Clinical trial NCT01932788
Conditions: Vitamin D3 Deficiency
Keywords: pregnancy Vitamin D status
Sponsor: Medical University of South Carolina
Start date: 2013-01-01; Completion date: 2018-04-30
Enrollment: 342 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The purpose of this study is to give all mothers the best chance for a healthy pregnancy through vitamin D supplementation. We will study women of diverse racial/ethnic backgrounds who will receive either the current vitamin D standard of 400 IU/day (in the prenatal vitamin) or 4000 IU/day (dose found in previous pregnancy studies to achieve vitamin D sufficiency).

This research is sponsored by the W.K. Kellogg Foundation and the Medical University of South Carolina. The purpose of this study is to examine the effectiveness and infection-fighting properties of the body in relationship to vitamin D levels. This study is being done at the Medical University of South Carolina (MUSC) clinics, and will involve approximately 450 volunteers.

Detailed description:
This project, comprised of discrete studies, expert evaluation and translation for public health campaign, promotes racial health equity for pregnant women. A strong racial disparity in the US surrounds vitamin D status: African American women have 20-fold and Hispanic women 2.4-fold greater risk of deficiency than Caucasian women. In South Carolina, greater than 70% of African American, 33% of Hispanic and 12% of Caucasian pregnant women meet the Institute of Medicine's (IOM's) 2010 definition of vitamin D deficiency (1-3). Such disparity on the basis of race/ethnicity represents a serious public health issue. Yet, some-including the IOM-argue that vitamin D deficiency minimally affects maternal and fetal health and that no vitamin D supplementation study has shown improved pregnancy outcome (3, 4). Results of our two recently completed vitamin D trials involving 510 pregnant women throughout pregnancy suggest otherwise (5-7). In the NICHD trial, women supplemented with 400 IU vitamin D/day (the amount in most prenatal vitamins) vs. 2000 or 4000 IU/day not only had worse vitamin D status throughout pregnancy, but combined higher risk of comorbidities of pregnancy (gestational diabetes, hypertensive disorders, infection, preterm labor/birth and periodontal inflammation) (7). In trial 2, women at two South Carolina community health centers were supplemented with vitamin D and again, higher vitamin D status was associated with lower comorbidities of pregnancy risk (6, 8).

A novel finding from the NICHD trial was that serum levels of active, hormonal vitamin D (1,25(OH)2D) during pregnancy were optimized at twice the level normally observed in non-pregnant women, levels that would be considered toxic in nonpregnant individuals. At no other time in life does this relationship exist. Is the hormone at such levels driving some vital process during pregnancy? If so, what is that process? There are hints as to why 1,25(OH)2D is elevated in pregnant women. During pregnancy, the maternal immune system undergoes drastic changes for fetal protection. For example, if a solid organ composed of 50% mismatched cell markers (antigens) was transplanted into a new host, it would be rejected within hours; however, a fetus that is 50% mismatched with paternal antigens is protected from immune destruction through a process known as immune privilege. During pregnancy the body increases immune suppressive cells, reduces highly activated natural killer and cytotoxic T-cells with capacity to destroy the fetus and still has the capacity to fight off foreign pathogens. While the mechanisms that orchestrate this complex balance are unknown, we hypothesize that due to vitamin D's ability to regulate immune cells, vitamin D is responsible for many immune alterations associated with fetal immune privilege. We further hypothesize that correction of vitamin D deficiency during pregnancy will protect against immune-mediated disorders such as recurrent miscarriage, preterm birth, bacterial infections, periodontal inflammation and gestational diabetes. This premise, tested and confirmed by this project, will lead to public policy changes and equality in vitamin D status during pregnancy. To test the validity of these hypotheses, the goals of this project are as follows:

The central goal of this project is to realize racial equity for all pregnant women and their developing babies in the US. To achieve this goal, there are 3 specific objectives:

1. Determine how vitamin D deficiency leads to immune imbalance and subsequent disparities in pregnancy health outcomes;
2. Determine how vitamin D supplementation prevents such imbalance and disparities; and
3. Translate such findings into public health policy. By comparing women of diverse racial/ethnic backgrounds who receive the current vitamin D IOM standard of 400 IU/day compared to 4000 IU/day \[daily dose shown to achieve optimal production of active vitamin D hormone (1,25(OH)2D)\], the clear effects of vitamin D will be realized. Furthermore, these effects of sufficient vitamin D during pregnancy transcend the racial differences of the women.

