Clinical trial NCT04629534
Conditions: Vitamin D Deficiency Nutritional Deficiency
Keywords: Vitamin D
Sponsor: Medical University of South Carolina
Start date: 2021-05-06; Completion date: 2022-04-01
Enrollment: 8 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The overall goal of this study is to assess maternal vitamin D status along with diet, and milk composition, and in turn, to evaluate if infant vitamin D status can be improved with increased maternal supplementation and diet education. The central hypothesis is that by maximizing maternal vitamin D status in breastfed, preterm infants (\<35 weeks), there will be an increase infant 25(OH)D status at 1,2, and 3 months of age.

Detailed description:

Arms: arm 1: None arm 2: None
Interventions: intervention 1: High dose Cholecalciferol intervention 2: Cholecalciferol
Intervention descriptions: intervention 1: Mothers in experimental group will receive 6,000 IU of vitamin D in addition to their prenatal vitamin, daily for 3 months. Infants in this group will receive placebo vitamin D drops intervention 2: Mothers in placebo control group will receive 600 IU of vitamin D in the form of their prenatal vitamin along with placebo vitamin D. Infants of the mothers in this group will receive standard 400 IU of vitamin D

Protocol documents:
Prot_002.pdf: 
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Mothers’ own Milk Optimization for Preterm Infants Project (MoMO 
PIP): Maternal Diet and Vitamin D Supplementation Effects on Preterm 
Infants, a Randomized Control Pilot Study. 
NCT # 04629534  
Date: 9/9/2021 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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PROTOCOL TITLE: 
 
Mothers’ own Milk Optimization for Preterm Infants Project (MoMO PIP): Maternal Diet and 
Vitamin D Supplementation Effects on Preterm Infants, a Randomized Control Pilot Study. 
 
PRINCIPAL INVESTIGATOR:  
Raechel Irons, MD 
 
1.0 
Objectives / Specific Aims 
 
The overall goal of this study is to assess maternal vitamin D status along with diet, and milk 
composition to, in turn, evaluate if infant vitamin D status can be improved with increased maternal 
supplementation and diet education. Our central hypothesis is that by maximizing maternal vitamin D 
status, there will be an increase infant 25(OH)D concentration in breastfed, preterm infants. This 
hypothesis will be tested by performing the following Specific Aims.  
 
Aim 1: Determine the efficacy, effectiveness, and safety of maternal and premature infant vitamin 
D supplementation as a function of maternal vitamin D status, lactation status, and ethnicity.  
H1: Maternal supplementation of high dose (6,600 IU) vitamin D3/day will be safe for lactating 
mothers. 
H2: It is anticipated that only high dose (6,600 IU) vitamin D3/day supplementation of lactating 
mothers will provide sufficient antirachitic activity in breast milk to prevent vitamin D deficiency in 
nursing premature infants, regardless of ethnicity/race.  
H3: High-dose maternal supplementation will be safe and more effective than infant vitamin D 
supplementation alone, without any adverse side effects or health consequences in the mother or infant. 
 
Consented lactating women who delivered preterm infants <35 weeks’ gestation admitted to the 
MUSC NICU will be randomized 3-7 days following delivery to either 1 of 2 study groups: Group A 
(standard therapy of 600 IU vitamin D3/day), Group B (high dose supplementation of 6,600 IU Vitamin 
D3/day). As such, infants whose lactating mothers are randomized to the 600-IU/day group will receive 
400-IU/day oral vitamin D3 (recommended practice). Those infants of lactating mothers assigned to 
Group B will receive placebo. Calcium homeostasis will be monitored in all groups. Safety will be 
assessed throughout the study on a case-rather than a treatment arm basis using cautionary levels as the 
stop rule with intervention prior to exceeding normal urinary calcium/creatinine ratios in mothers and 
serum calcium concentrations in both mothers and infants. Through these proposed studies, the 
prevalence of vitamin D deficiency among mothers and their infants, and the utility of maternal 
therapeutic intervention with vitamin D3 will be assessed. 
 
Aim 2: Aim 2: Evaluate and maximize maternal milk composition.  
For this aim, we will obtain maternal food dietary intake using the Diet History Questionnaire III 
(DHQ III). This is a validated tool that is based on a 24-hour dietary recall database from the National 
Health and Nutrition Examination Surveys (NHANES) in people 19 and older. This survey will be 
administered between post-partum days 3-7 at the time a milk sample is obtained and at 1-, 2-, and 3- 
month visits. We then will utilize a milk composition analyzer (name, city, state) in MUSC’s Milk Bank 
to evaluate maternal milk sample composition at the initial visit and again at 1, 2 and 3 months of infant 
age. Prior to discharge from the NICU, the infant will undergo a body mass analysis using the PEA POD 
and Bone ultrasound. Analysis will be performed on maternal diet, milk composition, infant body 


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composition, and vitamin D status for any correlations. Samples will also be archived for metabolomics 
and cytokine analysis.  
 
