Does nicotinamide riboside supplementation increase milk production in postpartum women with infants admitted to a neonatal intensive care unit? Protocol for the MOONRISE randomised, double-blind, placebo-controlled pilot feasibility trial

Abstract

Introduction

An adequate supply of a mother’s own milk plays a critical role in optimising health outcomes for at-risk hospitalised infants. Insufficient milk production disproportionately affects the mothers of these infants. In the USA, metoclopramide is the only approved drug available for off-label use as a galactagogue. Nicotinamide riboside (NR), a direct precursor to nicotinamide adenine dinucleotide (NAD⁺), has been shown in a murine model to augment high-quality milk production and enhance cognitive and physical development of pups. Human testing with NR has been limited, and studies in lactating individuals have not been performed. The aim of this study is to assess the feasibility of NR supplementation in mothers whose infants are admitted to the neonatal intensive care unit (NICU) for at least 4 weeks.

Method and analysis

This double-blind, randomised, placebo-controlled pilot feasibility trial aims to investigate NR supplementation in mothers of infants who are anticipated to be hospitalised in the NICU for at least 4 weeks. We plan to enroll 40 mother–infant pairs with the expectation that 30 will complete the study (n=15 in each group). The intervention period with maternal NR supplementation or placebo supplementation and maternal milk, urine and blood sampling will be 19 days, including an enrollment day.

The primary endpoint of the study includes feasibility of enrolling ≥50% of eligible mothers. Secondary endpoints include compliance, adherence or withdrawal from the protocol. Similarly, the study will assess the difference in mean milk volume, macronutrient and micronutrient content, human milk glycans, milk and urine metabolites and infant feeding practice between the two groups. Optional secondary endpoints include differences in serum prolactin, liver function, plasma lipidome, serum metabolites, serum cellular communication network factor 3 concentrations, serum NAD⁺-related precursors and intermediates between mothers of the two groups. Exploratory outcomes include changes in mothers’ weight and infants’ developmental and NICU outcomes. Safety endpoints include the frequency of adverse events.

Ethics and dissemination

The Institutional Review Board (IRB) protocol version date is 1 June 2026, approved by the IRB at the University of California Davis Medical Center (approval number: 1557473). Study investigators will communicate trial results to participants, healthcare professionals and the public through peer-reviewed publications and presentations at scientific conferences. Trial results will also be reported on ClinicalTrials.gov and made available through PubMed Central, in accordance with regulatory requirements. Deidentified participant data will be available to qualified researchers upon request after publication. Approved requests will be granted access within 12 months and are subject to study team and IRB approval and a data use agreement.

Trial registration number

NCT04614714.

STRENGTHS AND LIMITATIONS OF THIS STUDY.

• Randomisation stratified by parity and identical placebo capsules are used to minimise selection and performance bias.

• Standardised protocols for milk expression, sample collection and outcome assessment are used throughout the study to improve consistency.

• Predefined feasibility outcomes, including recruitment, adherence, retention and protocol completion, are used to evaluate trial feasibility.

• The study incorporates comprehensive assessments, including milk composition, metabolomics, lipidomics and optional maternal blood biomarkers, to evaluate the biological effects of supplementation.

• The single-centre design and pilot sample size may limit the generalisability of the findings, and the study is not powered to detect definitive treatment effects.

Introduction

Background

According to national data collected in 2023, approximately 10% of infants are born preterm.¹ Very preterm infants—those delivered at <32 weeks of gestation—typically remain in the neonatal intensive care unit (NICU) for several weeks and receive some combination of their mother’s own milk, donor human milk and premature infant formula. Additionally, many term infants are admitted to the NICU following surgical procedures or for the management of medical complications, including diaphragmatic hernia, gastroschisis, intestinal atresia, hypoxic-ischaemic encephalopathy, congenital heart disease and other genetic syndromes. An adequate supply of a mother’s own milk plays a critical role in optimising health outcomes for these at-risk infants. Nonetheless, insufficient milk production disproportionately affects mothers of infants in the NICU, particularly those who give birth to extremely and very preterm infants.² One study showed almost 50% of mothers in the USA, and two-thirds of women worldwide claimed ‘breastmilk insufficiency’ as a primary reason for discontinuing breastfeeding before 6 months.³

