient, and we are giving it at a level well below the upper threshold for recommended intake (37), we do not foresee a circumstance where unblinding will be necessary. However, if at any time unblinding is deemed medically necessary by a study physician, the study pharmacist will contact TIPT directly for the unblinding of that single study id. Data collection and management Plans for assessment and collection of outcomes {18a} Data will be entered directly into the study’s RedCap database via tablet. All data will be reviewed by a second study staff member before the data is released to the database. Paper copies of all case report forms (CRF’s) will also be available in case of tablet failure. All paper case report forms will be signed by the person filling the form and also by a second person who will verify that the information on the form is correct and complete. The study data manager will generate a data query log each week, with corrections and quality control made immediately. Neurobehavioral assessment: All neurobehavioral sessions will be video recorded. All assessments will be double scored by two Uganda-based neuropsychological testers. Additionally, 20% of videos will be scored/coded by master trainers for the Behavioral Rating Scale and the Elicited Imitation Task, to help ensure ongoing quality control and scoring consistency. Paper copies of all study CRF’s were approved by and on-file with the Mulago Hospital Research Ethics Committee (MREC). Plans to promote participant retention and complete follow-up {18b} All analysis will be intention-to-treat. We have biweekly contact with each participant via alternating physical home visits and phone calls to maintain connection and enthusiasm in the study. Ongoing access to health services and monthly growth monitoring help ensure retention and adherence. Data management {19} Aside from neuropsychological testing coding sheets, all study data will be entered directly into a RedCap database via direct data entry. Verification by a second data entrant is required for the data to be submitted to the database. The study data manager runs data quality reports each week, with any errors addressed immediately. The database will include health data, including blood markers for iron infection, physical examination findings, neurobehavioral findings, and information, e.g., diagnoses, physical findings, from sick visits to the health clinic. All variables have biologically plausible range checks built in to alert the entrant of a potential error. All data management procedures are codified in Standard Operating Procedure documents, available in hardcopy in the study office or upon request from the study principal investigators. Databases are kept on password-protected computers accessible only to authorized study project personnel and will be uploaded nightly onto UMN BOX, which requires duo authentication. Hard copies of any forms will be kept in the study data room in patient documents kept in locked filing cabinets. Final database files will be kept in the data room in the project offices in UMN computers and on a back-up drive. Data files will be kept on UMN BOX and accessible only by designated individuals with permission from the PI. A copy of the consent forms will be kept in the participant’s study file, but will not be included in medical, employment, or educational records. Confidentiality {27} No personal identification information such as names, address, or phone numbers will be included in the study database. All information in the database will be linked by the three-digit study id. Thus, only deidentified data are included in the database, and no pre-existing medical records will be accessed for study purposes, aside from a child’s vaccination card if brought to the hospital by the caretaker and shown to study personnel. We additionally have a data use sharing agreement as a separate consent form that explains that de-identified data is collected as part of the study and may be shared electronically within the study team. Plans for collection, laboratory evaluation and storage of biological specimens for genetic or molecular analysis in this trial/future use {33} All blood samples collected as part of the study will be labeled with only 3-digit study id and laboratory accession number for storage. Any plasma that remains after iron and inflammatory marker testing will be stored for up to ten years at -80 degrees C in the Paediatrics Laboratory at Mulago National Referral Hospital, Kampala. Permission to bank blood for future use will be included as a consent form. Personal, identifying information will not be kept in the study database. Permission will be required by the U.S. and Ugandan PI to access any banked specimen. Statistical methods Statistical methods for primary and secondary outcomes {20a} Primary analyses will be intention-to-treat. Primary outcome : Elicited Imitation outcomes (number of steps correctly recalled and number of steps recalled in the correct order for both immediate and delayed recall) at age 15 months, i.e., after nine months of daily supplementation with choline or placebo. For each of these outcomes, we will compare groups using a one-way analysis of covariance (ANCOVA). Secondary outcomes. For each secondary outcome (Composite Z-score on Mullen, scores on each scale of the BRS, and proportion of time the child views an animation viewing across a computer screen on the Early Childhood Vigilance Test), we will do the same analyses as for the primary outcome, using the baseline value of each test as a covariate to absorb variation in the outcome and thus increase power. Adjusted analysis will examine the effect of covariates including sex, age, iron status, CRP concentration, and home environment. Any covariate that appears to be a confounder will be included in the final analysis. While primary analyses will be intention-to-treat, we will also conduct per protocol analyses to include only children with more than 90% adherence to the study supplement to assess any difference in effect size. We will also conduct analyses with and without children with CRP > 10 mg/L. If we find a difference in the relationship between choline and neurobehavioral outcomes that is modified by having a CRP > 10 mg/L, we will exclude children in this group. Interim analyses {21b} The study has a five-member Data and Safety Monitoring Board that will meet at the start of enrollment and every six months thereafter for the duration of the study. Once half (n=150) of the full sample size has completed follow-up, official interim analysis will occur. The interim meeting will consist of an open and closed session. In the open session, matters of study procedure, including supplement compliance, and data quality will be discussed. This open session will be open to all members of the monitoring board, study investigators, and any other interested parties. Only DSMB members will attend the closed session. At this session, randomization will be broken, and morbidity/side effect and outcome data will be presented by treatment group. After each meeting, the DSMB will prepare a report for investigators that includes recommendations for any amendments to study protocol. Copies of the reports will be submitted to the Mulago Hospital Research Ethics Committee (MREC), Uganda National Council of Science and Technology (UNCST), and the Uganda National Drug Authority (NDA). If at any time the DSMB recommends study termination (see stopping rules, below), a full vote of the DSMB is required, with majority vote (3 of 5) needed to enact the proposed change. The DSMB chairperson may convene ad hoc DSMB meetings any time he/she feels that discussion is required to make important decisions regarding revision of study procedures or suspending study activities. Stopping rules The stopping rules for this trial will incorporate stopping rules to monitor serious adverse events and stopping rules to determine early success . The DSMB may also stop the study based on futility analysis. Interim analyses will use the group-sequential testing method of Lan and DeMets, with the O’Brien-Fleming alpha-spending function. The study team proposes an interim analysis, after half of the participants have completed follow up, but the DSMB will determine how many interim analyses it will examine and when during follow up they will be done. The LanDeMets method is flexible enough to accommodate that. For determining whether to continue or terminate the study, the DSMB may also request computation of conditional power using the method of stochastic curtailment (also known as futility analysis). Safety review if study is stopped If the study is stopped for any reason, a full safety review will be completed by the study team and the DSMB. The RECs of both Mulago National Referral Hospital and the University of Minnesota must be informed in the event of study stopping, as well as NDA and UNCST. The study will restart only upon completion of the safety review by the DSMB and DSMB approval. Methods for additional analyses (e.g. subgroup analyses) {20b} Addressed in statistical methods for primary and secondary outcomes. Methods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c} While primary analyses will be intention-to-treat, we will also conduct per protocol analyses to include only children with more than 90% adherence to the study supplement to assess any difference in effect size. We will also conduct analyses with and without children with CRP > 10 mg/L. If we find a difference in the relationship between choline and neurobehavioral outcomes that is modified by having a CRP > 10 mg/L, we will exclude children in this group. Plans to give access to the full protocol, participant level-data and statistical code {31c} Access to the full protocol, de-identified dataset, and statistical code will be considered by the PIs upon receipt of a written request describing the intended use of information. Oversight and monitoring Composition of the coordinating centre and trial steering committee {5d} The day-to-day operations of the SupCHO study are overseen by US-based and a Uganda-based PIs. The field team includes a study-specific pediatrician, three nurses (one who serves as the home visitor), two neuropsychological testers, a pharmacist, a dispenser, two data entrants, a data manager, and a laboratory technician. We also will employ some staff, including immunization clinic nurses and HIV-testing counselors on a locum basis. The field team meets every other week with the US-based PI via Zoom. Minutes for all meetings are kept securely online and in the study regulatory binder. Composition of the data monitoring committee, its role and reporting structure {21a} A Data and Safety Monitoring Board (DSMB) has been established to provide external, objective advice regarding the safety and efficacy of the administered interventions. The Board is comprised of five members: an expert in clinical trials/biostatistics, an expert in global pediatrics/child health, an expert in choline, an expert in child development, and one non-medical scientist. Four board members are Ugandan. The Board will be formed to ensure the safety of study participants and the validity of collected data though review of subject recruitment, retention and follow-up, data forms, protocol adherence, and data quality. The committee will meet with investigators shortly after the start of the study (a month into the study) to review the protocol, DSMB charter, proposed interim analysis tables, and any new pertinent data and literature not included in the original protocol. Members will meet every six months for the duration of the study, including an interim analysis meeting once half of the total sample size has completed follow-up. Board members are independent from the sponsor and have signed conflict of interest forms and confidentiality agreements. The DSMB charter has been submitted to and approved by the Uganda National Drug Authority and is available upon request to the study PI. Adverse event reporting and harms {22} Adverse events and Serious adverse events An adverse event (AE) is any untoward medical occurrence in a subject undergoing a study-related procedure or consuming a study-related supplement that is believed reasonably to be caused by that procedure or supplement. The only procedure performed in this study is blood sampling by venipuncture or finger prick collection. Adverse events associated with this procedure could include prolonged bleeding, persistent pain, development of infection, and development of a large blood collection in the skin (hematoma) . We anticipate that such AEs from this study procedure will be infrequent (<2%). The only known potential adverse event of