2.0  
Background 
 
Human milk remains the favorable form of nutrition for the neonate and infant (1). Neonatal 
vitamin D stores are inversely related to gestational age (2). Along with this, two-thirds of fetal calcium is 
accumulated in the third trimester. Postnatal risk factors for vitamin D deficiency (VDD) of preterm 
neonates include decreased sunlight exposure, difficulties with adequate enteral nutrition, and higher 
requirements for vitamin D. All these factors along with use of diuretics and steroids, lead to an increased 
risk. Many studies have shown the importance of vitamin D in neonatal and infant health. Munshi et al. 
found that 80% of ELBW and VLBW infants at Albany Medical Center were deficient or insufficient in 
vitamin D at 1 month of age (3). Along with deficiency, the incidence of metabolic bone disease is still 
elevated at 32% of VLBW and 54% of ELBW (4, 5). There is also a significant association between 
respiratory distress syndrome (RDS) and vitamin D deficiency, especially in infants ≤ 30 weeks(6).  
Maternal breast milk is the recommended source of nutrition for both full and preterm infants; 
however, if mother is deficient in vitamin D, her milk provides limited vitamin D, with roughly 3-4 
IU/kg/day provided (7, 8). It has also been found that mothers and their preterm infants were more likely 
to be vitamin D deficient than mothers and their infants who carried to term. To further worsen this, those 
preterm infants who are not fed mother’s own milk (MoM) are given donor breast milk. Unfortunately, 
through pasteurization there is 10-20% less vitamin D (9). 
Maternal vitamin D status as measured by total circulating 25(OH)D concentration is influenced 
by many factors including ethnicity, season, high BMI, gestational diabetes, socioeconomic status (poor 
diet), and prenatal intake (10). From an ethnicity standpoint, African American, Middle Eastern, and 
Asian women are at highest risk for vitamin D deficiency. To no surprise, these are also the populations at 
increased risk of preterm delivery. In South Carolina in 2010, our group showed that vitamin D deficiency 
[defined as 25(OH)D <20 ng/mL (50 nmol/L)] was around 48% overall, with 68.3% of African-American 
women being deficient (11). In our NICHD vitamin D supplementation trial, there were similar findings 
with significant deficiency in African American and Hispanic women at baseline (12).   
There are several studies evaluating various supplementation regimens in preterm infants; 
however, considerable attention has not been given to the role of maternal vitamin D supplementation and 
its effects on preterm neonate’s vitamin D status. Many groups have recommendations on vitamin D 
supplementation that have failed to form a consensus or have differing recommendations for preterm 
neonates. The AAP Committee on nutrition recommends 200-400 IU of vitamin D for very low 
birthweight infants (VLBW). On the other hand, the European Society of Pediatric Gastroenterology and 
Nutrition (ESPGAN) recommends 800-1600 IU daily (13). Due to the lack of substantial data, these 
various groups fail to make recommendations on arguably those at highest risk--preterm, very low 
birthweight (VLBW) infants, and extremely low birthweight (ELBW) infants. Along with variations in 
supplementation recommendations, there are also variations among scientific groups as to an ideal target 
25(OH)D level. The AAP recommends 25(OH)D  > 20 ng/mL (50 nmol/L) (14) and in the same year 
another group published a paper with recommended 25(OH)D > 30 ng/mL (75 nmol/L) (15). The 
Endocrine Society targets >30 mg/mL (75 nmol/L) (16) while ESPGAN >32 ng/mL (80 nmol/L) (13). 
Previous groups have looked at the effects of various vitamin D supplementation regimens in preterm 
infants, many of which have not shown significant changes in infant 25(OH)D concentrations, but have 
shown that higher supplementation leads to improved length, head circumference, and possibly immune 
function (17).  Previous studies have shown safety of increased vitamin D supplementation with 800 IU 
and 1000 IU without significant toxicity (18-20).  
In a previous randomized control trial performed at this institution by Dr. Carol Wagner and 
colleagues, maternal supplementation with 6400 IU/day of vitamin D3 was more effective in achieving 


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vitamin D sufficiency in mothers without increased risk to infants or mothers (21, 22). This present pilot 
study plans to replicate this in mothers and their preterm infants (22). See IRB application attachments for 
this paper that is in press.  
To our knowledge, there are no studies specifically investigating maternal vitamin D 
supplementation on preterm infant vitamin D status. There has been investigation into maternal vitamin D 
supplementation on term infant vitamin D stores and data does suggest increased infant serum 25(OH)D 
levels with increased maternal supplementation (21, 23, 24).  The objective of this study is to not only 
improve preterm infant vitamin D status but also maternal vitamin D status, as this continues to be an area 
of need in the Charleston, SC area.  
The proposed pilot study will aim to improve 25(OH)D concentrations in mothers and their 
preterm infants, and therefore, allow us to further investigate and understand effects of maternal 
supplementation in preterm infants.  
 
3.0  
Intervention to be studied 
Cholecalciferol or vitamin D3 is an FDA approved medication for treatment and prevention 
of hypovitaminosis D.  
Per Lexicomp, “Dosing: Adult 
Note: 1 mcg = 40 units 
Hypoparathyroidism (off-label use): Note: Active vitamin D preparations (ie, alfacalcidol, calcitriol) in 
conjunction with calcium supplementation are recommended therapy. Addition of cholecalciferol (or 
ergocalciferol) may be considered for supplemental therapy (25). 
Osteoporosis, prevention (off-label use): Adults ≥50 years of age: Oral: 800 to 1,000 units/day is 
recommended, through dietary sources and/or supplementation if needed (26).  
Vitamin D insufficiency/deficiency treatment (off-label use): Note: Repletion strategies may vary 
depending on desired target serum 25(OH)D levels as well as the clinical status of the patient. The 
optimal serum 25(OH)D level is controversial; the Institute of Medicine recommends a 25(OH)D 
level >20 ng/mL (50 nmol/L) as sufficient in nearly all persons (27), whereas others have suggested 
targeting a level of ~30 ng/mL (75 nmol/L) to minimize the risk of fractures, particularly in patients 
with osteoporosis (28); (26). During pregnancy, the conversion of 25(OH)D to 1,25(OH)2D, the 
active hormone of vitamin D is optimized at 40 ng/mL (100 nmol/L) and greater. However, some 
data suggest levels >40 ng/mL (>100 nmol/L) (median level in one trial: ~48 ng/mL) are associated 
with increased risk of falls in postmenopausal women (29, 30). 
Therefore, some experts recommend a range of 20 to 40 ng/mL (50-100 nmol/L) as a reasonable 
target in most patients (31). In patients with normal absorption, for every 100 units/day of 
cholecalciferol, the serum 25(OH)D concentration is expected to increase by ~0.7 to 1 ng/mL 
(1.75 to 2.5 nmol/L) after a few weeks (32); (31). The dose-response declines as the 25(OH)D 
concentration increases above 40 ng/mL (100 nmol/L) (31). The following recommendations 
are based primarily on expert opinion and clinical experience: 
Initial dosing (according to baseline serum 25(OH)D level): 
Serum 25(OH)D 20 to 30 ng/mL (50 to 75 nmol/L): Initial: Supplementation dosing: Oral: 600 
to 800 units once daily; a repeat serum 25(OH)D concentration is not required 
(31) or 1,000 to 2,000 units once daily; may consider a repeat serum 25(OH)D 
concentration in ~3 months to determine if the target level has been achieved (33). 
Serum 25(OH)D 10 to <20 ng/mL (25 to 50 nmol/L): Initial: 