Rationale

To address inadequate milk production, it is important to first address the influence of maternal and neonatal health, suck-swallow-breathe coordination, proper latch and frequency of milk expression and to take steps to correct these issues. Once these foundational factors have been addressed and milk supply remains insufficient, oral galactagogues, which include pharmacologic or non-pharmacologic bioactive substances intended to stimulate milk production, may be considered. Pharmacological galactagogues can be prescribed for initiation and maintenance of lactation and include domperidone and metoclopramide, which are dopamine antagonists that facilitate milk production by increasing prolactin levels.⁴ In the USA, metoclopramide is the only Food and Drug Administration (FDA)-approved drug available for off-label use as a galactagogue. While the data supporting domperidone’s effect on increasing milk production are robust,^{5 6} domperidone is not approved by the FDA for any indication. Metoclopramide is approved by the FDA for gastro-oesophageal reflux and gastroparesis but not as a galactagogue, and current FDA information sheets warn against use while breastfeeding. The evidence for metoclopramide is conflicting, with recent systematic reviews concluding there is insufficient evidence that metoclopramide enhances milk volume.^{7 8} Weaker still is the evidence behind the efficacy, long-term safety or mechanism of action of botanicals such as fenugreek and moringa, which remain popular among holistic health providers and many within lactation support communities.⁹

The limitations of currently available galactagogues and the weak evidence supporting the use of many galactagogues on the market highlight the need for novel interventions that target the fundamental metabolic biology of lactation. A compelling body of evidence indicates that postpartum and lactation constitute unique metabolic states characterised by an increase in systemic energy demands and whole-body mobilisation of cellular resources. In this context, nicotinamide riboside (NR), a key precursor to nicotinamide adenine dinucleotide (NAD⁺), the central regulator of cellular energy metabolism, emerges as a biologically plausible intervention to support lactation.

In animal studies, NR supplementation during pregnancy has been shown to significantly increase milk volume and augment milk composition, suggesting that NR may be a therapeutic target for reducing the risk of lactation insufficiency. In a sow model, maternal NR supplementation resulted in an increased milk yield of 31% and was associated with upregulation of lactogenesis genes and increased mammary gland alveolar lumen area, thereby allowing for greater milk accumulation or greater storage capacity within the alveoli.¹⁰ Findings from a murine model further support this concept, demonstrating that mammary tissue has high NAD⁺ demands during lactation and that NR supplementation enhances lactational behaviour and promotes greater transfer of maternal nutrients into milk, resulting in improved milk quality and enhanced cognitive and physical development in offspring.¹¹ Human testing with NR has been limited, and studies in lactating individuals have not been performed.^{12 13} Although some trials have evaluated NR supplementation for metabolic and cardiovascular outcomes, its application during human lactation has not yet been investigated.^14–17 Mothers of infants in the NICU are particularly vulnerable to decreased milk production and thus demonstrate the greatest potential for benefiting from a supplement aimed at improving milk quality and lactation efficacy. If NR enhances milk production by increasing prolactin or beneficially changes milk composition in lactating mothers, this simple intervention could provide a safe and effective means of improving growth outcomes in the NICU.

Objective

Primary objective

We will test the feasibility of conducting a supplementation study in mothers of hospitalised infants, with enrollment feasibility defined as enrolling ≥50% eligible mothers.

Secondary objectives

Secondary objectives include assessing feasibility metrics (supplement compliance), milk sample collection adherence and withdrawal rates; protocol adherence and the impact of NR supplementation on milk volume, milk composition (including macronutrient and micronutrient composition, glycans (eg, glycome), metabolites, lipidome and cellular communication network factor 3 (CCN3) and urinary metabolites. Remnant infant blood samples will be used to examine the relationship between infant feeding practices and neonatal insulin, glucose and amino acid concentrations.