choline is fishy body odor which has been reported with much higher doses. We will assess the frequency of this event as well as all other illnesses or physical symptoms through parental reports in monthly home visits and will discontinue therapy if discomfort persists. Home-visit forms will be returned to the study office each day, and the medical officer will monitor the physical symptoms/illnesses reported on this form. Tallied lists of physical symptoms will be submitted to the DSMB for the biannual meetings. A serious adverse event (SAE) is any event regardless of relationship to the intervention that: 1) Results in death; 2) Is life-threatening; 3) Requires hospitalization or prolongation of existing hospitalization; 4) is an important medical condition in the opinion of the investigator and in relation to our intervention In the event of an SAE, the participant will be managed accordingly. In case of a life-threatening event our team will promptly attend to the patient in the Paediatric emergency unit (Acute Care Unit) giving them the appropriate first aid. It is well equipped with airway and cardiorespiratory resuscitation equipment and an Intensive Care Unit with life support equipment and well-trained staff. If a participant develops an important medical condition, we will attend to them still in the hospital with our teams based in Mulago National Referral Hospital. An SAE form will be filled out by the attending physician, and the site PI (Dr. Mupere) will be notified within 48 hours. Dr. Cusick (PI) will be notified in no more than 7 days, and she will relay the SAE to the DSMB chairperson within 15 calendar days of the event. All SAEs will be reported to the ethical and regulatory bodies in accordance with their institutional policies. Any unanticipated problem that results in unexpected risk to subject participants and requires changes in the study protocol will be immediately report by the PI to both the Ugandan and US IRBs/RECs.A five-member Data and Safety Monitoring Board, as described above, will be responsible for overseeing the overall safety of trial participants and ensuring adherence to study protocol. Frequency and plans for auditing trial conduct {23} An independent study monitor will audit the trial annually. Each audit will include a 3-4-day visit to the study site, all study rooms, evaluation of completeness of the regulatory binder, study SOP’s, and study database integrity. Plans for communicating important protocol amendments to relevant parties (e.g. trial participants, ethical committees) {25} All protocol amendments will be submitted to MREC and the University of Minnesota IRB for approval. Once MREC approval for the change is given, we will notify UNCST and NDA. The requested amendment will not be carried out until full approval for the amendment is received. If the requested amendment affects the wellbeing of participants, caretakers will be informed of the change and the reason for the change immediately. Dissemination plans {31a} Trial results will be published in a peer-reviewed manuscript. There are no publication restrictions. Depending on their significance, findings may also be presented to Ministry of Health officials or other policy makers. Study findings that are available immediately, e.g., participant height and weight, temperature, HemoCue hemoglobin value, malaria rapid test results, will be communicated to the caretaker immediately. Discussion We are not measuring polymorphisms in choline-related genes which may affect endogenous synthesis and/or transport of choline. Choline-related SNP rs12325817 is a variant of the PEMT enzyme that is associated with a reduction in the endogenous synthesis of choline (59). We do not know the prevalence of this SNP in our population, but it was reported to be ~20% among South African women (60). If we assume the same prevalence in our sample and remove 20% of the children, our detectable difference for EI score would increase from 0.35 to 0.40 and retain clinical significance. We also did not include biomarkers of choline status. Based on the preliminary studies among children with FASD (34), however, serum choline increased in children who received choline supplementation, as expected. The limited information expected to be gained thus did not justify the high cost of choline analysis. We can assess this analyte in stored blood samples, if determined to be scientifically meaningful. A consideration when interpreting our results is that because choline and DHA work together in brain development, we may miss a choline effect if sufficient DHA is not present (61,62). We designed the current study as an efficacy study to test the effect of choline alone, as we did in our FASD study. However, if we do see an effect of choline in this study, we will include DHA in future studies to see if this effect is enhanced. Alternatively, if we see no choline effect, we can include DHA in future studies to see if an effect emerges. Finally, iron therapy in a malaria-endemic area has been associated with a potential risk of increased episodes of malaria or other infectious illness (63). However, iron remains the standard of care treatment for children with iron deficiency. Multiple Cochrane Reviews have concluded that iron can be given safely to children in areas where malaria prevention and management services are provided, as they are in the context of the proposed trial (64–66). Current WHO guidelines for universal iron supplementation reflect these results, stating that prophylactic iron therapy should be given to children in malaria-endemic areas where anemia prevalence is 40% or greater if public health measures to prevent, diagnose, and treat malaria are in place (67). All children in our study will be under active surveillance for illness and will be given an insecticide-treated bednet at enrollment. Any child found to have malaria or other illness will receive treatment according to Mulago Hospital and Uganda National guidelines free of charge. A finding of benefit in neurobehavioral outcomes when choline is given with iron in the current study may support future research to see if the recommended dose of iron for the correction of IDA can be reduced in malaria-endemic areas, potentially lessening infection risk. Trial status Protocol version 1.1 dated May 8, 2025. Approximate date of start of recruitment: August 1, 2025 Approximate date of trial completion: June 2027 Abbreviations AE: Adverse Event BRS: Behavior Rating Scales CBC: Complete Blood Count CRF: Case report form CRP: C-reactive protein DSMB: Data and Safety Monitoring Board ECVT: Early Childhood Vigilance Test EI: Elicited Imitation FASD: Fetal alcohol spectrum disorders Hb: Hemoglobin ID: Iron deficiency IDA Iron deficiency anemia IRB: Institutional Review Board NB: Neurobehavioral NDA: Uganda National Drug Authority MREC: Mulago Hospital Research Ethics Committee MSEL: Mullen Scales of Early Learning PI: Principal Investigator RDT: Rapid Diagnostic Test SAE: Serious adverse event SOP: Standard operating procedure SupCHO: Supplemental choline to prevent and treat learning and memory deficits of early iron deficiency TIPT: Toronto Institute of Pharmaceutical Technology UMN: University of Minnesota UNCST: Uganda National Council of Science and Technology WHO: World Health Organization ZPP: Zinc protoporphyrin Declarations Acknowledgements Not applicable Authors’ contributions {31b} SC is the principal investigator and led the protocol development. SC, JW, and MG conceived the study and provided preliminary data; EM is the site principal investigator, is the liaison with regulatory authorities, ensures adherence to protocol and SOPs, and supervises day-to-day trial operations. PB oversees onsite neurobehavioral testing; RB is the study manager and pediatrician and oversees study logistics and the study team; CC conducted the training for the Elicited Imitation task, provides ongoing quality control and external validation for this assessment, and will help with interpretation of results. MK developed the behavioral rating scale and will oversee behavioral coding. All authors read and approved the final manuscript Funding {4} E.W. “AL” Thrasher Award, Thrasher Research Fund (Primary funder) University of Minnesota Masonic Institute for the Developing Brain Interdisciplinary Faculty Research Award (Supplemental funder) The funding bodies will have no role in the design of the study, the collection, analysis, and interpretation of data, or in the writing of the trial manuscripts. Availability of data and materials {29} SC and EM will have access to the final trial dataset. No contractual agreements limit access for investigators. Ethics approval and consent to participate {24} University of Minnesota Institutional Review Board # STUDY00022408 version 1 date 6/18/2024; version 1.1 date 7/2/2025 Mulago Hospital Research Ethics Committee Date # MHREC 2024-158 version 1 5/5/2024; version 1.1 6/24/2025 Uganda National Council of Science and Technology: HS4915ES 11/13/2024 Uganda National National Drug Authority: CTA 0288/2025 Written, informed consent to participate will be obtained from the caretakers of all participants. Consent for publication {32} Attached. Competing interests {28} The authors declare that they have no competing interests Authors’ information (optional) Not applicable References Lönnerdal B, Hernell O. Iron: Physiology, Dietary Sources, and Requirements. In: Encyclopedia of Human Nutrition [Internet]. Elsevier; 2013 [cited 2025 May 27]. p. 39–46. Available from: https://linkinghub.elsevier.com/retrieve/pii/B9780123750839001641 Kuziga F, Adoke Y, Wanyenze RK. 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Available from: https://www.who.int/publications-detail-redirect/9789241596107 Mupere E, Parraga IM, Tisch DJ, Mayanja HK, Whalen CC. Low nutrient intake among adult women and patients with severe tuberculosis disease in Uganda: a cross-sectional study. BMC Public Health. 2012 Dec 5;12:1050. Patterson KY, Bhagwat SA, Williams JR, Howe JC, Holden JM, Zeisel SH, et al. USDA Database for the Choline Content of Common Foods, Release 2 (2008) [Internet]. Nutrient Data Laboratory, Beltsville Human Nutrition Research Center, ARS, USDA; 2015 [cited 2022 May 30]. Available from: https://data.nal.usda.gov/dataset/usda-database-choline-content-common-foods-release-2-2008 Ssemata AS, Hickson M, Ssenkusu JM, Cusick SE, Nakasujja N, Opoka RO, et al. Delayed iron does not alter cognition or behavior among children with severe malaria and iron deficiency. Pediatr Res. 2020 Sep;88(3):429–37. Bangirana P, Opoka RO, Boivin MJ, Idro R, Hodges JS, Romero RA, et al. Severe malarial anemia is associated with long-term neurocognitive impairment. Clin Infect Dis. 2014 Aug 1;59(3):336–44. Familiar I, Sikorskii A, Chhaya R, Weiss J, Seffren V, Ojuka JC, et al. Early Childhood Vigilance Test (ECVT) of attention in younger HIV-exposed Ugandan children predicts Tests of Variables of Attention (TOVA) at school age. Neuropsychology. 2022 Mar;36(3):185–94. Brahmbhatt H, Boivin M, Ssempijja V, Kigozi G, Kagaayi J, Serwadda D, et al. Neurodevelopmental benefits of antiretroviral therapy in Ugandan children aged 0-6 years with HIV. J Acquir Immune Defic Syndr. 2014 Nov 1;67(3):316–22. Riggins T, Cheatham CL, Stark E, Bauer PJ. Elicited Imitation Performance at 20 Months Predicts Memory Abilities in School-Age Children. J Cogn Dev. 2013 Jan 1;14(4):593–606. Bauer PJ, Hertsgaard LA, Dow GA. After 8 months have passed: long-term recall of events by 1- to 2-year-old children. Memory. 1994 Dec;2(4):353–82. DeBoer T, Wewerka S, Bauer PJ, Georgieff MK, Nelson CA. Explicit memory performance in infants of diabetic mothers at 1 year of age. Dev Med Child Neurol. 2005 Aug;47(8):525–31. de Haan M, Bauer PJ, Georgieff MK, Nelson CA. Explicit memory in low-risk infants aged 19 months born between 27 and 42 weeks of gestation. Dev Med Child Neurol. 2000 May;42(5):304–12. Riggins T, Miller NC, Bauer PJ, Georgieff MK, Nelson CA. Consequences of low neonatal iron status due to maternal diabetes mellitus on explicit memory performance in childhood. Dev Neuropsychol. 2009;34(6):762–79. Fagan JF. Infants’ recognition memory for faces. Journal of Experimental Child Psychology. 1972 Dec 1;14(3):453–76. Mullen EM, American Guidance Service. Mullen Scales of Early Learning. Circle Pines, Minnesota: AGS; 1995. Fuglestad AJ, Kroupina MG, Johnson DE, Georgieff MK. Micronutrient status and neurodevelopment in internationally adopted children. Acta Paediatr. 2016 Feb;105(2):e67-76. Goldman DZ, Shapiro EG, Nelson CA. Measurement of vigilance in 2-year-old children. Dev Neuropsychol. 