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Supplementation dosing: Oral: 800 to 1,000 units once daily (31) or 2,000 units once daily 
(33); a repeat serum 25(OH)D level should be drawn after ~3 months. If target serum 
25(OH)D level has not been achieved, may increase to 2,000 units once daily or 
administer therapeutic dosing of 50,000 units once weekly for 6 to 8 weeks (31). 
OR 
Therapeutic dosing (ie, high-dose cholecalciferol): Oral: 50,000 units once weekly (or 
5,000 to 7,000 units once daily) for ~8 weeks, followed by decreased maintenance 
dosing as needed to maintain target serum 25(OH)D level (34); (26). 
Serum 25(OH)D <10 ng/mL (< 25 nmol/L) or in patients with deficiency symptoms: Initial: 
Therapeutic dosing (ie, high-dose cholecalciferol): Oral: 50,000 units once weekly (or 
5,000 to 7,000 units once daily) for 6 to 8 weeks to achieve target serum 25(OH)D level; a 
repeat serum 25(OH)D level should be drawn after ~3 months to assure target serum 
25(OH)D level has been met (34); (31);(26). 
Maintenance dosing: Maintenance dosing is highly patient specific and dependent on target 
25(OH)D level, and may range from: 600 to 800 units/day (31) to 1,000 to 2,000 units/day 
(34);(26). 
Special populations (obese patients, patients on medications known to affect vitamin D metabolism, 
patients with malabsorption syndromes or gastrectomy): Higher doses or longer durations may 
be necessary for adequate repletion (34); (31). 
Vitamin D deficiency/insufficiency in patients with chronic kidney disease (off-label use): Oral: 
Note: In patients without severe and progressive hyperparathyroidism, including chronic kidney 
disease stages G3 to G5 and dialysis or transplant patients, KDIGO guidelines recommend 
correcting vitamin D deficiency and insufficiency with treatment strategies recommended for 
the general population using cholecalciferol (or ergocalciferol) while avoiding hypercalcemia 
and ensuring phosphate levels are in the normal range. An individualized monitoring approach 
to direct treatment is also recommended (35);(36). In patients in whom serum parathyroid 
hormone levels are progressively rising and remain persistently elevated despite correction of 
modifiable factors (eg, hyperphosphatemia, vitamin D deficiency), calcitriol or vitamin D 
analogs are suggested instead of cholecalciferol (or ergocalciferol) (37). 
 
Dosing: Geriatric 
Refer to adult dosing. 
 
Dosing: Renal Impairment: Adult 
There are no dosage adjustments provided in the manufacturer's labeling 
 
Dosing: Hepatic Impairment: Adult 
There are no dosage adjustments provided in the manufacturer's labeling. 
 
 
 
 


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Dosing: Pediatric 
 
Vitamin D deficiency, prevention (eg, Rickets prevention): AAP (14); (38):  
Oral: 
Breastfed infants (fully or partially): Oral: 400 units/day beginning in the first few days of life. 
Continue supplementation until infant is weaned to ≥1,000 mL/day or 1 qt/day of vitamin D-
fortified formula or whole milk (after 12 months of age) 
Formula-fed infants ingesting <1,000 mL of vitamin D-fortified formula: Oral: 400 units/day 
Children and Adolescents without adequate intake: Oral: 400 to 600 units/day. Note: Children with 
increased risk of vitamin D deficiency (chronic fat malabsorption, maintained on chronic 
antiseizure medications) may require higher doses; use laboratory testing [25(OH)D, PTH, bone 
mineral status] to evaluate 
Vitamin D deficiency, treatment: Oral: Note: Treatment should also include calcium and phosphorus 
supplementation; some patients with chronic fat malabsorption, obesity, or who are maintained on 
chronic antiseizure medications, glucocorticoids, HIV medications, or antifungals may require higher 
doses of cholecalciferol (39); monitor vitamin D status closely. 
Infants: Oral: 2,000 units daily for 6 weeks to achieve a serum 25(OH)D level >20 ng/mL (>50 
nmol/L); followed by a maintenance dose of 400 to 1,000 units daily. Note: For patients at high 
risk of fractures a serum 25(OH)D level >30 ng/mL (75 nmol/L) has been suggested (39). 
Children and Adolescents: Oral: 2,000 units daily for 6 to 8 weeks to achieve serum 25(OH)D level 
>20 ng/mL (> 50 nmol/L); followed by a maintenance dose of 600 to 1,000 units 
daily. Note: For patients at high risk of fractures a serum 25(OH)D level >30 ng/mL 
(>75nmol/L) has been suggested (39). 
 
Vitamin D deficiency in cystic fibrosis, prevention and treatment: Oral: 
CF guidelines (40): 
Recommended initial daily intake to maintain serum 25(OH)D level ≥30 ng/mL (>75 nmol/L): 
Infants: Oral: 400 to 500 units/day 
Children ≤10 years: Oral: 800 to 1,000 units/day 
Children >10 years and Adolescents: Oral: 800 to 2,000 units/day 
Dosing adjustment for serum 25(OH)D level between 20 to 30 ng/mL (50 to 75 nmol/L) and 
patient adherence established (Step 1 increase): 
Infants: Oral: 800 to 1,000 units/day 
Children ≤10 years: Oral: 1,600 to 3,000 units/day 
Children >10 years and Adolescents: Oral: 1,600 to 6,000 units/day 
Dosing adjustment for serum 25(OH)D level <20 ng/mL (<50 nmol/L) or persistently between 
20 to 30 ng/mL (50 to 75 nmol/L) and patient adherence established (Step 2 increase): 
Infants: Increase up to a maximum 2,000 units/day 
Children ≤10 years: Increase to a maximum of 4,000 units/day 