Optional secondary objective

An optional component of the study involves the collection of maternal blood to assess the impact of NR supplementation on the concentration of serum prolactin, Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), metabolites and CCN3; plasma lipidome and whole blood concentration of NAD⁺-related precursors. Throughout lactation, calcium is mobilised from maternal skeletal reserves to promote infant skeletal growth and metabolic functions. However, unchecked calcium mobilisation can compromise maternal bone health. Recent research in rodent models has shown that CCN3 is a key regulator of maternal skeletal remodelling during lactation and functions to maintain maternal calcium homeostasis.¹⁸ An optional component of this study will involve collecting maternal blood samples to measure CCN3 and compare them to calcium levels in breast milk to further explore maternal calcium regulation during lactation.

Methods

Study design

Milk Outcomes Following Oral Nicotinamide Riboside Supplementation in the NICU Environment (MOONRISE) is a double-blind, randomised, placebo-controlled pilot feasibility study investigating NR supplementation in mothers of hospitalised infants. The study is planned to be conducted over approximately 24 months, with enrolment starting on 1 July 2026 and to be continued through 1 July 2028. The intervention period will last 19 days, including the enrolment day, and will involve a 14-day supplementation period (+2 days of extra capsules for loss or extending sample collection) with NR or a placebo containing microcrystalline cellulose, along with measurement of milk volume and collection of maternal milk, urine and blood (optional) at baseline and after intervention. The 14-day supplementation period was chosen out of practical necessity, representing the minimum number of days prior to NICU discharge.

Study setting

The MOONRISE study will take place at the University of California, Davis, a large academic medical centre with a 49-bed NICU that accepts patients from over 33 counties within a 65 000-square-mile region that includes Northern California, Southern Oregon and Nevada. According to internal data, there is an average of 1911 infants delivered at this institution, with a total of 718 admissions to the NICU per year.

Eligibility criteria

Participants will include mothers aged ≥18 years who deliver a singleton infant at 24–32 weeks’ gestation, or an infant of any gestational age anticipated to require ≥4 weeks of NICU hospitalisation (eg, gastroschisis, congenital cardiac defects or intestinal atresia). Infants must be delivered at the University of California Davis Medical Center (UCDMC) or transferred to UCDMC within the first 7 days of life. Eligible mothers must plan to provide breast milk for at least 3 months, initiate milk expression within 12 hours of delivery and express milk at least six times per 24-hour period from 72 hours postpartum until the enrollment visit.

During the study period, participants will be asked to refrain from tandem feeding another child, enrolling in other interventional trials, using pseudoephedrine and using non-study supplements containing NR or other galactagogues. Participants will be required to remove nipple piercings and to maintain milk expression at least six times per 24-hour period with no interval exceeding 5 hours. Participants must also be willing to express, weigh, record and collect breast milk over a 24-hour period at specified study time points.

Mothers will be enrolled between 4 and 8 days post partum. They will be asked to complete their first 24-hour milk collection after the onset of transitional milk production, defined as a score of ≥3 on the Onset of Milk Production Questionnaire (OMPQ).

Participants will be excluded if they do not meet the eligibility criteria outlined above. Mothers who were pregnant with multiple infants will be excluded. Additional exclusion criteria include a history of breast surgery or breast trauma that may impair lactation (eg, procedures involving areolar incision), current cancer treatment, the receipt of lithium or gold therapies, current hormonal birth control use or plans to initiate hormonal birth control during the study period. Mothers who are involuntarily confined, unable to provide informed consent or whose temporary residence during their infant hospitalisation at the UCDMC NICU is located more than 50 miles from UCDMC will be excluded. Participants may also be excluded at the discretion of the investigators if deemed unfit for study participation.

Recruitment

To ensure adequate participant enrollment, we will use several strategies, including active screening of eligible patients, clear communication about the study to prospective participants and close collaboration with the UC Davis NICU team to identify potential participants. The prescreening, screening and consent processes will be streamlined, and participants will receive both video instructions and written educational materials. The study team will hold weekly meetings to address recruitment challenges, monitor enrollment progress and implement additional outreach as needed to reach the target sample size, based on feedback from both the team and participants.

Informed consent

Initial eligibility (prescreening) will be determined by clinical trial physicians and trained study personnel through review of medical records using the study’s inclusion and exclusion criteria. Mothers who are not excluded during prescreening will be approached prospectively at UCDMC at the time of delivery or on transfer from a referring hospital and will be informed about the purpose, goals and potential benefits of the study. Women who express interest in the study will undergo additional screening to confirm eligibility. If a mother is eligible and chooses to participate, she will be scheduled for an enrollment visit and provided with a copy of the consent form to review in advance. The enrollment visit will take place on-site at UCDMC and will be considered day 0 of the study. Written informed consent will be obtained at the enrollment visit. Mothers may enrol between 96 hours (postnatal day 4) and postnatal day 8; enrollment will not occur after day 8 to ensure the collection of first transitional milk.