2004;25(3):227–50. The Republic of Uganda Ministry of Health. Question and Answer Booklet on Routine Immunisation [Internet]. Available from: https://www.unicef.org/uganda/media/2776/file/Routine%20Immunisation%20Q%20&%20A%20booklet.pdf Silver MJ, Corbin KD, Hellenthal G, da Costa KA, Dominguez-Salas P, Moore SE, et al. Evidence for negative selection of gene variants that increase dependence on dietary choline in a Gambian cohort. FASEB J. 2015 Aug;29(8):3426–35. Jacobson SW, Carter RC, Molteno CD, Stanton ME, Herbert JS, Lindinger NM, et al. Efficacy of Maternal Choline Supplementation During Pregnancy in Mitigating Adverse Effects of Prenatal Alcohol Exposure on Growth and Cognitive Function: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial. Alcohol Clin Exp Res. 2018 Jul;42(7):1327–41. Cheatham CL. Nutritional Factors in Fetal and Infant Brain Development. Ann Nutr Metab. 2019;75 Suppl 1:20–32. Cheatham CL, Sheppard KW. Synergistic Effects of Human Milk Nutrients in the Support of Infant Recognition Memory: An Observational Study. Nutrients. 2015 Nov 3;7(11):9079–95. Sazawal S, Black RE, Ramsan M, Chwaya HM, Stoltzfus RJ, Dutta A, et al. Effects of routine prophylactic supplementation with iron and folic acid on admission to hospital and mortality in preschool children in a high malaria transmission setting: community-based, randomised, placebo-controlled trial. Lancet. 2006 Jan 14;367(9505):133–43. Neuberger A, Okebe J, Yahav D, Paul M. Oral iron supplements for children in malaria-endemic areas. Cochrane Database Syst Rev. 2016 Feb 27;2(2):CD006589. Ojukwu JU, Okebe JU, Yahav D, Paul M. Oral iron supplementation for preventing or treating anaemia among children in malaria-endemic areas. Cochrane Database Syst Rev. 2009 Jul 8;(3):CD006589. Okebe JU, Yahav D, Shbita R, Paul M. Oral iron supplements for children in malaria-endemic areas. Cochrane Database Syst Rev. 2011 Oct 5;(10):CD006589. World Health Organization. Daily Iron Supplementation in infants and children [Internet]. 2016 [cited 2023 Apr 15]. Available from: https://apps.who.int/iris/bitstream/handle/10665/204712/9789241549523_eng.pdf Cite Share Download PDF Status: Published Journal Publication published 19 Nov, 2025 Read the published version in Trials → Version 1 posted Reviewers agreed at journal 08 Oct, 2025 Reviewers invited by journal 19 Aug, 2025 Editor assigned by journal 12 Aug, 2025 First submitted to journal 11 Aug, 2025 Editorial decision: Minor revision 10 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7041039","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":502372637,"identity":"24a02369-65ba-4ded-9ee5-01a0734b6a2d","order_by":0,"name":"Sarah 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Hill","correspondingAuthor":false,"prefix":"","firstName":"Carol","middleName":"","lastName":"Cheatham","suffix":""},{"id":502372643,"identity":"5f1add58-4b77-4bd0-bc1c-d06881587a31","order_by":6,"name":"Jeffrey Wozniak","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Jeffrey","middleName":"","lastName":"Wozniak","suffix":""},{"id":502372644,"identity":"e381bd91-5bab-4033-8c58-03e8a4c61163","order_by":7,"name":"Michael Georgieff","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Georgieff","suffix":""}],"badges":[],"createdAt":"2025-07-03 19:26:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7041039/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7041039/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13063-025-09246-2","type":"published","date":"2025-11-19T15:59:03+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90003772,"identity":"068fc5a4-ffef-456e-888b-2d2db8203adf","added_by":"auto","created_at":"2025-08-27 09:14:39","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":92444,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStudy Design\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7041039/v1/f484027279f17ae50c991542.jpg"},{"id":90002515,"identity":"80ad313b-def3-47ed-b45f-3c32cb6cd5d3","added_by":"auto","created_at":"2025-08-27 09:06:39","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":93698,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7041039/v1/2cfc94aa75203a2865010b68.jpg"},{"id":96650406,"identity":"3ef9e9f6-0d59-4cc1-8e49-1ea22e39a0eb","added_by":"auto","created_at":"2025-11-24 16:12:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1870430,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7041039/v1/61728d56-f8c9-4d67-bf40-55c9b0d1703f.pdf"}],"financialInterests":"","formattedTitle":"Supplemental choline to prevent and treat learning and memory deficits of early-life iron deficiency (The SupCHO Study): study protocol for a randomized, placebo-controlled trial in Ugandan infants with iron deficiency anemia","fulltext":[{"header":"Administrative information","content":"\u003cp\u003eNote: the numbers in curly brackets in this protocol refer to SPIRIT checklist item numbers. The order of the items has been modified to group similar items (see http://www.equator-network.org/reporting-guidelines/spirit-2013-statement-defining-standard-protocol-items-for-clinical-trials/).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"639\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eTitle {1}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 421px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eSupplemental choline to prevent and treat learning and memory deficits of early-life iron deficiency (The SupCHO Study): study protocol for a randomized, placebo-controlled trial in Ugandan infants with iron deficiency anemia\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eTrial registration {2a and 2b}.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 421px;\"\u003e\n \u003cp\u003eClinical trials.gov#\u0026nbsp;NCT06527391; Registered 24 July 2024\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eProtocol version {3}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 421px;\"\u003e\n \u003cp\u003e2\u003csup\u003end\u003c/sup\u003e May 2025; Protocol version 1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eFunding {4}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 421px;\"\u003e\n \u003cp\u003eE.W. \u0026ldquo;AL\u0026rdquo; Thrasher Award, Thrasher Research Fund (Primary funder)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eUniversity of Minnesota Masonic Institute for the Developing Brain Interdisciplinary Faculty Research Award (Supplemental funder)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eAuthor details {5a}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 421px;\"\u003e\n \u003cp\u003eSarah E. Cusick, PhD, Department of Pediatrics, University of Minnesota\u003c/p\u003e\n \u003cp\u003eEzekiel Mupere MBChB, MMED, MS, PhD, Department of Paediatrics and Child Health, Makerere University\u003c/p\u003e\n \u003cp\u003ePaul Bangirana, PhD, Department of Psychiatry, Makerere University\u003c/p\u003e\n \u003cp\u003eReagan I. Baluku, Global Health Uganda\u003c/p\u003e\n \u003cp\u003eMaria Kroupina PhD, Department of Pediatrics, University of Minnesota\u003c/p\u003e\n \u003cp\u003eCarol Cheatham, PhD, Department of Psychology and Neuroscience, University of North Carolina at Chapel Hill\u003c/p\u003e\n \u003cp\u003eJeffrey Wozniak, PhD, Department of Psychiatry and Behavioral Science, University of Minnesota\u003c/p\u003e\n \u003cp\u003eMichael K. Georgieff MD, Department of Pediatrics, University of Minnesota\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eName and contact information for the trial sponsor {5b}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 421px;\"\u003e\n \u003cp\u003eThrasher Research Fund, Allison Martinez, tel +1 (801) 240-4753, email [email protected]\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eUniversity of Minnesota Masonic Institute for the Developing Brain, Hannah Shryer, tel +1 (612) 365-8400, email: [email protected]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 218px;\"\u003e\n \u003cp\u003eRole of sponsor {5c}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 421px;\"\u003e\n \u003cp\u003eStudy sponsors/funders have no role in study design; collection, management, analysis, and interpretation of data; writing of the report; or submission for publication and will have no authority over any of these activities.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Introduction","content":"\u003cp\u003e\u003cstrong\u003eBackground and rationale {6a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp id=\"_Toc130410035\"\u003eIron deficiency (ID) remains the most common nutritional deficiency in the world despite prevention and food fortification efforts\u0026nbsp;(1). In\u0026nbsp;regions\u0026nbsp;of\u0026nbsp;the\u0026nbsp;world\u0026nbsp;such\u0026nbsp;as\u0026nbsp;Uganda,\u0026nbsp;iron deficiency anemia (IDA)--the most severe stage of ID--affects\u0026nbsp;one in every four children under five years of age (2). Early-life ID is\u0026nbsp;a primary cause of\u0026nbsp;long-term\u0026nbsp;brain impairment because of cognitive deficits that persist for decades, despite diagnosis and iron\u0026nbsp;treatment (3–6).\u0026nbsp;Thus,\u0026nbsp;early-life\u0026nbsp;ID leads to unmet educational and job potential, thereby perpetuating an entrenched, transgenerational cycle of poverty and low societal productivity (7). The neurobehavioral pathology frequently occurs before a child is diagnosed as iron-deficient by hemoglobin because in all developing mammals including humans, the brain becomes iron-deficient before the red cells (8). In many low-income countries, including Uganda, there is no screening program for anemia, and ID is not diagnosed until clinical symptoms of severe ID anemia develop--well past the time that therapeutic iron can fully protect the brain (9)\u003cem\u003e.\u0026nbsp;\u003c/em\u003eBecause the global prevalence of IDA among children 6-59 months of age is approximately 30% (10), the need to treat the brain dysfunction caused by this early-life ID that is not fully remediable with iron therapy is urgent.\u003c/p\u003e\n\u003cp\u003ePreclinical\u0026nbsp;models\u0026nbsp;show\u0026nbsp;that\u0026nbsp;a\u0026nbsp;major\u0026nbsp;factor\u0026nbsp;contributing\u0026nbsp;to\u0026nbsp;long-term\u0026nbsp;deficits\u0026nbsp;is\u0026nbsp;the\u0026nbsp;timing\u0026nbsp;of\u0026nbsp;iron\u0026nbsp;repletion.\u0026nbsp;When repletion\u0026nbsp;does\u0026nbsp;not\u0026nbsp;precede\u0026nbsp;or\u0026nbsp;coincide\u0026nbsp;with\u0026nbsp;the\u0026nbsp;developing\u0026nbsp;brain’s\u0026nbsp;peak\u0026nbsp;need\u0026nbsp;for\u0026nbsp;iron,\u0026nbsp;the\u0026nbsp;brain\u0026nbsp;is\u0026nbsp;impacted\u0026nbsp;long term,\u0026nbsp;despite\u0026nbsp;subsequent\u0026nbsp;iron\u0026nbsp;repletion.\u0026nbsp;The\u0026nbsp;hippocampus\u0026nbsp;is\u0026nbsp;particularly\u0026nbsp;vulnerable\u0026nbsp;to\u0026nbsp;early\u0026nbsp;life\u0026nbsp;ID because of its high metabolic demand (11–14).\u0026nbsp;It\u0026nbsp;is\u0026nbsp;the\u0026nbsp;brain’s\u0026nbsp;major processor for recognition learning and memory. In humans, it grows and differentiates particularly rapidly in the last trimester of pregnancy through the first two years of life, encompassing two peak risk periods for ID--the neonatal period\u0026nbsp;and toddlerhood (9,15,16).\u0026nbsp;Iron is essential to support\u0026nbsp;the very high energy (ATP) neuronal energy demands of structural development of the hippocampus through its critical role in mitochondrial cytochrome function and ATP generation (11–14). Iron also regulates JARID histone demethylase activity of Brain Derived Neurotrophic Factor IV (BDNF-IV), a critical growth factor involved in neuronal growth and differentiation and synaptic plasticity (17–19).\u0026nbsp;Without\u0026nbsp;adequate\u0026nbsp;iron\u0026nbsp;to\u0026nbsp;support\u0026nbsp;this\u0026nbsp;rapid\u0026nbsp;growth\u0026nbsp;during\u0026nbsp;its\u0026nbsp;critical\u0026nbsp;growth\u0026nbsp;spurt,\u0026nbsp;permanent\u0026nbsp;structural\u0026nbsp;damage, characterized by simplified and abnormal dendritic arborization, occurs\u0026nbsp;to\u0026nbsp;not\u0026nbsp;only\u0026nbsp;the neurons in\u0026nbsp;the\u0026nbsp;hippocampus,\u0026nbsp;but\u0026nbsp;also\u0026nbsp;to\u0026nbsp;subsequent\u0026nbsp;developing\u0026nbsp;higher-order\u0026nbsp;structures\u0026nbsp;in\u0026nbsp;the\u0026nbsp;brain that depend on proper connections from the hippocampus during development (20,21). These include frontal lobe structures supporting attention and behavior. Because this damage frequently occurs before a child is diagnosed as iron-deficient by hemoglobin, iron therapy is often started too late to fully protect the brain (9).