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Children >10 years and Adolescents: Increase to a maximum of 10,000 units/day 
Alternate dosing (41): 
Initial dose: Serum 25(OH)D level ≤30 ng/mL (≤ 75 nmol/L) 
Infants: Oral: 8,000 units/week 
Children and Adolescents: Oral: 800 units/day 
Medium-dose regimen: Serum 25(OH)D level remains ≤30 ng/mL (≤ 75 nmol/L) and patient 
compliance established 
Infants and Children <5 years: Oral: 12,000 units/week for 12 weeks 
Children ≥5 years and Adolescents: Oral: 50,000 units/week for 12 weeks 
High-dose regimen: Repeat 25(OH)D level remains ≤30 ng/mL (≤ 75 nmol/L) and patient 
compliance established 
Infants and Children <5 years: Oral: 12,000 units twice weekly for 12 weeks 
Children ≥5 years and Adolescents: Oral: 50,000 units twice weekly for 12 weeks 
 
Vitamin D insufficiency or deficiency associated with CKD (stages 2 to 5, 5D), treatment; serum 25 
hydroxyvitamin D [25(OH)D] level ≤30 ng/mL (≤ 75 nmol/L) (42): Oral: 
Serum 25(OH)D level 16 to 30 ng/mL (40 to 75 nmol/L): Infants, Children, and Adolescents: 2,000 
units/day for 3 months or 50,000 units every month for 3 months 
Serum 25(OH)D level 5 to 15 ng/mL (12.5 to 37.5 nmol/L): Infants, Children, and Adolescents: 
4,000 units/day for 12 weeks or 50,000 units every other week for 12 weeks 
Serum 25(OH)D level <5 ng/mL (< 12.5 nmol/L): Infants, Children, and Adolescents: 8,000 
units/day for 4 weeks then 4,000 units/day for 2 months for total therapy of 3 months or 50,000 
units/week for 4 weeks followed by 50,000 units 2 times/month for a total therapy of 3 months 
Maintenance dose [once repletion accomplished; serum 25(OH)D level >30 ng/mL (>75 nmol/L)]: 
Infants, Children, and Adolescents: 200 to 1,000 units/day 
 
Nutritional rickets, treatment: Limited data available (38): Administer in combination with calcium 
supplementation: 
Daily therapy (preferred): 
Infants: Oral: 2,000 units daily for ≥3 months, followed by maintenance dose of 400 units daily 
Children: Oral: 3,000 to 6,000 units daily for ≥3 months, followed by maintenance dose of 600 
units daily 
Adolescents: Oral: 6,000 units daily for ≥3 months, followed by maintenance dose of 600 units 
daily 
Single-dose therapy: 
Infants ≥3 months: Oral: 50,000 units once, or in divided doses over several days; after 3 
months, initiate maintenance dose of 400 units daily 
Children: Oral: 150,000 units once, or in divided doses over several days; after 3 months, 
initiate maintenance dose of 600 units daily 


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Adolescents: Oral: 300,000 units once, or in divided doses over several days; after 3 months, 
initiate maintenance dose of 600 units daily 
 
Dosing: Renal Impairment: Pediatric 
There are no dosage adjustments provided in the manufacturer's labeling; however, cholecalciferol is not 
renally eliminated to a significant extent and dosage adjustment is not necessary. 
 
Dosing: Hepatic Impairment: Pediatric 
There are no dosage adjustments provided in the manufacturer's labeling. 
 
Use: Labeled Indications 
 
Dietary supplement: As a vitamin D dietary supplement 
Doses for this study are under IND 66,346 and it’s safety has been proven in this 
referenced study that is currently in press in Breastfeeding medicine (22).  
Maternal and infant placebos will be used in this study. Maternal placebo will be sugar 
gummies from the same manufacturer (Church & Dwight). Infant placebo vitamin D drops will be 
from the same company (D Drops) and contain the eluent without the vitamin D.  
 
4.0  
Study Endpoints 
• 
Primary outcomes will be: 
o Infant total circulating 25(OH)D concentration at 3 months  
o Mother total circulating 25(OH)D concentration at 3 months 
• 
Primary or secondary safety endpoints 
o Maternal serum calcium concentration initially and at months 1-, 2-, and 3 
o Maternal urine calcium/creatinine ratios initially and at months 1-, 2-, and 3 
o Infant serum calcium concentration at time points initially and at months 1-, 2-, and 
3 
 
5.0  
Inclusion and Exclusion Criteria/ Study Population 
 
Only women 18-45 years who are lactating and their preterm infants (<35 weeks) within the first 
week postpartum will be eligible for the study. Men will be excluded on the basis of the study criterion of 
lactation. Infants of both genders will be followed along with their mothers. Women who deliver at MUSC 
will be eligible for participation.  
a. Inclusion Criteria for Lactating Women: Women with documentation of fully 
breastfeeding who present within one month of delivery, who are 18-45 years old and in 
good general health will be eligible for participation. 
b. Exclusion Criteria for Lactating Women: Women with preexisting type I or type II 
diabetes, hypertension requiring medication or parathyroid disease will not be eligible for 
participation in the study. Those women with active thyroid disease will not be eligible for 
inclusion in the study; however, women who have normal thyroid parameters on thyroid 
replacement hormone will be eligible for participation in the study. In addition, because of 


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the potentially confounding effect of multiple gestations/births (e.g. twins, triplets), women 
will multiple gestations/births will not be eligible for participation in the study. 
 
No racial or ethnic groups will be excluded from this study.  
 
6.0  
Number of Subjects 
For this initial Pilot study, we will enroll 50 mothers/infant dyads in total, 25 in the control 
and 25 in the placebo group. 
 
7.0  
Setting 
Mothers and their infants will be initially seen in the MUSC Shawn Jenkins Children’s 
Hospital NICU. If still inpatient for the remainder of their 2-3 visits, they will continue to be 
seen there. Following discharge, study visits will either be conducted in infant High-Risk Clinic 
or the Research Nexus clinic (MUSC Clinical Sciences Building, suite 214). The infant High-
Risk Clinic has exam rooms, registration personnel and nursing staff. The NEXUS outpatient 
clinic has exam rooms, nursing staff, registration staff and lab personnel. We will meet the 
subjects at any of these sites and conduct the visits. There is only potential for 3 outpatient visits. 
 