Following consent, mothers will be screened to confirm onset of milk production using the OMPQ, which assesses breast fullness. If a mother does not report a score of ‘3’ (breasts feel noticeably fuller) or higher at this visit, study personnel will ask her to complete the questionnaire on subsequent day(s). Once a score of ‘3’ or higher is reported, indicating that transitional milk production has begun, the mother will be instructed to collect her baseline samples.

At enrollment, mothers will receive a study kit containing a pumping manual, study notebook, pumping kit, pumping bra and milk sample collection supplies labelled with a unique subject ID. Participants will receive both written and verbal instructions on study procedures, including pumping both breasts, weighing and recording 24-hour milk volumes, collecting milk and urine samples and completing daily and weekly logs. Mothers will also be provided with freezer packs and a cooler bag for sample transport. The study team will ensure that all mothers have met with a lactation consultant and are comfortable using the Medela Symphony dual breast pump, which will be used exclusively during the study.

There is optional blood sampling on two occasions: once at the randomisation visit (or prior to starting supplementation) and once at the end-of-study visit.

Mothers will complete one brief electronic weekly log that includes questions about menstruation, contraception, breast health and use of lactation resources. The enrollment visit will last approximately 60 min. If time permits, mothers will also be asked to complete two additional electronic questionnaires, the Health History Questionnaire (HHQ) and the Pregnancy and Reproductive Health Questionnaire (PRHQ), which review health history, diet, lifestyle and the most recent pregnancy and delivery.

Randomisation

The UCDMC investigational drug pharmacy (IDS) will generate randomisation sequences. Groups will be stratified by parity (primiparous or multiparous) in a 1:1 ratio for each arm of the study. The UCDMC investigational pharmacy will randomise and provide the study product and placebo in presorted blocks with randomisation codes for each stratification group. IDS will have access to the random allocation sequence.

Blinding

Study participants, clinical trial physicians, outcome assessors and data analysts will be blinded to the study. The study medication and placebo will be identical in appearance and coded by IDS. The randomisation code will be revealed only after data collection is completed or in case of a medical emergency. In case of a serious adverse event where supplementation knowledge is essential for management, investigators may request unblinding from IDS, with documentation of the reason.

Intervention

The MOONRISE study period will be 19 days, including the enrolment day (day 0), and the intervention period (NR supplement or placebo consumption) will be 14 days (two additional capsules will be provided to accommodate missed doses or extension of sample collection when necessary). Niagen nicotinamide riboside chloride 250 mg capsules (Chromadex) were chosen for investigation because of their demonstrated galactagogue and metabolic effects in rodents as well as their efficacy in humans for increasing circulating NAD⁺ without adverse effects of long-term use. Tru Niagen is commercially available for purchase through the distributor and online retail sites.

NR was produced from D -ribofuranose tetra-acetate in accordance with cyclic guanosine monophosphate at a certified contract manufacturing organisation (Chromadex). The product of a two-step synthesis was vacuum-dried, milled, sieved and passed through a magnet to yield nicotinamide-D-riboside chloride. Structural determination was achieved by means of NMR and mass spectral analysis. Capsules contain 250 mg of pure product and are shelf-stable. Mothers will be asked to keep bottles in a cool, dry area away from direct sunlight.

During the randomisation visit (day 3), mothers will be rescreened to ensure they meet final study criteria and will be randomised to a treatment arm based on parity. Randomisation will be performed by the UCDMC investigational pharmacy, which will provide the study product and placebo as opaque capsules in 14-count bottles (+2 extra capsules for loss or extending sample collection). Mothers will begin the assigned study supplement on day 4 (±1 day) and continue for 14 consecutive days. The supplementation period may extend through day 19 to accommodate missed doses or delayed sample collection. To assess compliance, mothers will be instructed to bring the supplement bottle and any remaining pills to their end-of-study visit on day 18+2 days (D18–20).