\u003c/p\u003e\n\u003cp\u003eDevelopmentally informed, timed supplementation with the essential nutrient choline as an adjunct therapy to iron in children with\u0026nbsp;IDA\u0026nbsp;may be part of the solution to protect the brain. We have shown\u0026nbsp;that compared to iron alone, choline given with iron to iron-deficient rat pups during critical periods of hippocampal development improves hippocampus-dependent learning and memory function across the lifespan(22,23). Mechanistically, choline counteracts early-life ID’s long-term harmful effects on the hippocampus by reversing negative epigenetic marks in synaptic plasticity genes, improving energy efficiency in neurons, and stabilizing neuronal structure (11,24,25). Timed choline is effective in\u0026nbsp;models of other conditions characterized by hippocampal damage (26–28), including fetal alcohol spectrum disorders (FASD), where brain ID is part of the neuropathology (29–32).\u003c/p\u003e\n\u003cp\u003eCholine’s positive effects on hippocampal function seen in preclinical models of FASD has\u0026nbsp;been\u0026nbsp;successfully\u0026nbsp;translated\u0026nbsp;to\u0026nbsp;young\u0026nbsp;children. We recently led the first clinical trial of choline supplementation in young children with FASD(33,34)and found that nine months of daily choline supplementation as 500 mg choline bitartrate in a fruit-flavored drink was feasible for parents to administer and was well-tolerated with no serious adverse events. Choline improved memory performance in 2-3-year-old children more so than in 4-5-year-old children, as measured by the Elicited Imitation (EI) task (a hippocampus-dependent recognition memory task), suggesting a critical window early in postnatal development (34). Follow-up studies after four years showed that choline improved memory function, non-verbal intelligence, behavioral symptoms of Attention Deficit Hyperactivity Disorder, and visual-spatial skills (35). After seven years, choline-supplemented children had better of executive functioning and speed of processing, with better white matter organization on MRI scan (36). These circuit- and structure-specific improvements observed in this study and subsequent similar studies guide the selection of the neurobehavioral (NB) tests used in this protocol, which include those that assess hippocampal function (working memory), behavior, and attention.\u003c/p\u003e\n\u003cp\u003eCholine supplementation has not been tested in children with IDA, yet it may be a low-cost adjunct therapy to iron that could be used immediately. To fill this gap, we propose a randomized, placebo-controlled trial of choline in children with IDA living in Uganda. We will shift enrollment age earlier than in the FASD study to age 6 months to maximize NB impact by capturing more of the epoch of rapid hippocampal growth and differentiation. This trial is significant because of the high prevalence of early-life ID globally, its long-term individual and intergenerational consequences, and the possibility that an inexpensive and immediately available nutritional intervention may lessen those consequences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjectives {7}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur \u003cstrong\u003eprimary objective\u003c/strong\u003e is to conduct a randomized, placebo-controlled clinical trial\u0026nbsp;to test whether nine months of daily supplementation with the essential nutrient choline along with standard iron therapy improves hippocampus-dependent neurobehavioral outcomes in 6-month-old infants with iron deficiency anemia. We \u003cstrong\u003ehypothesize\u003c/strong\u003e that infants with iron deficiency anemia who receive iron and choline will have better scores on specific neurobehavioral tests of recognition memory than infants who receive iron and placebo.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrial design {8}\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Supplemental Choline to Prevent and Treat Learning and Memory Deficits of Early Iron Deficiency (SupCHO) study is a randomized, double-blinded, placebo-controlled, superiority clinical trial testing if 6-month-old infants with IDA who receive iron with choline have better scores on NB tests of hippocampal and global function at 15 months of age compared to infants who receive iron with placebo. The study will be conducted using a 1:1 allocation ratio, two arms, with parallel groups. We will recruit and enroll from immunization clinics at Mulago National Referral Hospital and Kawempe National Referral Hospital, both in Kampala, Uganda. Study randomization and baseline and follow-up assessments will be based at Mulago Hospital. The SupCHO protocol adheres to the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines and was registered at www.clinicaltrials.gov (NCT06527391).\u003c/p\u003e"},{"header":"Methods: Participants, interventions and outcomes","content":"\u003cp\u003e\u003cstrong\u003eStudy setting {9}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChildren will be enrolled from the immunization clinics at Mulago National Referral Hospital and Kawempe National Referral Hospital in Kampala, Uganda. All follow-up visits will take place at Mulago National Referral Hospital.\u0026nbsp;These hospitals are approximately 3.5 km apart and are both staffed by study medical officers and co-investigators.\u0026nbsp;Uganda is the only country where data will be collected.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEligibility criteria {10}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy-specific, protocol-trained medical officers and nurses will conduct the assessments required to determine eligibility according to the inclusion and exclusion criteria:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInclusion\u0026nbsp;criteria:\u0026nbsp;\u003c/strong\u003e1) Age 6 months +/- 28 days; 2) Hemoglobin (Hb) 7 - \u0026lt; 11.0 g/dL; 3) Zinc protoporphyrin (ZPP) \u0026gt; = 80; 4) T\u0026lt;37.5\u0026deg;C; 5) Malaria-negative based on Rapid Diagnostic Test (RDT); 6) Mother is HIV-negative by HIV RDT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExclusion criteria:\u003c/strong\u003e 1) Developmental disorder; 2) Severe malnutrition (severe wasting or bipedal edema); 3) Known sickle cell disease; 4) Neurologic disorder, brain injury, or other condition affecting brain development; 5) Not currently breastfeeding; 6) Birthweight \u0026lt; 2000 g.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWho will take informed consent? {26a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe screening consent process will take place in a private, partitioned-off space near the immunization clinic at Kawempe National Referral Hospital or Mulago National Referral Hospital, depending on where the child presents. All study nurses and medical officers conducting the screening are trained in Good Clinic Practice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eChildren found to be eligible for the full study after screening will be enrolled in the study office in either Kawempe\u0026nbsp;National Referral Hospital\u0026nbsp;or Mulago\u0026nbsp;National Referral Hospital\u0026nbsp;in a study room that is separated by the rest of the unit by a closed door. Once the medical officer/nurse determines that a potential participant meets all the inclusion criteria and has none of the exclusion criteria, he or she will go through the consent process for the full study with the caregiver.\u0026nbsp;Both the screening and full-study consent forms will be available in English and Luganda, the local language.\u003c/p\u003e\n\u003cp\u003eThe medical officer or nurse will read through the entire consent forms with the caregiver of the potential participant child. All questions will be answered completely. At the end of the full-study consent discussion, the parent/guardian will demonstrate understanding by answering 10 comprehension questions. If the caregiver answers fewer than eight questions correctly, the informed consent process will be repeated until at least eight questions can be answered correctly.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIf the caregiver successfully completes the questionnaire and agrees for the child to participate in the study, he/she will sign the consent form or provide a thumb print if he or she cannot sign his or her name. An impartial witness will also sign the form. The impartial witness will be present during the entire consent discussion if the participant\u0026rsquo;s caregiver is unable to read.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional consent provisions for collection and use of participant data and biological specimens {26b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn addition to the consent forms for screening and enrollment in the full study, we have separate consent forms for storage and future use of biological specimens and a data-use agreement. All consent forms are available in English and Luganda, and the informed consent process will be conducted in whichever of the two languages the caregiver prefers.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInterventions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExplanation for the choice of comparators {6b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll children will receive three months of liquid oral ferrous sulfate (2 mg/kg/day) as treatment for iron-deficiency anemia. Additionally, each child will be randomized to 200 mg of choline bitartrate or identical placebo tablet as a liquid-dispersible tablet. The benefit of choline for brain development in young children has not been established, nor has a potential role for choline as an adjunct therapy with iron to treat iron deficiency anemia. Thus, the comparator for choline is a placebo tablet.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntervention description {11a}\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRandomization and study supplement:\u003c/strong\u003e On Day 0, children will be randomized to nine months of daily choline bitartrate or identical placebo tablet as a 200 mg dispersible tablet all manufactured under GMP by Toronto Institute of Pharmaceutical Technology. Treatment assignment (choline or placebo) will be made in a 1:1 ratio by permuted randomization in random block lengths. Investigators, field staff, and participants will be blinded to treatment assignment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplement instruction:\u003c/strong\u003e For the choline/placebo tablets, caregivers will be instructed to put the tablet in a small amount (60 mL or \u0026frac14; C) of liquid, e.g., breastmilk, water, or juice, to be dispersed. An initial one-month (28 days) supply of the study tablets will be sent home with each mother at enrollment along with a dosage calendar where days can be ticked off as well as written instructions for use. The supplements and calendar will be replenished each month at a home visit. Home visitors will check the preceding month\u0026rsquo;s calendar and verify adherence by pill counts. Iron syrup will be given the first three months of follow-up, while choline and placebo tablets will be given for all nine months of follow-up.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCriteria for discontinuing or modifying allocated interventions {11b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCholine is an essential nutrient and occurs naturally in foods (37). Choline is found in abundance in an animal-based diet, but many Ugandans, especially if iron-deficient, may not consume sufficient choline.\u0026nbsp;Choline can cause vomiting, heavy sweating and salivation, low blood pressure, and liver damage when given in high doses (\u0026gt;1000 mg)\u0026nbsp;(37\u0026ndash;39), but there are no known adverse reactions to the nutrient supplement at the dose given in this trial. Children 2-3 y with FASD who consumed 500 mg of choline bitartrate daily for nine months had a greater incidence of fishy body odor, but this odor did not lead any of the children to discontinue the supplement\u0026nbsp;(33, 34). We will actively monitor all physical symptoms at home visits and phone calls, tallying the symptoms weekly. If at any time the study pediatrician deems it medically necessary for a child, the supplement will be stopped. The supplement may also be stopped at any time by the caregiver. We will track adherence with pill counts during home visits and provide counsel as needed, but a decision to stop the supplement by the caregiver will not preclude the child from participation in study follow-up. Tablet doses and allocations will not be modified over the course of the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrategies to improve adherence to interventions {11c}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll caretakers will be given a calendar with nine 28-day months on which to tick off each daily dose of the study supplement. Starting with the Day 0, continuing monthly at the home visits, and ending at the Month 9 clinic visit, we will take a printable color photo of the child at each age that can be pasted into the calendar, with the nine months of the study collectively showing the child\u0026rsquo;s monthly development from age 6 \u0026ndash; 15 months. The calendar also has the World Health Organization (WHO) height and weight charts, and these growth measures will be tracked at each home visit, so that there is also a visual depiction of the child\u0026rsquo;s growth.