8.0  
Recruitment Methods 
Mothers of infants and their infants who both meet entry criteria will be approached by 
someone directly involved in the infant’s care which could potentially also be a study personnel. 
The mother will then be consented by the study personnel within the first week of their infants’ 
NICU stay. This could be by telephone if mom has been discharged or in person. Chart review 
will be used to determine eligibility.  
9.0  
Consent Process 
Before performing any procedure, informed consent will be obtained from the mother 
enrolled in this study for both her and her infant child. This consent will be obtained by the PI, co-
investigators, or the Study Coordinator. Consent will be given following an informative discussion 
period and then either by reading the Informed Consent Agreement to the subject or by allowing 
the subject to read the consent, then reviewing it with the subject. This will take place at MUSC, 
either in the NICU or on Labor and Delivery. There could potentially be no waiting period 
between informing the prospective subject and obtaining the consent.  
 
10.0  Study Design / Methods 
 
 
 
 
 
 
 
 
 
 
 
Group A: Standard Supplementation: N=25 
Mother: Prenatal Vitamin (600 IU 
vitamin D3/day) (n=25) 
Infant: 400 IU vitamin D3/day (n=25) 
 
Group B: High-dose supplementation: N=25 
Mother: Prenatal Vitamin + 6,000 IU vitamin 
D3 (6,600 IU vitamin D3/day) (n=25) 
Infant: and 0 IU/day (placebo) (n=25) 


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Mother 
1. Visits: Each mother accompanied by her breastfeeding infant will be instructed to either present to the 
infant’s room in the NICU, Infant High-Risk Clinic, or NEXUS clinic for evaluation.  
 
2. Sociodemographic  and  Medical  History: Following a woman’s informed consent, each woman will 
have generated a study record that will include identifying information such as  name, address, 
sociodemographic, ethnic and racial background, and medical  information that will include past medical 
history and history about the most recent pregnancy, delivery, and postpartum state. This information will 
be collected from the mother via interview and from her clinical medical record and will be collected for 
research purposes only. 
 
3. Questionnaires:  Each woman will be asked to complete a series of questionnaires throughout the 
study that pertain to her health status, activity level, clothing use, dietary intake, and labor/delivery and 
breastfeeding history. 
Initial Visit 
1 Month Follow Up 
2 Month Follow Up 
3 Month Follow Up 
Sociodemographic (Q1)  
 
 
Pregnancy & Delivery 
(Q2) 
 
 
 
Newborn History (Q3) 
 
 
 
Physical Activity (Q4) 
Physical Activity (Q4) 
Physical Activity (Q4) 
Physical Activity (Q4) 
Maternal Clothing (Q5) 
Maternal Clothing (Q5) 
Maternal Clothing (Q5) 
Maternal Clothing (Q5) 
Breast Feeding Log 
(Q6) 
Breast Feeding Log 
(Q6) 
Breast Feeding Log 
(Q6) 
Breast Feeding Log 
(Q6) 
 
Breastfeeding Review 
(Q7) 
Breastfeeding Review 
(Q7) 
Breastfeeding Review 
(Q7) 
 
Infant Health/Diet (Q8) 
Infant Health/Diet (Q8) 
Infant Health/Diet (Q8) 
Maternal/Infant Health 
Assessment (Q9) 
Maternal/Infant Health 
Assessment (Q9) 
Maternal/Infant Health 
Assessment (Q9) 
Maternal/Infant Health 
Assessment (Q9) 
Edinburgh PPD Screen 
(Q10) 
Edinburgh PPD Screen 
(Q10) 
Edinburgh PPD Screen 
(Q10) 
Edinburgh PPD Screen 
(Q10) 
Online DHQ-III 
Online DHQ-III 
Online DHQ-III 
Online DHQ-III 
 
4. Body Mass Index (BMI): Based on a woman’s weight and her height, a woman’s body mass index 
(BMI) will be calculated at her initial visit and all subsequent visits, and will be recorded in Redcap 
 
5. Daily Vitamin D and Prenatal Vitamins: Using a web-based computer program, each mother will be 
randomized to receive one of two vitamin D3 regimens (600 or 6,600 IU/day). The 600 IU of vitamin D 
group will receive this from their prenatal vitamin. The experimental group will continue their prenatal 
vitamin (600IU vitamin D), and 6,000 IU vitamin D which will be from taking six, 1,000IU Vitamin D 
gummies.    
 
6. Laboratory Data from Blood and Urine Samples: At initiation of the study and at each monthly visit 
(1, 2, and 3), each woman will have blood sample drawn (20 mL) for detailed analysis of vitamin D and 
calcium homeostasis. The total blood to be drawn from woman during the 3-month study period will be 
60mL (<1/2 cup). In addition, she will be asked to provide a urine sample to measure her urinary 
calcium/creatinine. A total of up to 4 blood and urine samples will be obtained from each mother, 
depending on continued participation in the study. 


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7. Milk Samples: At time of initiation of the study and at each monthly visit (1, 2, and 3), each mother 
will provide a milk sample from her left breast. The skin will be washed with warm water prior to 
pumping. The amount obtained will be less than 20 mL at each visit. This milk can be pumped that day 
and kept refrigerated or pumped at time of visit. This will then be run on the Calais Milk Analyzer to be 
tested for Fat, Total Protein, True Protein, Lactose, and Calorie/ounce. 
 
8. Degree of Skin Pigmentation will be measured at each visit using the Smart Probe 400, a 
commercially available spectrometer, which is placed lightly on the skin. Sites to be measured include the 
inner and outer lower arm and the buttocks. 
 
9. Depression/Anxiety Screening: Each mother enrolled   in   the   study   will   complete   the Edinburgh 
Postnatal Depression Scale (EPDS) at each monthly study visit. Women who score above the threshold 
consistent with possible depression will be referred to the appropriate trained professional. Any woman 
who responds “yes” to the suicide related statements in the EPDS will be immediately referred to the 
Emergency Room for further evaluation and treatment. 
 
Infant  
1. Outpatient Visits: Each mother will be asked to bring her infant as a scheduled outpatient at her 
monthly visits. If still admitted to the NICU, these monthly visits will be held in the MUSC NICU. If 
discharged, visits will be held at Infant High-Risk Clinic or NEXUS clinic. These visits will be the initial 
at approximately 3-7 days of life, then at months 1, 2, and 3.  
 
2. Questionnaires: We will ask each mother to complete a questionnaire about her baby’s dietary type 
(breastfeeding, formula-supplementation), and feeding frequency for the week prior to each outpatient 
visit. Each questionnaire will take approximately 5 minutes to complete. See maternal section table.  
 