Criteria for discontinuing or modifying allocated interventions

The study intervention may be modified or discontinued for any of the following reasons: a participant request, the occurrence of a rare or serious adverse event related to the study supplement or placebo or clinical judgement by the study physicians. Participants may also be withdrawn if the family lives at a distance from UC Davis Children’s Hospital that creates a significant hardship to continued participation. If the infant is discharged from the NICU before study completion or in the event of the infant’s death, parents will be offered the choice to either continue participation through study completion or withdraw from the study. Participants who withdraw from the study before completing all required activities will be asked to complete the first portion of the End-of-Study Questionnaire to document the reason for withdrawal.

Strategies to improve adherence to interventions

Strategies to promote adherence include providing participants with clear instructional videos, as well as written and verbal instructions. Flexible scheduling will be offered whenever possible to accommodate participants’ needs. Direct access to clinical trial physicians and study personnel will be available throughout the study period to address questions or concerns as they arise. Adherence will be assessed at the end-of-study visit on day 18+2 days by counting any remaining capsules in the supplement bottle, with compliance defined as consuming at least 75% of the assigned supplement.

Comparator

The comparator group will receive placebo supplementation for 14 consecutive days (+2 days of extra capsules for loss or extending sample collection) with microcrystalline cellulose. Placebo capsules consist of 250 mg/day of microcrystalline cellulose and will be generated by Chromadex. The placebo capsule is visually identical to the NR supplement capsule.

Relevant concomitant care permitted or prohibited during the trial

Participants will be asked to refrain from using pseudoephedrine (often found in Sudafed, Theraflu, Claritin-D, etc), pharmacologic and non-pharmacologic galactagogues or non-study supplements that contain NR (or similar derivatives, including nicotinamide mononucleotide) during the study period.

Data collection

Feasibility objectives will be assessed by tracking the number of eligible mothers approached, enrollment rates, adherence to the protocol and withdrawal rates.

Mothers will complete two electronic health questionnaires (HHQ and PRHQ) via Qualtrics after informed consent has been obtained. The OMPQ, daily logs, weekly logs and 24-hour milk collection log will also be completed electronically via Qualtrics. The programme requires that each question be answered in its entirety before allowing a participant to continue, thus preventing missing data from being entered. Deidentified subject IDs will be used on all questionnaires, study notebook logs, sample labels, collection kit labels and data files. Sample containers will be prelabelled by the study team and recorded in a spreadsheet with the subject IDs that are maintained on a secure shared drive. Data for both groups will be collected prospectively from the electronic health record (EHR).

Statistical analysis will be performed to analyse mean milk volume per day and macronutrient content following the end of supplementation. Secondary outcomes will include maternal milk fat, protein, carbohydrate, fat and calcium content of individual milk samples. We will measure CCN3 levels using ELISA. Maternal liver enzymes will be measured via a comprehensive metabolic panel. Maternal NR will be measured by NADMED,¹⁹ and the lipidome will be measured via Metabolon. Milk macronutrient analysis will be conducted using the MIRIS Human Milk Analyzer following established protocols, while the micronutrient analysis will be done by inductively coupled plasma mass spectroscopy (ICP-MS). Samples of milk and urine will be run in duplicate or triplicate. If remnant infant plasma is available, insulin and glucose will be measured by the Center for Diagnostic Innovation Laboratory via the Roche analyzer. If any plasma remains, amino acids and metabolites will be analysed following established protocols.

Outcomes

The primary outcome is to assess the feasibility of enrolling mothers of hospitalised infants in a double-blind, placebo-controlled supplementation trial, defined as obtaining consent from at least 50% of eligible mothers.

Secondary outcomes include feasibility metrics, such as supplement compliance of at least 75%, adherence to milk sample collection of at least 75% and participant withdrawal of 20% or less. Another secondary outcome is the proportion of enrolled mothers who follow the study protocol without major deviations. Differences in 24-hour milk volume between NR and placebo will be assessed over time. Differences in milk analytes between NR and placebo will also be evaluated over time, including human milk protein, carbohydrate and fat concentrations (measured by Miris HMA); micronutrient concentrations (measured by ICP-MS); the human milk glycome (including Human Milk Oligosaccharides, glycoproteins and glycolipids); milk metabolites (measured by NMR metabolomics); the milk lipidome (Complex Lipids Targeted Panel, Metabolon) and CCN3 concentration. In infant blood, the relationship between infant feeding practices and plasma levels of insulin, glucose, amino acids and metabolites (measured by NMR metabolomics) will be examined.