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition to talking to the caretaker and examining the dosing calendar, we will count pills remaining in the supplement bottle and compare it to the number of pills expected at each home visit. Percent adherence for the month will be calculated as (doses taken/doses prescribed) * 100. Counsel will be given as needed to address any concerns with the supplement.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRelevant concomitant care permitted or prohibited during the trial {11d}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll children will receive a three-month course of oral iron therapy. Any child found to be anemic during the finger-prick hemoglobin check at the month 6 (age 12 months) follow-up home visit will be given an additional three-month course of iron. Any medical care needed by the child while enrolled in the study will be permitted, and no medically prescribed care will be prohibited.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Provisions for post-trial care {30}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no provisions for post-trial care. Each participant is covered by Sanlam Insurance while enrolled in the trial, with provision for up to $10,000 for any research-related injury in accordance with applicable Ugandan laws.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOutcomes {12}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePrimary\u0026nbsp;outcome\u003c/em\u003e: Elicited Imitation outcomes (number of steps correctly recalled and number of steps recalled in the correct order for both immediate and delayed recall) at age 15 months, i.e., after nine months of daily supplementation with choline or placebo. For each of these outcomes, we will compare groups using a one-way analysis of covariance (ANCOVA).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSecondary\u0026nbsp;outcomes.\u003c/em\u003e Mean composite Z-score on Mullen, scores on each scale of the BRS, and proportion of time the child views an animation viewing across a computer screen on the Early Childhood Vigilance Test at age 15 months in the choline vs. placebo groups.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003cstrong\u003eParticipant timeline {13}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll 300 participants will be enrolled at age 6 months, will be given a 3-month course of oral ferrous sulfate (2 mg/kg/day), and will be randomized to receive a dispersible tablet of choline (200 mg as choline bitartrate) or placebo daily for nine months of follow-up, i.e., until 15 months of age (\u003cstrong\u003eFigure 1\u003c/strong\u003e). We will conduct biweekly surveillance via home visits and phone calls for adherence and illness throughout the nine-month-follow-up period and will compare neurobehavioral test scores between the choline and placebo groups at age 15 months \u003cstrong\u003e(Figure 2).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePre-Screening\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe will identify potentially eligible participants during their routine visits at age 14 weeks and 6 months to the immunization clinics at Kawempe National Referral Hospital\u0026nbsp;or Mulago National Referral Hospital. These hospitals are approximately 3.5 km apart and are both staffed by study medical officers and co-investigators.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e14-week visit (most children will likely be identified at this visit):\u003c/em\u003e Study staff will identify age-eligible children who attend the clinic for their 14-week routine immunizations. The caregiver of the child will be told about the study and invited to bring the child for study screening during their 6-month routine visit. The caregiver will be reminded that the biological mother must be present at screening at 6 months.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e6-month visit:\u003c/em\u003e Age-eligible children who were not identified at their 14-week visit can be screened directly at their 6-month routine immunization visit if they are with their biological mother. In this case, study staff will ask the mother if she is interested in participating, and if yes, she will be invited for screening that very day. The biological mother must be present at screening because she must be screened for HIV by RDT as part of the inclusion criteria.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy screening (at 6-month visit):\u0026nbsp;\u003c/strong\u003eAll official screening will be at the child\u0026rsquo;s 6-month visit and will take place at Mulago National Referral Hospital or Kawempe National Referral Hospital, depending on where they were first identified. At this visit, mothers of age-eligible, afebrile infants will provide written screening consent for a finger-prick blood sample from their child and themself. The child\u0026rsquo;s blood sample will be used to measure zinc protoporphyrin (ZPP), hemoglobin, and for a malaria Rapid Diagnostic Test (RDT). The maternal sample will be used for an HIV RDT test. If the child meets the inclusion criteria below and has none of the exclusion criteria, he or she is eligible for enrollment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiagnosis of IDA by two point-of-care tests:\u003c/strong\u003e We will measure hemoglobin (Hb) by HemoCue and ZPP by hematofluorometer to diagnose IDA. Per WHO recommendations, anemia will be defined as Hb \u0026lt; 11.0 g/dL in children 6 months of age (40). ZPP is a hemoglobin precursor that accumulates in the developing red blood cells when iron is not available to synthesize hemoglobin (41,42). Thus, a high ZPP (\u0026ge; 80\u0026micro;mol/mol heme) reflects iron-deficient erythropoiesis. We will have front-face hematofluorometers onsite at both Kawempe National Referral Hospital and Mulago National Referral Hospital (Protofluor-Z, Helena Biosciences) to measure ZPP in real time. If a child has a hemoglobin concentration of \u0026lt; 7.0 g/dL, h fetal alcohol spectrum disorders he/she is not eligible for the current study and will be referred for treatment at the pediatric clinic at Mulago National Referral Hospital or Kawempe National Referral Hospital.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnrollment:\u0026nbsp;\u003c/strong\u003eOnce a child meets all inclusion criteria, he/she can be enrolled. Informed consent for the full study will take place on the day of screening and thus can be at either Mulago National Referral Hospital or Kawempe National Referral Hospital. After consent and enrollment, the child will be scheduled for a neuropsychological testing/Randomization Day (Day 0) at Mulago National Referral Hospital within 10 days of screening/enrollment. Participants are informed on the full consent form that if for any reason they are not able to come for the Day 0 visit within 10 days, they cannot remain in the study. All children will be started on iron on the day of enrollment. For the iron syrup, the study pharmacist will instruct each mother how to draw up the correct amount of iron syrup (ferrous sulfate, 2/mg/kg-day) in a medication syringe.\u003c/p\u003e\n\u003cp\u003eAny child found to be ill at screening will be referred immediately to the Mulago Paediatric Assessment Center or to the Kawempe Outpatient Department, depending on the screening site, for diagnosis and treatment. Emergencies will be referred to the Acute Care Unit at each hospital.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDay 0 Testing/Randomization Day (all at Mulago National Referral Hospital):\u003c/strong\u003e Each enrolled child\u0026rsquo;s official Day 0 will take place at Mulago National Referral Hospital. On this day, they will have baseline neuropsychological testing (see below), a physical exam, dietary assessment, and a venous blood draw for measurement of iron status biomarkers. At the end of this visit, the child will be given a blinded randomized treatment assignment for choline or placebo (Figure 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDietary Assessment:\u003c/strong\u003e We will assess dietary consumption of choline on Day 0 and at the end of the follow-up period to verify that this consumption does not differ between treatment groups. Trained study staff will administer a 24-hour food recall developed specifically for the Ugandan diet (43) to each mother to answer for their child at enrollment and at the follow-up visit when the child is 15 months of age. Estimates of portion size will be made using standard Ugandan plates, cups, and spoons. We will calculate the choline content of the foods using the USDA database (44).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNeurobehavioral\u0026nbsp;testing:\u0026nbsp;\u003c/strong\u003eEnrolled children will be evaluated by hippocampus-specific and general behavioral tests at Day 0 (6 months of age) and after nine months of daily supplementation, at 15 months of age (45\u0026ndash;48). Measurements at enrollment will be used to characterize overall function of the groups pre-intervention, while the study outcome measures will be NB test scores at 15 months of age. \u003cem\u003eTask descriptions\u0026nbsp;\u003c/em\u003e 1) \u003cu\u003eElicited Imitation (EI)\u003c/u\u003e: This test provides a comprehensive understanding of hippocampal function by interleaving components of immediate and delayed recall to assess working memory, a more advanced hippocampus-dependent task (49\u0026ndash;52). At age 15 months, children will be presented with multi-piece props and are able to manipulate them to provide a baseline measure of spontaneous occurrences of the target behaviors. After this period, a tester will model a multi-step sequence using the props. The sequence will be modeled twice with narration. Infants will be allowed to model the sequence immediately (immediate recall, a measure of encoding) and after 15 minutes (delayed recall, a measure of memory). All sessions will be recorded and coded by trained observers. To ensure reliability, 20% of videos will be coded by a second trained observer. Performance will be scored as the number of target actions recalled and number of actions produced in the correct order for both immediate and delayed conditions. We have extensively used and published on this paradigm to examine hippocampal function in infants with prenatal metabolic and nutritional deficiencies (34,53). Ongoing quality control for EI scoring will occur via biweekly Zoom calls for the duration of the trial, with the EI trainer independently scoring 20% of the videos. EI is not a reliable measure of hippocampal function at age 6 months, but we will capture baseline hippocampal function in the global cognition tests that we will perform(50,54). 2) \u003cu\u003eThe Mullen Scales of Early Learning (MSEL):\u003c/u\u003e MSEL is a global cognition test, measuring the domains of gross motor, fine motor, visual reception, receptive language, and expressive language (55). We have used the Mullen multiple times in our studies in Uganda (45,46). 3) \u003cu\u003eThe Behavior Rating Scales\u003c/u\u003e developed by Lozoff (56) for use in studying ID will be coded from videotaped Mullen testing sessions. These scales have three composite scores that assess behaviors (fearfulness, impulsivity, and low positive affect) secondary to the monoaminergic effects of ID. Two testers will observe each testing session. One will administer the Mullen, while the other will observe behavior. At the end of the session, both testers will complete a BRS scoresheet and compare scores for quality control. Approximately one-fifth of videos from all sessions will be coded independently by a Ugandan master coder to ensure reliability. 4) The Early Childhood Vigilance Test (ECVT) is a computerized test to evaluate attention that was modeled after Ruff\u0026rsquo;s puppet vigilance paradigm (57). The total time required for all tests is approximately 1.5-2 hours. Rest and snack breaks will be given as needed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVenipuncture:\u0026nbsp;\u003c/strong\u003eAfter neurobehavioral testing, we will collect 3-5 mL of whole blood from each child. We will use whole blood for CBC. The remaining blood will be centrifuged, and plasma/RBC pellet will be collected, aliquoted, and frozen onsite at -80\u0026deg;C for analysis of plasma iron and inflammatory biomarkers (onsite by ELISA assay).