3. Daily Vitamin D Supplementation: Infants of mothers who receive 600 IU of vitamin D3 will receive 
400 IU of vitamin D3 in the form of drops. Infants of mothers who receive 6600 IU of vitamin D3 will 
receive placebo drops. 
 
3. General Health Assessment of Infant: At 1 week after delivery and then monthly until 3-months of 
age, each enrolled infant will undergo a growth (measuring head circumference, weight, length, head 
circumference, Ponderal index, body mass index [BMI]) and general health assessment.  
 
4. PEAPOD Assessment: Prior to discharge from the NICU, the infant will also be placed in the 
PEAPODÒ for body mass composition analysis. The material and data will be specifically obtained for 
research purposes. There is minimal risk. This is a safe device in which the infant lays in for 2 minutes.  
 
5. Blood Sample from Infant: At initial visit, 1-, 2- and 3-months of age, each infant will have a blood 
sample 4mL (<1tsp) taken from either a vein (infant’s hand, arm or foot) or the heel. Total amount 
obtained will be less than 12mL (<1 1/2 tablespoon). The serum sample will be analyzed for vitamin D 
and calcium concentration. Parents will be counseled that if their infant is vitamin D deficient, the infant’s 
primary care physician and they will be contacted regarding the finding(s) with recommended follow-up. 
 
11.0  Specimen Collection and Banking 
Blood, urine and milk specimens will be collected. The specimen will be marked with 
patient name, subject ID, date and time of collection as well as MRN. This is necessary to 
ensure integrity of the data is maintained. These specimens will be stored in a double 


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locked laboratory maintained by Dr. Carol Wagner and Dr. John Baatz in the Darby 
Children’s Research Institute (DCRI), Room 304. The specimens will be stored for up to 5 
years. The PI and the co-investigators as listed on the IRB application will have access to 
the specimens and associated data and will be responsible for receipt or transmission of 
them. Specimens will not be sent to an outside facility.  
Specimen/Banking for Future Use 
• 
Describe the linkages to subjects and who will have access to subject identities. 
o Specimens will be stored in -80oC freezer with study ID and 
participant name, MRN on samples. The samples will be located in 
the double locked lab of Drs. Carol Wagner and John Baatz. These 
samples will be destroyed after 5 years.  
o This information will be maintained in a database and all other data, 
including results of the study will be in a separate database that is 
password protected with the results by subject ID. 
• 
List the data to be stored or associated with each specimen. 
o In one password-protected database: subject ID, participant name, 
MRN 
o In a separate password-protected database: subject ID, results 
• 
State whether samples could be used for genetic analysis. 
o Yes, blood samples will be used for genetic polymorphisms of 
vitamin D binding proteins as this can affect vitamin D metabolism 
and processing and well as overall vitamin D status.  
• 
Can specimens be withdrawn? If so, what is the process.   
o Yes, if a mother wants her samples withdrawn, she will inform the 
PI in writing of her request and sample(s) will be destroyed. This 
will be documented in the database and in the participant research 
record.  
12.0  Data Management  
 
All maternal and infant blood and urine samples obtained will be labeled with the patient’s name, 
subject ID number, and medical record number to ensure proper tracking.  Each study subject and her 
infant then will be assigned a specific study number without any reference to the woman or infant’s name, 
which will be used to enter all data into RedCap. We will follow all HIPAA guidelines to maintain 
protection of patient/subject information. With these precautions in place, there is a remote risk that the 
samples and data could be linked to the woman and her infant; however, the study number and the name 
will be kept in a separate file in the locked office of Dr. Irons. The data will be entered into a secured 
database with only the study number entered, thus ensuring that the number and data will be kept 
separately from the woman and infant’s names.  
• 
Describe the data analysis plan, including any statistical procedures. 


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Power Analysis
 
F tests - ANOVA: Repeated measures, within-between interaction based on effect size calculated from figure 
above. 
Analysis:   A priori: Compute required sample size  
Input:        Effect size f = 0.834 
                   α err prob = 0.05 
                   Power (1-β err prob) = 0.80 
                   Number of groups = 2 
                   Number of measurements = 3 
                   Corr among rep measures = 0.5 
                   Nonsphericity correction ε = 1 
Output:     Noncentrality parameter λ   = 25.0400160 
                   Critical F = 4.4589701 
                   Numerator df = 2.0000000 
                   Denominator df   = 8.0000000 
                   Total sample size = 6 (3 per group) 
                   Actual power = 0.9620629 
 
13.0  Provisions to Monitor the Data to Ensure the Safety of Subjects 
 
A. Creation of a Data and Safety Monitoring Board (DSMB): We will have a Data and Safety 
Monitoring Committee (DMSC) in place. Two of the members of the DSMB are physician scientists 
external to MUSC well known in the field of neonatology and nutrition. Dr. Sarah Taylor, MD a 
neonatologist at Yale University, and Dr. Rita Ryan, MD, a neonatologist at Rainbow Babies and Children’s 
Hospital. One member will be a biostatistician at MUSC. He will maintain the database of the study, follow 


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HIPAA guidelines, and conduct ongoing interim analyses to ensure that the risk: benefit analyses remain in 
favor of benefit to the subjects. 
 
B. Conduct ongoing monitoring of interventional trial by those who have appropriate 
expertise to accomplish the trial’s mission: The PI will be responsible for checking laboratories of the 
women within 24 hours of their reporting and then entering the laboratory data into the computer. She will 
be provided with a set of normative laboratory values as a reference. The PI will review values weeklyor 
will be notified if a value falls outside of the referent value range. All data will be verified independently 
by Renee Washington, MA. The DSMB will be notified via email with source documents and adverse 
report sheets if a subject’s value falls outside the referent value range. In addition, all adverse events will 
be reviewed quarterly by the DSMC, whose report will be forwarded to the IRB on a quarterly basis. The 
Investigators will report all Serious Adverse Events by email to the IRB and the DSMC; in addition, the 
IRB will receive a email report within 10 days of the Clinical Investigators’ knowledge of the Event. In 
addition, the investigators will generate a quarterly report to the DSMC regarding subject enrollment, 
subject completion, adverse events and serious adverse events. The DSMC will review the report and a 
summary letter with their findings sent to the IRB. 
 