Maternal urine will be analysed for differences in concentration of urinary metabolites (measured by NMR metabolomics), including NAD⁺-specific metabolites as a proxy for NR supplementation, between NR and placebo over time.

Optional outcomes will be measured only in participants who provide blood sampling at both the randomisation visit (baseline) and the end-of-study visit (day 18±2 days). These outcomes will include differences in serum prolactin; liver function markers (AST, ALT and calcium measured as part of a comprehensive metabolic panel); plasma lipidome (Complex Lipids Targeted Panel, Metabolon); serum metabolites (measured by NMR metabolomics); serum CCN3 and whole-blood NAD⁺-related precursors and intermediates in K2 EDTA whole blood (measured by NADMED, Helsinki, Finland). Participants who decline the optional blood collection or are unable to complete one or both blood draws (eg, scheduling constraints, illness or early study withdrawal) will remain enrolled in the study and contribute data to all non-blood study outcomes. Optional blood analyses will be performed only on participants with available blood samples.

Exploratory outcomes include safety outcomes such as the frequency of adverse events; changes in maternal weight over time; diet, health and anthropometric outcomes reported by mothers when their child turns 1, 2 and 3 years of age; NICU outcomes obtained from the electronic medical record (EMR), including morbidity, death, necrotising enterocolitis, chronic lung disease, retinopathy of prematurity and sepsis and developmental outcomes for infants followed up at the UC Davis Medical Investigation of Neurodevelopmental Disorders (MIND) Institute (qualifying infants admitted to UC Davis NICU may receive developmental follow-up at the UC Davis MIND Institute until age 3).

Data management

Study data will be collected directly from the EHR and entered into a password-protected Excel file accessible only to the research staff at the study site. All data will be deidentified to protect participant privacy.

Participant timeline

The intervention period, including maternal supplementation or placebo and collection of maternal milk, urine and blood samples, will last 19 days, beginning with the enrolment visit. The participant study timeline with sample collection is shown in figure 1 and online supplemental table 1.

Figure 1. Flowchart illustrating participant enrolment, randomisation and end-of-study visit. The 24-hour pooled milk sample is collected at the randomisation and end-of-study visit along with a urine sample and optionally blood samples if the participant consents to do so. Additionally, participants will maintain daily logs and fill out weekly questionnaires during the study.

Remnant infant blood and maternal milk sampling will continue until the infant is discharged from the NICU. Data collection will be considered complete after neurodevelopmental follow-up at 36 months, if applicable. Enrolment of all participants is expected to take place tentatively over 24 months, from 1 July 2026 to 1 July 2028.

Sample size

The number of mother–infant dyads was chosen based on the feasibility of enrollment in the proposed period. We plan to enrol 40 mother–infant pairs with the expectation that 30 will complete the study (n=15 in each group). No formal power calculation was performed because this is a pilot feasibility study.

Feasibility will be defined as obtaining consent from ≥50% of eligible mothers. Feasibility metrics include supplement compliance of ≥75%, milk sample collection adherence of ≥75% and withdrawal of ≤25%.

Data analysis

Statistical analysis will be performed to analyse mean milk volume per day and macronutrient content following 14 days of supplementation. Secondary outcomes will include maternal milk fat, protein, carbohydrate, fat and calcium content of individual milk samples, also compared using two-sample t-tests.

We will conduct two sets of analyses for all endpoints: intent-to-treat (ITT) and per-protocol (PP). The ITT dataset will include all randomised participants, analysed in their originally assigned groups, regardless of adherence, dropout or protocol deviations. The PP data set will include only participants who sufficiently adhered to the protocol and completed the study according to prespecified criteria. The PP dataset will exclude participants who have not met the minimum supplement compliance (eg, ≥75% of supplements), missed key visits without make-up visits or missed outcome measurements, had major protocol violations or stopped the intervention early.