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFollow-up Clinic Visit at age 15 months (+/- 2 weeks)\u003c/strong\u003e\u003cem\u003e:\u003c/em\u003e All children will have a scheduled follow-up clinic visit after nine months of follow up, i.e., at 15 months of age. This follow-up visit will include neurobehavioral tests, a venous blood draw of 3-5 mL, and a 24-hour food recall as at the enrollment visit.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurveillance and Follow-up:\u0026nbsp;\u003c/strong\u003e\u003cem\u003eHome visits:\u003c/em\u003eAll enrolled children will receive an insecticide-treated bednet. For the next nine months of follow-up, we will have alternating biweekly (every two weeks +/- 3 days) home visits and phone calls for each enrolled study child to assess adherence to the study tablets (tablet counts for entire nine-month follow-up period) and iron syrup (volume check, for first three months of follow-up) and to inquire about any illnesses or side effects. Any ill child with history of fever, persistent diarrhea or vomiting, lethargy, or anorexia, will be transported to the clinic for diagnosis and treatment. Mothers will also be instructed to call the study clinic any time their child is ill and will be given transportation refund to attend the clinic for treatment. The home visitor will assess the home environment using the Infant-toddler version of the HOME Inventory (The Home Observation for the Measurement of the Environment Inventory) form at the first visit, and\u0026nbsp;at\u0026nbsp;each home visit, dietary strategies to prevent future ID through increased consumption of locally available food that is high in bioavailable iron will be presented using local materials and methods. Hemoglobin will be measured by finger-prick blood sample using HemoCue at the home visit at 6 months follow-up (12 months of age). Any child with hemoglobin \u0026lt; 11.0 g/dL will receive an additional 3-month course of iron therapy. Receipt of a second course of iron therapy will be controlled for in final analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample size {14}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is powered for the primary\u0026nbsp;outcome, Elicited imitation scores (continuous) at 15 months of age in the choline vs. placebo group. Our sample size gives a detectable difference (80% power) in a t-test of Cohen\u0026apos;s d =\u0026nbsp;0.3, i.e., roughly one-third a SD unit, where the SD describes variation of the\u0026nbsp;outcome\u0026nbsp;within\u0026nbsp;group. Allowing\u0026nbsp;15%\u0026nbsp;lost\u0026nbsp;to\u0026nbsp;follow-up\u0026nbsp;gives\u0026nbsp;150 children\u0026nbsp;per\u0026nbsp;group\u0026nbsp;(choline\u0026nbsp;and\u0026nbsp;placebo), 300 children total.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRecruitment {15}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe will identify eligible children approximately 2.5 months prior to enrollment at their 14-week visit to the immunization clinic when they receive their polio and pneumococcal conjugate immunizations (58). The Immunization Clinics at Mulago and Kawempe National Referral Hospitals are among the largest in the country. Approximately 140 children attend the clinics each month for their 14-week visit (E. Mupere, personal communication). Since roughly one in four Ugandan children has IDA, we anticipate enrolling the 300 children with IDA comfortably in 12-18 months, with a target of 4-5 children enrolled each week.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssignment of interventions: allocation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequence generation {16a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tablet manufacturer, Toronto Institute of Pharmaceutical Technology (TIPT), will generate the allocation sequence (1:1; choline:placebo) by permuted randomization in random block lengths using 3-digit numbers, ranging from 101 - 400. These 3-digit numbers will correspond to participants\u0026rsquo; study ids. Each study id (linked to a treatment assignment) will be printed directly onto nine supplement (tablet) bottles, representing one bottle for each of the nine months of supplementation. The link between treatment assignment and study id will be kept at TIPT only. Study staff, investigators, and caregivers will all be blinded to treatment assignment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConcealment mechanism {16b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy id numbers that are linked to the randomly assigned treatment assignment will be printed directly onto supplement bottles. Each id will be printed onto nine separate bottles of study tablet, along with the bottle number, e.g., Study id 101, bottle 1; Study id 101, bottle 2, up to Study id bottle 9. Study staff on the ground will only give out the regimens according to sequential study id number and will not know or have access to the link between the id number and treatment assignment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImplementation {16c}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe allocation sequence will be generated by TIPT, the tablet manufacturer. Participants will be enrolled by study nurses and pediatricians at Mulago and Kawempe National Referral Hospitals. Assignment of study id\u0026rsquo;s will be done by the study dispenser/pharmacist, thus assigning each participant a blinded treatment assignment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssignment of interventions: Blinding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWho will be blinded {17a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTrial participants, care providers, outcome assessors, data analysts, and investigators will all be blinded to treatment assignment until all outcomes for the trial, including plasma biomarkers, have been collected, all trial data have been entered into the study database and verified, and the final analytical data set is generated and locked.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;Procedure for unblinding if needed {17b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBecause choline is an essential nutrient, and we are giving it at a level well below the upper threshold for recommended intake (37), we do not foresee a circumstance where unblinding will be necessary. However, if at any time unblinding is deemed medically necessary by a study physician, the study pharmacist will contact TIPT directly for the unblinding of that single study id.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData collection and management\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlans for assessment and collection of outcomes {18a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be entered directly into the study\u0026rsquo;s RedCap database via tablet. All data will be reviewed by a second study staff member before the data is released to the database. Paper copies of all case report forms (CRF\u0026rsquo;s) will also be available in case of tablet failure. All paper case report forms will be signed by the person filling the form and also by a second person who will verify that the information on the form is correct and complete. The study data manager will generate a data query log each week, with corrections and quality control made immediately. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNeurobehavioral assessment: All neurobehavioral sessions will be video recorded. All assessments will be double scored by two Uganda-based neuropsychological testers. Additionally, 20% of videos will be scored/coded by master trainers for the Behavioral Rating Scale and the Elicited Imitation Task, to help ensure ongoing quality control and scoring consistency.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePaper copies of all study CRF\u0026rsquo;s were approved by and on-file with the Mulago Hospital Research Ethics Committee (MREC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlans to promote participant retention and complete follow-up {18b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll analysis will be intention-to-treat. We have biweekly contact with each participant via alternating physical home visits and phone calls to maintain connection and enthusiasm in the study. Ongoing access to health services and monthly growth monitoring help ensure retention and adherence. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData management {19}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAside from neuropsychological testing coding sheets, all study data will be entered directly into a RedCap database via direct data entry. Verification by a second data entrant is required for the data to be submitted to the database. The study data manager runs data quality reports each week, with any errors addressed immediately. The database will include health data, including blood markers for iron infection, physical examination findings, neurobehavioral findings, and information, e.g., diagnoses, physical findings, from sick visits to the health clinic. All variables have biologically plausible range checks built in to alert the entrant of a potential error. All data management procedures are codified in Standard Operating Procedure documents, available in hardcopy in the study office or upon request from the study principal investigators.\u0026nbsp;Databases are kept on password-protected computers accessible only to authorized study project personnel and will be uploaded nightly onto UMN BOX, which requires duo authentication. Hard copies of any forms will be kept in the study data room in patient documents kept in locked filing cabinets. Final database files will be kept in the data room in the project offices in UMN computers and on a back-up drive. Data files will be kept on UMN BOX and accessible only by designated individuals with permission from the PI. A copy of the consent forms will be kept in the participant\u0026rsquo;s study file, but will not be included in medical, employment, or educational records.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConfidentiality {27}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo personal identification information such as names, address, or phone numbers will be included in the study database. All information in the database will be linked by the three-digit study id. Thus, only deidentified data are included in the database, and no pre-existing medical records will be accessed for study purposes, aside from a child\u0026rsquo;s vaccination card if brought to the hospital by the caretaker and shown to study personnel. \u0026nbsp;We additionally have a data use sharing agreement as a separate consent form that explains that de-identified data is collected as part of the study and may be shared electronically within the study team.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlans for collection, laboratory evaluation and storage of biological specimens for genetic or molecular analysis in this trial/future use {33}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll blood samples collected as part of the study will be labeled with only 3-digit study id and laboratory accession number for storage. Any plasma that remains after iron and inflammatory marker testing will be stored for up to ten years at -80 degrees C in the Paediatrics Laboratory at Mulago National Referral Hospital, Kampala. Permission to bank blood for future use will be included as a consent form. Personal, identifying information will not be kept in the study database. Permission will be required by the U.S. and Ugandan PI to access any banked specimen.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical methods for primary and secondary outcomes {20a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimary analyses will be intention-to-treat.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePrimary\u0026nbsp;outcome\u003c/em\u003e: Elicited Imitation outcomes (number of steps correctly recalled and number of steps recalled in the correct order for both immediate and delayed recall) at age 15 months, i.e., after nine months of daily supplementation with choline or placebo. For each of these outcomes, we will compare groups using a one-way analysis of covariance (ANCOVA). \u003cem\u003eSecondary outcomes.