C. Interim Data Analyses & Monitoring: Interim analyses for the evaluation of safety and efficacy 
will be conducted based on the recommendations of the Data Safety & Monitoring Board for two reasons: 
more than 10% of subjects in a given arm had achieved urinary calcium/creatinine ratios outside the 
acceptable range or there is evidence to the DSMC that there is significant clinical benefit to a given 
treatment arm over the other arm or arms of the study. The DSMC will serve to monitor for safety and 
efficacy. DSMB reports will include summary statistics: on mother-child recruitment (expected vs. actual); 
mother-child follow-up visits (expected vs. actual); data form quality (completion and timeliness of forms); 
tracking of data editing; demographics of the randomized mothers-children; aggregate safety; aggregate 
efficacy; and related information. The DSMB also will monitor the trial from the standpoint of futility using 
the techniques of Lan and Wittes (43). 
 
14.0  Withdrawal of Subjects 
• 
Describe anticipated circumstances under which subjects will be withdrawn from 
the research without their consent, including stopping participation for safety 
reasons. 
o Mother and infant move away from the study site  
o Infant is transferred to another facility 
o If mother had baseline hypercalcemia or hypercalciuria or baby had 
hypercalcemia, they would exit the study  
• 
Describe any procedures for orderly termination of subjects by investigator. 
o The PI would notify the subjects in both writing and verbally if their 
participation is terminated.  
• 
Describe procedures that will be followed when subjects voluntarily withdraw from 
the research, including partial withdrawal from procedures with continued data 
collection. 
o For voluntary withdrawal, the participant would notify the PI in 
writing. If mother requests to withdrawal either her or the infant, 
they will both be removed from the study without further data 
collection.  


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15 Risks to Subjects 
 
1. The Risks of Phlebotomy: 
a. Maternal: The total amount of blood to be drawn at each maternal visit is 15 mL. Using 
aseptic technique, blood will be drawn monthly for a maximum of 4 blood draws (initial visit 
and monthly beginning at month 1 through month 3) during the study period and the total 
amount of blood over the study period will not exceed 60mL (<½ cup). Momentary 
discomfort may occur when skilled technicians or nurses perform phlebotomy with slight pain 
when the needle is inserted into a vein. Pressure will be applied to the arm following the 
procedure to minimize the risk of bruising; however, despite these efforts, a bruise may form 
at the site. Lastly, there is a slight chance of inflammation of the vein and/or blood clot 
formation, but this is extremely rare. 
b. Infant: The total amount of blood to be drawn during the study period (initial visit and 
months 1, 2, and 3) will be 2 mL at each visit (less than a teaspoon or less than 0.6% of the 
infant’s total blood volume) or 10 mL (<2 tablespoons) over the course of 3 months. Using 
aseptic technique, blood will be drawn from the infant’s hand, arm or heel either via 
venipuncture or heel stick. As with any blood draw, there may be a slight pain when the 
needle is inserted into a vein. Momentary discomfort may occur when skilled technicians or 
nurses perform phlebotomy. Pressure will be applied to the arm following the procedure to 
minimize the risk of bruising; however, despite these efforts, a bruise may form at the site. 
Lastly, there is a slight chance of inflammation of the vein and/or blood clot formation, but 
this is extremely rare.  
 
2. The Risks of Randomization in the Study:  
a. There is a possibility that the treatment that each subject receives may prove to be less 
effective or to have more side effects than the other study treatment(s) or other available 
treatments. Because the ideal dose of vitamin D during lactation for mothers of preterm 
infants is unknown presently, the only way to determine the optimal dose is through a 
well-controlled, well designed scientific study that we are attempting to initiate.  
 
3. The Risks of High Dose Maternal Vitamin D Supplementation are hypercalciuria and 
hypercalcemia. There is a small risk that the woman’s blood calcium level would increase in 
response to higher dose vitamin D supplementation. The body responds by excreting calcium in 
the urine to maintain normal calcium levels. If calcium levels in the blood continue to rise, then 
serum calcium will increase leading to hypercalcemia. This process takes weeks to months to 
occur. It takes approximately 3 months to reach steady state on an increased vitamin D 
intake/regimen. In related studies conducted at MUSC comparing maternal vitamin D 
supplementation with 400, 2400, or 6400 IU of vitamin D3/day and their infants for 7 months, 
there were no differences in infant or maternal safety parameters (22). There were no reported 
episodes of either hypercalciuria or hypercalcemia in any of the treatment groups. In fact, maternal 
25 (OH)D levels were higher in the 6400 IU/day treatment group compared to the 400 IU group. 
In breastfed infants, there was no statistical difference in the average circulating 25 (OH)D in 
either treatment arm (22).  
a. To ensure that each lactating mother and her breastfeeding infant and each nonlactating 
mother (and her formula-feeding infant as a control) maintain normal vitamin D status 
throughout the study period and not develop hypervitaminosis D, both mother and infant 
will be screened monthly for hypercalciuria, the first indicator of hypervitaminosis D. All 
study subjects will be monitored closely for hypervitaminosis D. Operationally, we define 
caution limits as urinary Ca/creatinine ratio > 0.8 mmol/mmol. Whenever any patient 


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exceeds the caution limit, a specific case study will be initiated by the Data Safety and 
Monitoring Committee (DSMC) to examine the contribution of sunlight exposure and 
other confounding factors. Operationally, we define upper limits as a urinary 
calcium/creatinine ratio > 1.0 mmol/mmol Ca/creatinine ratio. This is a conservative upper 
limit as circulating levels achieved in individuals living in sun-rich environments achieve 
this amount without causing toxicity. The DSMC will be convened to review any patient 
who exceeds any predefined toxic limit for any outcome. If the elevated levels are 
unexplained, the patient will be withdrawn from supplementation but will continue to be 
monitored. 
 