All analyses will follow an ITT principle. The extent and pattern of missing data will be examined by the treatment group and time point. Primary analyses will assume data are missing-at-random (MAR). Missingness is expected to be related to observed participant characteristics and logistical factors rather than unobserved outcomes, supporting a MAR assumption. For longitudinal outcomes, mixed-effects models will be used to incorporate all available data without requiring imputation. When appropriate, multiple imputations will be used to address missing outcome data, with imputation models including treatment assignments and relevant baseline covariates. Sensitivity analyses will be conducted to assess the robustness of results to alternative missing data assumptions. An interim analysis will not be conducted because NR supplementation is considered low risk in lactating women.

Primary analyses will follow an ITT principle, including all randomised participants analysed according to their original group assignment, regardless of protocol adherence. Protocol non-adherence will be explored in secondary PP analyses restricted to participants who meet predefined adherence criteria and have no major protocol deviations. Missing outcome data will be handled using ITT-consistent methods, including mixed-effects models for longitudinal outcomes and multiple imputation when appropriate, rather than exclusion-based approaches.

Protocol and statistical analysis plan

Descriptive statistics with Confidence Intervals (CIs) will describe the primary outcome components:

1. Recruitment rate (# enrolled / # eligible approached).

2. Retention (# completing end visit / # randomised).

3. Adherence (pill counts, self-report, whole blood NAD⁺ metabolites).

4. Completeness of sample collection (milk).

5. Protocol deviations and adverse events.

NR treatment effects for outcomes measured at baseline and at the end of the intervention will use analysis of covariance (ANCOVA) for each endpoint (milk macronutrients, micronutrients, CCN3; blood/serum NAD⁺ metabolites, prolactin, ALT/AST).

Milk volume is measured across three time points and will be analysed using a linear mixed effects model for all three time points.

To derive a global (lipidome/metabolome/glycome) shift score, principal components analysis will be applied to the standardised delta (end-baseline) of the endpoints. The first principal component (PC1), representing the dominant pattern of multivariate change, will be analysed as a continuous summary measure using ANCOVA with adjustment for multiple testing using the Benjamini–Hochberg false discovery rate procedure within each ‘omics’ platform.

Plans to promote participant retention and complete follow-up

Strategies to promote participant retention and complete follow-up include automated text message reminders for completion of daily and weekly logs and for scheduled study visits. Flexible scheduling will be offered whenever possible to accommodate participants’ needs. Direct access to clinical trial physicians and study personnel will be available throughout the study period to address questions or concerns as they arise. In addition, NICU staff will be educated about the study, and direct feedback will be obtained from the clinical care teams to support participant engagement. Daily logs will also be reviewed for completeness before the randomisation visit, and participants will receive additional instruction if logs are missing or completed incorrectly. Mothers who complete the study will receive a 150 US$ electronic Amazon gift card at the end-of-study visit on Day 18 as a token of appreciation. Mothers who consent to provide blood samples and complete both blood draws on days 3 and 18 within approximately the same time (±2 hours) will receive an additional 50 US$ Amazon gift card after confirmation of the day 18 draw.

Additionally, during enrolment, mothers may opt to participate in three brief (10–15 min) phone interviews when their child turns 1, 2 and 3 years old. Each call, conducted by study personnel following the child’s annual well-child visit, will include a semistructured survey on the child’s diet, health and growth (weight, height, etc). Participants will receive a 25 US$ Amazon gift card for each completed interview, for a total of up to 75 US$. Mothers may choose to complete one, two or three interviews.

Confidentiality

Once a participant has been enrolled, she will be assigned a deidentified subject ID. This ID will be used on all questionnaires, study notebook logs, sample labels, collection kits and data files. Sample containers will be prelabelled by the study team and tracked in a spreadsheet with subject IDs stored on a secure shared drive.

Signed consent forms containing participant-identifying information will be kept in a locked filing cabinet separate from all other study files at the UCDMC NICU and later transferred to a secure, locked location for long-term storage. All questionnaires will be administered electronically via Qualtrics. Any questionnaire data containing personal health information will be coded and stored on password-protected Excel spreadsheets accessible only to the study personnel listed. Following study completion, all forms will be shredded according to UC Davis Institutional Review Board (IRB) guidelines.