\u003c/em\u003e For each secondary outcome (Composite Z-score on Mullen, scores on each scale of the BRS, and proportion of time the child views an animation viewing across a computer screen on the Early Childhood Vigilance Test), we will do the same analyses as for the primary outcome, using the baseline value of each test as a covariate to absorb variation in the outcome and thus increase power. Adjusted analysis will examine the effect of covariates including sex, age, iron status, CRP concentration, and home environment. Any covariate that appears to be a confounder will be included in the final analysis. While primary analyses will be intention-to-treat, we will also conduct per protocol analyses to include only children with more than 90% adherence to the study supplement to assess any difference in effect size. We will also conduct analyses with and without children with CRP \u0026gt; 10 mg/L. If we find a difference in the relationship between choline and neurobehavioral outcomes that is modified by having a CRP \u0026gt; 10 mg/L, we will exclude children in this group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInterim analyses {21b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study has a five-member Data and Safety Monitoring Board that will meet at the start of enrollment and every six months thereafter for the duration of the study. Once half (n=150) of the full sample size has completed follow-up, official interim analysis will occur. The interim meeting will consist of an open and closed session. In the open session, matters of study procedure, including supplement compliance, and data quality will be discussed. This open session will be open to all members of the monitoring board, study investigators, and any other interested parties. Only DSMB members will attend the closed session. At this session, randomization will be broken, and morbidity/side effect and outcome data will be presented by treatment group. After each meeting, the DSMB will prepare a report for investigators that includes recommendations for any amendments to study protocol. Copies of the reports will be submitted to the Mulago Hospital Research Ethics Committee (MREC), Uganda National Council of Science and Technology (UNCST), and the Uganda National Drug Authority (NDA). If at any time the DSMB recommends study termination (see stopping rules, below), a full vote of the DSMB is required, with majority vote (3 of 5) needed to enact the proposed change. The DSMB chairperson may convene ad hoc DSMB meetings any time he/she feels that discussion is required to make important decisions regarding revision of study procedures or suspending study activities.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStopping rules\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe stopping rules for this trial will incorporate stopping rules to \u003cem\u003emonitor serious adverse events\u003c/em\u003e and stopping rules to \u003cem\u003edetermine early success\u003c/em\u003e. The DSMB may also stop the study based on futility analysis.\u003c/p\u003e\n\u003cp\u003eInterim analyses will use the group-sequential testing method of Lan and DeMets, with the O\u0026rsquo;Brien-Fleming alpha-spending function. The study team proposes an interim analysis, after half of the participants have completed follow up, but the DSMB will determine how many interim analyses it will examine and when during follow up they will be done. The LanDeMets method is flexible enough to accommodate that. For determining whether to continue or terminate the study, the DSMB may also request computation of conditional power using the method of stochastic curtailment (also known as futility analysis).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSafety review if study is stopped\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIf the study is stopped for any reason, a full safety review will be completed by the study team and the DSMB. The RECs of both Mulago National Referral Hospital and the University of Minnesota must be informed in the event of study stopping, as well as NDA and UNCST. The study will restart only upon completion of the safety review by the DSMB and DSMB approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods for additional analyses (e.g. subgroup analyses) {20b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAddressed in statistical methods for primary and secondary outcomes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhile primary analyses will be intention-to-treat, we will also conduct per protocol analyses to include only children with more than 90% adherence to the study supplement to assess any difference in effect size. We will also conduct analyses with and without children with CRP \u0026gt; 10 mg/L. If we find a difference in the relationship between choline and neurobehavioral outcomes that is modified by having a CRP \u0026gt; 10 mg/L, we will exclude children in this group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlans to give access to the full protocol, participant level-data and statistical code {31c}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccess to the full protocol, de-identified dataset, and statistical code will be considered by the PIs upon receipt of a written request describing the intended use of information.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOversight and monitoring\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComposition of the coordinating centre and trial steering committee {5d}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe day-to-day operations of the SupCHO study are overseen by US-based and a Uganda-based PIs. The field team includes a study-specific pediatrician, three nurses (one who serves as the home visitor), two neuropsychological testers, a pharmacist, a dispenser, two data entrants, a data manager, and a laboratory technician. We also will employ some staff, including immunization clinic nurses and HIV-testing counselors on a locum basis. The field team meets every other week with the US-based PI via Zoom. Minutes for all meetings are kept securely online and in the study regulatory binder.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComposition of the data monitoring committee, its role and reporting structure {21a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA Data and Safety Monitoring Board (DSMB) has been established to provide external, objective advice regarding the safety and efficacy of the administered interventions. The Board is comprised of five members: an expert in clinical trials/biostatistics, an expert in global pediatrics/child health, an expert in choline, an expert in child development, and one non-medical scientist. Four board members are Ugandan. The Board will be formed to ensure the safety of study participants and the validity of collected data though review of subject recruitment, retention and follow-up, data forms, protocol adherence, and data quality. The committee will meet with investigators shortly after the start of the study (a month into the study) to review the protocol, DSMB charter, proposed interim analysis tables, and any new pertinent data and literature not included in the original protocol. Members will meet every six months for the duration of the study, including an interim analysis meeting once half of the total sample size has completed follow-up. Board members are independent from the sponsor and have signed conflict of interest forms and confidentiality agreements. \u0026nbsp;The DSMB charter has been submitted to and approved by the Uganda National Drug Authority and is available upon request to the study PI.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdverse event reporting and harms {22}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAdverse events and Serious adverse events\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAn adverse event (AE) is any untoward medical occurrence in a subject undergoing a study-related procedure or consuming a study-related supplement that is believed reasonably to be caused by that procedure or supplement. The only procedure performed in this study is blood sampling by venipuncture or finger prick collection. \u003cem\u003eAdverse events associated with this procedure could include prolonged bleeding, persistent pain, development of infection, and development of a large blood collection in the skin (hematoma)\u003c/em\u003e. We anticipate that such AEs from this study procedure will be infrequent (\u0026lt;2%).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe only known potential adverse event of choline is fishy body odor which has been reported with much higher doses.\u003c/em\u003e We will assess the frequency of this event as well as all other illnesses or physical symptoms through parental reports in monthly home visits and will discontinue therapy if discomfort persists. Home-visit forms will be returned to the study office each day, and the medical officer will monitor the physical symptoms/illnesses reported on this form. Tallied lists of physical symptoms will be submitted to the DSMB for the biannual meetings.\u003c/p\u003e\n\u003cp\u003eA serious adverse event (SAE) is any event regardless of relationship to the intervention that: 1) Results in death; 2) Is life-threatening; 3) Requires hospitalization or prolongation of existing hospitalization; 4) is an important medical condition in the opinion of the investigator and in relation to our intervention\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc53202851\"\u003eIn the event of an SAE, the participant will be managed accordingly. In case of a life-threatening event our team will promptly attend to the patient in the Paediatric emergency unit (Acute Care Unit) giving them the appropriate first aid. It is well equipped with\u0026nbsp;airway and cardiorespiratory resuscitation equipment and an\u0026nbsp;Intensive Care Unit with life support equipment and well-trained staff. If a participant develops an important medical condition, we will attend to them still in the hospital with our teams based in Mulago\u0026nbsp;National Referral Hospital. An SAE form will be filled out by the attending physician, and the site PI (Dr. Mupere) will be notified within 48 hours. Dr. Cusick (PI) will be notified in no more than 7 days, and she will relay the SAE to the DSMB chairperson within 15 calendar days of the event. All SAEs will be reported to the ethical and regulatory bodies in accordance with their institutional policies. Any unanticipated problem that results in unexpected risk to subject participants and requires changes in the study protocol will be immediately report by the PI to both the Ugandan and US IRBs/RECs.A five-member Data and Safety Monitoring Board, as described above, will be responsible for overseeing the overall safety of trial participants and ensuring adherence to study protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFrequency and plans for auditing trial conduct {23}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn independent study monitor will audit the trial annually. Each audit will include a 3-4-day visit to the study site, all study rooms, evaluation of completeness of the regulatory binder, study SOP\u0026rsquo;s, and study database integrity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlans for communicating important protocol amendments to relevant parties (e.g. trial participants, ethical committees) {25}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll protocol amendments will be submitted to MREC and the University of Minnesota IRB for approval. \u0026nbsp;Once MREC approval for the change is given, we will notify UNCST and NDA. The requested amendment will not be carried out until full approval for the amendment is received. If the requested amendment affects the wellbeing of participants, caretakers will be informed of the change and the reason for the change immediately.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDissemination plans {31a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTrial results will be published in a peer-reviewed manuscript. There are no publication restrictions. Depending on their significance, findings may also be presented to Ministry of Health officials or other policy makers. Study findings that are available immediately, e.g., participant height and weight, temperature, HemoCue hemoglobin value, malaria rapid test results, will be communicated to the caretaker immediately.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe are not measuring polymorphisms in choline-related genes which may affect endogenous synthesis and/or transport of choline. Choline-related SNP rs12325817 is a variant of the PEMT enzyme that is associated with a reduction in the endogenous synthesis of choline (59). \u0026nbsp;We do not know the prevalence of this SNP in our population, but it was reported to be ~20% among South African women (60). If we assume the same prevalence in our sample and remove 20% of the children, our detectable difference for EI score would increase from 0.35 to 0.40 and retain clinical significance.