4. The Risk of Maternal Vitamin D Supplementation on the Infant: The developing infant’s 
vitamin D levels are 50% of maternal serum levels at birth and rise gradually over the next 2-3 
months if there is adequate delivery or synthesis (by skin). Because mothers who are breastfeeding 
and who are deficient in vitamin D produce milk that is deficient in vitamin D, there is a need to 
supplement either the infant directly or the mother herself. This study is designed to increase 
maternal circulating 25(OH)D levels of lactating women through the use of high dose 
supplementation. Approximately ~20% of maternal vitamin D passes into the milk, and therefore, 
the risk of hypervitaminosis D in infants whose mothers are receiving the 6,600 IU/day vitamin D3 
regimen—based on predicted maternal circulating 25(OH)D is extremely low (22). There is a risk 
of hypovitaminosis D to the infant whose mother is receiving the 600 IU/day D3 regimen if that 
infant is not receiving the infant vitamin D3 drops that are prescribed as part of that treatment arm 
(Group A).  
Minimizing Risk of Hypovitaminosis D Identified by an Infant’s Participation in this 
Study: As an additional safety measure, if at visit 4, any infant in any treatment arm, 
circulating 25(OH)D concentration is found to be below normal [defined a priori as <20 
ng/mL (<50 nmol/L)], will be supplemented with the 400 IU vitamin D/day. If an infant’s 
circulating 25(OH)D concentration is <10ng/mL (<25 nmol/L) and is associated with a serum 
calcium level of <7.5 mg/dL (<18.75 nmol/L) at 3 months or older, then compliance of mother 
with her prescribed vitamin D regimen and that of the infant will be assessed. If mother has 
been compliant with the prescribed infant vitamin D3 supplementation and the infant continues 
to have evidence of vitamin D deficiency with hypocalcemia, the infant will be referred to 
their pediatrician for further evaluation. 
 
5. The Risk of Milk Collection: There is minimal physical risk in donating a milk sample either by 
using a manual or electric pump.  
 
6. The Risk of Breach of Confidentiality through Collection of Medical History and Clinical 
Laboratory Data: As with any research study, there is a small risk that medical information, 
laboratory reports and data could become available to individuals outside of the study. All study 
personnel will adhere to HIPAA guidelines and regulations. 
 
7. The Risk of Measuring Degree of Skin Pigmentation using a skin tone chart and the Smart 
Probe 400, a commercially available spectrometer, is minimal. The probe, from which a beam of 
visual spectrum light is emitted, and the absorbance measured, lightly touches the skin. Readout of 
the absorbance corresponds to the degree of pigmentation on a scale from 1 to 12. This technology 
is similar to what is currently used to measure bilirubin levels in neonates (transcutaneous 
bilirubin spectrometer) without any known side-effects. The information will be entered as a 
number into the database that will not be linked to the individual’s name or medical record number 
such that confidentiality is maintained.  
 


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8. The Risk of Postnatal Depression/Anxiety Screening: The benefits of postnatal 
depression/anxiety screening far outweigh the risks. There is a risk that if the women are not 
screened for postpartum depression, they will not receive adequate evaluation and treatment. 
Asking questions about sadness and depression, anxiety and sleep problems can raise 
uncomfortable feelings that could precipitate suicidal ideation; however, any woman with clinical 
depression who goes without medical assessment and treatment is at greater risk. Studies have 
shown that early intervention has a positive impact on the outcome of the woman and her family. 
 
Based on the EPDS screening tests, those women who score in the high range for anxiety and 
depression will be referred to their primary care provider or if having suicidal ideation, to the 
Emergency Department (as outlined below). Those women who qualify for further evaluation and 
treatment will receive both written and verbal information regarding postpartum depression and 
the ill effects it can produce for themselves, their infant and other family members. Women will be 
referred to therapists at MUSC who specialize in postpartum depression. Any woman responding 
positively to the suicidal questions will be escorted to MUSC’s Emergency Department for further 
evaluation and treatment. 
 
9. Unknown Risks: The experimental treatments may have unknown side-effects that previously 
have not been reported (unexpected and unanticipated adverse events or effects). 
 
16.0  Potential Benefits to Subjects or Others 
 
Mothers enrolled in the study who will be taking the 6,600 IU/day vitamin D3 supplementation 
regimen are predicted to have improved calcium and vitamin D homeostasis. In addition, it is hypothesized 
that mothers taking 6,600 IU/day vitamin D will have higher circulating 25(OH)D concentrations, and thus, 
greater transfer of vitamin D in their milk, rendering their milk superior in its antirachitic activity compared 
to the concentration in their milk at baseline or compared to those mothers randomized to the 600 IU vitamin 
D3/day regimen.  
 
Another potential benefit of this study is that data will be generated that will translate into 
recommendations for optimal vitamin D status for the mother-infant breastfeeding dyad and postpartum 
women in a controlled fashion. As was mentioned earlier, increasing numbers of Americans are becoming 
aware of the risks of hypovitaminosis D, and have begun to purchase vitamin D through mail-order vendors; 
thus, higher dose vitamin D supplements are easily available. Without monitoring, the potential for 
overdosing—although small, exists. There needs to be a controlled trial that will serve as a guide for 
practitioners and health care policy makers when recommending the recommended dietary intake for 
vitamin D of lactating women who deliver premature infants.  
 
On a separate issue, women who are suffering from postpartum depression will be identified early 
and referred appropriately for evaluation and treatment. Early identification carries a better prognosis than 
if the symptoms persist without intervention. 
 
17.0  Sharing of Results with Subjects 
At the end of the study we will share with each participant her vitamin D status at baseline, 
and if it has improved or worsened over the course of the study. We will do the same for their 
infants’ vitamin D status. Infant body composition results will also be shared with the mother. 
Maternal milk calorie/oz, protein, carbs, and fats will also be shared.  
 
18.0  Drugs 
 


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The vitamin D gummies (1000 IU/gummy) along with placebo gummies, vitamin D drops, and 
placebo drops will be dispensed by Investigational Drug Services (IDS). At the start of the trial, they will 
dispense 4 bottles of gummies, and 1 bottle of drops. At each of the 3 following visits, they will bring 
their opened bottles, which will be checked for compliance through pill counts. This will also be done at 
the end of the study. All leftover medication will be returned to Investigational Drug Services for 
confiscation. Cholecalciferol, vitamin D3 has an IND 1571 held by Dr. Bruce Hollis and Dr. Carol 
Wagner. 
 
 
 


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