Oversight and monitoring

Composition of the data monitoring committee, its role and reporting structure

An independent data monitoring committee was not established for this study because the intervention is considered minimal risk and does not involve a new drug, high-risk device, gene therapy or vaccine. The study is designed as a pilot and feasibility trial with a small sample size and a short follow-up, and no significant safety issues are anticipated based on the existing evidence. All the participants, including infants admitted to the NICU, will be overseen by principal investigators.

Harms

NR is a dietary supplement that is generally recognised as safe, with no known significant side effects, and we do not anticipate adverse effects in this population. Infants in the NICU are at risk for complications such as sepsis, necrotising enterocolitis, poor neurodevelopment and death related to their underlying medical conditions. These infants remain under the care of the neonatologist throughout the study and are continuously monitored. While the NICU team is blinded to the specific study assignment, they will know that the mother is receiving either NR or a placebo and will notify clinical trial physicians of any unexpected outcomes.

Physical, psychological, privacy, legal, social and economic risks from participation are minimal. Slight discomfort or soreness may occur from breast pumping. NR may increase milk volume, which could theoretically reduce nutrient content per mL, but this is unlikely to affect infant nutrition, as growth is closely monitored in all NICU patients. Adjustments such as increasing feeding volume or supplementing with commercial human milk fortifier can be implemented if needed. Blood drawn by heel stick, venipuncture or arterial puncture can be painful. Many NICU infants have arterial catheters, making blood collection painless. There is a small risk of anaemia, and very premature infants may require transfusions for routine laboratory blood loss. Because only remnant blood samples will be examined, no additional blood will be collected for the study beyond what is clinically required. Given the low risk of side effects, no data safety monitoring committee, unblinded statistician or interim safety analysis will be conducted.

Adverse event reporting

Adverse events will be actively monitored, documented and reviewed throughout the study by principal investigators. Serious adverse events will be reported to the IRB according to the regulatory requirements.

Frequency and plans for auditing trial conduct

Trial conduct will be reviewed on an ongoing basis by principal investigators using a risk-based approach. The study is subject to audit by the IRB or institutional bodies independent of the investigators. Further, the university ensures that the human subjects are protected and that the trial conduct is in compliance with the currently approved protocol and regulatory requirements.

Plans for communicating important protocol amendments to relevant parties

All protocol amendments must receive IRB approval before implementation, unless an emergent change is needed to eliminate a hazard to participants. If important protocol changes require participant notification, the consent form modification will be submitted to the IRB.

Patient and public involvement

Patients and the public were not involved in the conception and design of this study.

Ethical consideration and dissemination plans

Ethical approval for this study was obtained from the University of California, Davis IRB (approval number: 1557473). IRB at the University of California, Davis, can be contacted at irbadmin@health.ucdavis.edu. All protocol amendments must receive IRB approval before implementation, unless an emergent change is needed to eliminate a hazard to participants. If important protocol changes require participant notification, the consent form modification will be submitted to the IRB.

Each prospective participant will receive a signed copy and an electronic copy of the consent form via email to review and discuss with her doctor, family and friends. A signed copy of the consent document for each participant will be filed in their EMR, if applicable. The signed form, containing identifiable information, will be stored in a locked cabinet in one of the principal investigators’ offices accessible only to study staff. Participants will be asked to sign a UC Davis HIPAA authorisation form for both the mother and child at the same time as consent to permit access to EMRs, which may become part of the research record and be reviewed by the sponsor of this study and government agencies or other groups associated with the study. Participants will receive a signed copy of their records. Study investigators will communicate trial results to participants, healthcare professionals and the public through peer-reviewed publications and presentations at scientific conferences. Trial results will also be reported on ClinicalTrials.gov and made available through PubMed Central, in accordance with regulatory requirements.

Protocol version

IRB protocol version date is 1 June 2026. Recruitment is anticipated to start in July 2026 and is anticipated to tentatively complete around July 2028.

Availability of data and materials

After publication, deidentified participant data will be available to qualified researchers on request, subject to a data use agreement, study team review and IRB approval. Access will be granted within 12 months for requests meeting scientific and ethical standards.