\u003c/p\u003e\n\u003cp\u003eWe also did not include biomarkers\u0026nbsp;of choline status. Based on the preliminary studies\u0026nbsp;among children with FASD (34),\u0026nbsp;however,\u0026nbsp;serum choline increased in children who received choline supplementation, as expected. The limited information expected\u0026nbsp;to\u0026nbsp;be\u0026nbsp;gained\u0026nbsp;thus\u0026nbsp;did\u0026nbsp;not\u0026nbsp;justify\u0026nbsp;the\u0026nbsp;high\u0026nbsp;cost\u0026nbsp;of\u0026nbsp;choline\u0026nbsp;analysis.\u0026nbsp;We can\u0026nbsp;assess\u0026nbsp;this\u0026nbsp;analyte\u0026nbsp;in stored blood samples, if determined to be scientifically meaningful.\u003c/p\u003e\n\u003cp\u003eA consideration when interpreting our results is that because choline and DHA work together in brain development, we may miss a choline effect if sufficient DHA is not present (61,62). We designed the current study as an efficacy study to test the effect of choline alone, as we did in our FASD study. However, if we do see an effect of choline in this study, we will include DHA in future studies to see if this effect is enhanced. Alternatively, if we see no choline effect, we can include DHA in future studies to see if an effect emerges.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, iron therapy in a malaria-endemic area has been associated with a potential risk of increased episodes of malaria or other infectious illness (63). However, iron remains the standard of care treatment for children with iron deficiency. Multiple Cochrane Reviews have concluded that iron can be given safely to children in areas where malaria prevention and management services are provided, as they are in the context of the proposed trial (64–66). Current WHO guidelines for universal iron supplementation reflect these results, stating that prophylactic iron therapy should be given to children in malaria-endemic areas where anemia prevalence is 40% or greater if public health measures to prevent, diagnose, and treat malaria are in place (67). All children in our study will be under active surveillance for illness and will be given an insecticide-treated bednet at enrollment. Any child found to have malaria or other illness will receive treatment according to Mulago Hospital and Uganda National guidelines free of charge. A finding of benefit in neurobehavioral outcomes when choline is given with iron in the current study may support future research to see if the recommended dose of iron for the correction of IDA can be reduced in malaria-endemic areas, potentially lessening infection risk. \u0026nbsp;\u003c/p\u003e"},{"header":"Trial status","content":"\u003cp\u003eProtocol version 1.1 dated May 8, 2025.\u003c/p\u003e\u003cp\u003eApproximate date of start of recruitment: August 1, 2025\u003c/p\u003e\u003cp\u003eApproximate date of trial completion: June 2027\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAE: Adverse Event\u003c/p\u003e\n\u003cp\u003eBRS: Behavior Rating Scales\u003c/p\u003e\n\u003cp\u003eCBC: Complete Blood Count\u003c/p\u003e\n\u003cp\u003eCRF: Case report form\u003c/p\u003e\n\u003cp\u003eCRP: C-reactive protein\u003c/p\u003e\n\u003cp\u003eDSMB: Data and Safety Monitoring Board\u003c/p\u003e\n\u003cp\u003eECVT: Early Childhood Vigilance Test\u003c/p\u003e\n\u003cp\u003eEI: Elicited Imitation\u003c/p\u003e\n\u003cp\u003eFASD: Fetal alcohol spectrum disorders\u003c/p\u003e\n\u003cp\u003eHb: Hemoglobin\u003c/p\u003e\n\u003cp\u003eID: Iron deficiency\u003c/p\u003e\n\u003cp\u003eIDA Iron deficiency anemia\u003c/p\u003e\n\u003cp\u003eIRB: Institutional Review Board\u003c/p\u003e\n\u003cp\u003eNB: Neurobehavioral\u003c/p\u003e\n\u003cp\u003eNDA: Uganda National Drug Authority\u003c/p\u003e\n\u003cp\u003eMREC: Mulago Hospital Research Ethics Committee\u003c/p\u003e\n\u003cp\u003eMSEL: Mullen Scales of Early Learning\u003c/p\u003e\n\u003cp\u003ePI: Principal Investigator\u003c/p\u003e\n\u003cp\u003eRDT: Rapid Diagnostic Test\u003c/p\u003e\n\u003cp\u003eSAE: Serious adverse event\u003c/p\u003e\n\u003cp\u003eSOP: Standard operating procedure\u003c/p\u003e\n\u003cp\u003eSupCHO: Supplemental choline to prevent and treat learning and memory deficits of early iron deficiency\u003c/p\u003e\n\u003cp\u003eTIPT: Toronto Institute of Pharmaceutical Technology\u003c/p\u003e\n\u003cp\u003eUMN: University of Minnesota\u003c/p\u003e\n\u003cp\u003eUNCST: Uganda National Council of Science and Technology\u003c/p\u003e\n\u003cp\u003eWHO: World Health Organization\u003c/p\u003e\n\u003cp\u003eZPP: Zinc protoporphyrin\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions {31b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSC is the principal investigator and led the protocol development. SC, JW, and MG conceived the study and provided preliminary data; EM is the site principal investigator, is the liaison with regulatory authorities, ensures adherence to protocol and SOPs, and supervises day-to-day trial operations. PB oversees onsite neurobehavioral testing; RB is the study manager and pediatrician and oversees study logistics and the study team; CC conducted the training for the Elicited Imitation task, provides ongoing quality control and external validation for this assessment, and will help with interpretation of results. MK developed the behavioral rating scale and will oversee behavioral coding. All authors read and approved the final manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding {4}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eE.W. \u0026ldquo;AL\u0026rdquo; Thrasher Award, Thrasher Research Fund (Primary funder)\u003c/p\u003e\n\u003cp\u003eUniversity of Minnesota Masonic Institute for the Developing Brain Interdisciplinary Faculty Research Award (Supplemental funder)\u003c/p\u003e\n\u003cp\u003eThe funding bodies will have no role in the design of the study, the collection, analysis, and interpretation of data, or in the writing of the trial manuscripts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials {29}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSC and EM will have access to the final trial dataset. No contractual agreements limit access for investigators.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate {24}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUniversity of Minnesota Institutional Review Board #\u0026nbsp;STUDY00022408 version 1 date 6/18/2024; version 1.1 date 7/2/2025\u003c/p\u003e\n\u003cp\u003eMulago Hospital Research Ethics Committee Date # MHREC 2024-158 version 1 5/5/2024; version 1.1 6/24/2025\u003c/p\u003e\n\u003cp\u003eUganda National Council of Science and Technology:\u0026nbsp;HS4915ES 11/13/2024\u003c/p\u003e\n\u003cp\u003eUganda National National Drug Authority: CTA 0288/2025\u003c/p\u003e\n\u003cp\u003eWritten, informed consent to participate will be obtained from the caretakers of all participants.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication {32}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAttached.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests {28}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003einformation (optional)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eL\u0026ouml;nnerdal B, Hernell O. 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Daily Iron Supplementation in infants and children [Internet]. 2016 [cited 2023 Apr 15]. Available from: https://apps.who.int/iris/bitstream/handle/10665/204712/9789241549523_eng.pdf\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"trials","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trls","sideBox":"Learn more about [Trials](http://trialsjournal.biomedcentral.com/)","snPcode":"13063","submissionUrl":"https://www.editorialmanager.com/trls","title":"Trials","twitterHandle":"MedicalEvidence","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Choline, iron deficiency, brain development, nutrients and brain, hippocampus","lastPublishedDoi":"10.21203/rs.3.rs-7041039/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7041039/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground:\u003c/h2\u003e\u003cp\u003eIron deficiency (ID) limits the neurodevelopmental potential of more than 200\u0026nbsp;million children each year. Iron therapy started when IDA is first diagnosed\u0026mdash;typically by screening for anemia at or detection of clinical symptoms of IDA at 12 months of age\u0026mdash;does not fully correct earlier ID-mediated brain dysfunction, underscoring the need for low-cost, easily implementable adjunct therapies to iron to treat or prevent this dysfunction in high-risk populations. Supplementation with the essential nutrient choline lessens damage done to the developing hippocampus when given with iron in pre-clinical rodent models, and choline supplementation improves hippocampus-mediated memory and learning in 2-3-year-old children with Fetal Alcohol Spectrum Disorders, a condition associated with hippocampal damage and one for which ID is a component of the neuropathology. Choline has not been tested in children with IDA. Our overall aim is to conduct a randomized, placebo-controlled clinical trial to test whether nine months of daily choline supplementation along with standard iron therapy improves hippocampus-dependent neurobehavioral outcomes in Ugandan infants with IDA.\u003c/p\u003e\u003ch2\u003eMethods:\u003c/h2\u003e\u003cp\u003eThree hundred 6-month-old infants with IDA who present to immunization clinics at Mulago and Kawempe National Referral Hospitals in Kampala, Uganda, will be randomized to iron plus choline or iron plus placebo. Iron (oral ferrous sulfate 2 mg/kg/day) will be given for the first three months of follow-up, and a dispersible tablet of choline (200 mg as choline bitartrate) or identical placebo will be given daily for all nine months of follow-up. We will conduct neurobehavioral tests assessing hippocampus-specific memory and attention and global cognition at enrollment (when each infant is 6 months of age) and after nine months of follow-up (when each infant is 15 months of age).\u003c/p\u003e\u003ch2\u003eDiscussion:\u003c/h2\u003e\u003cp\u003eIf we find a neurobehavioral benefit when choline is given along with iron, choline could be added immediately to standard of care treatment for IDA. This low-cost intervention could safely mitigate the brain dysfunction of early-life ID that is often not diagnosed until the hippocampal critical window is closing, providing life-long benefit for both the individual and the economic and social prosperity of entire regions.\u003c/p\u003e\u003ch2\u003eTrial registration:\u003c/h2\u003e\u003cp\u003eClinical trials.gov# NCT06527391; Registered 24 July 2024\u003c/p\u003e","manuscriptTitle":"Supplemental choline to prevent and treat learning and memory deficits of early-life iron deficiency (The SupCHO Study): study protocol for a randomized, placebo-controlled trial in Ugandan infants with iron deficiency anemia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-27 09:06:34","doi":"10.21203/rs.3.rs-7041039/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-10-08T16:01:39+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-19T07:02:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-12T05:46:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Trials","date":"2025-08-11T12:30:41+00:00","index":"","fulltext":""},{"type":"decision","content":"Minor revision","date":"2025-08-11T03:31:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"trials","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trls","sideBox":"Learn more about [Trials](http://trialsjournal.biomedcentral.com/)","snPcode":"13063","submissionUrl":"https://www.editorialmanager.com/trls","title":"Trials","twitterHandle":"MedicalEvidence","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"82132c23-a92d-4d23-a111-e17aa47df690","owner":[],"postedDate":"August 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-24T16:07:47+00:00","versionOfRecord":{"articleIdentity":"rs-7041039","link":"https://doi.org/10.1186/s13063-025-09246-2","journal":{"identity":"trials","isVorOnly":false,"title":"Trials"},"publishedOn":"2025-11-19 15:59:03","publishedOnDateReadable":"November 19th, 2025"},"versionCreatedAt":"2025-08-27 09:06:34","video":"","vorDoi":"10.1186/s13063-025-09246-2","vorDoiUrl":"https://doi.org/10.1186/s13063-025-09246-2","workflowStages":[]},"version":"v1","identity":"rs-7041039","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7041039","identity":"rs-7041039","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}