Study Design: This is a randomized, placebo-controlled clinical trial of 450 pregnant women who will be enrolled between 10-14 weeks of gestation and followed until delivery. Following written, informed consent, each mother will be randomized to receive either placebo or 4000 IU/day vitamin D3 plus the standard prenatal vitamin (containing 400 IU vitamin D3). She will be followed monthly for a total of nine study visits. These visits will include the initial recruitment of mother at 10-14 weeks of pregnancy, monthly study visits, delivery study visit, and 18-month post-partum follow-up visit for mother and child. The primary outcome variable is maternal and neonatal health status as a function of maternal and infant vitamin D status. Secondary outcome variables to be analyzed, as a function of maternal vitamin D status during pregnancy will include: T-lymphocyte profile, immune function indicators, neonatal growth, inflammatory cytokine profile, methylation patterns of DNA of both mother and her neonate,

1. Subject Selection Criteria and Recruitment: Any mother (18-45 years of age) who presents to her obstetrician or midwife at the Medical University of SC (MUSC), Charleston, SC; Our Lady of Mercy, Johns Island, SC; or Franklin C. Fetter, Charleston, SC obstetrical facilities within the first 14 weeks after her last menstrual period (LMP) with confirmation of a singleton pregnancy will be eligible for enrollment in the study. Mothers of diverse ethnic backgrounds (African-American, Asian, Caucasian and Hispanic) will be actively recruited. A database will be generated weekly of undelivered patients for the labor and delivery staff continue to alert the staff of impending admissions.
2. Exclusion Criteria: Mothers with pre-existing calcium, uncontrolled thyroid disease, parathyroid conditions, or who require chronic diuretic or cardiac medication therapy including calcium channel blockers will not be eligible for enrollment into the study. Mothers with pre-existing sickle cell disease (not trait only), sarcoidosis, Crohn's disease, or ulcerative colitis may not participate in the study. In addition, because of the potentially confounding effect of multiple fetuses, mothers with multiple gestations will not be eligible for participation in the study. A sub-group of approximately 100 subjects with known diabetes, hypertension, HIV, or morbid obesity (body mass index \> 49) will participate in the study.
3. Subject Enrollment: The potential study subject's primary care provider will make first contact on behalf of the study and refer interested mothers to study personnel for further information and invitation. Upon enrollment into the study, expectant mothers at approximately 10-14 weeks' gestation will be randomized to receive one of two treatment regimens of vitamin D3. They will be randomized to 1 of 2 groups: (1) Group A: 400 IU vitamin D3/day-Standard dose treatment of placebo (0 IU vitamin D3) plus prenatal vitamin (400 IU/day); or (2) Group B: 4,400 IU/day (4,000 IU/2 gummies/day + 400 IU/day in prenatal). If, after 3 months of vitamin D supplementation, a mother is still frankly vitamin D deficient (less than 15 ng/ml), she will receive open label active vitamin D gummies for the remainder of her pregnancy. To attain equal group numbers (n=150) and balanced numbers by racial/ethnic group (African-American, Caucasian and Hispanic, which represent the predominant groups delivering at MUSC), a stratified block randomization will be used.
4. Race/Ethnicity Defined: Each mother will be asked to define the racial/ethnic group to which she belongs: (a) African-American, (b) Caucasian, (c) Hispanic, (d) American Indian, (e) Asian, and (f) other. Because few mothers of American Indian (0.08% of all deliveries) and Asian (1% of all deliveries) descent deliver at MUSC, there is insufficient power to include those groups in this study. In addition, the mother, by self-report, will be asked to define the race/ethnicity of her parents and the father of the baby and his parents.
5. Study Site Population: The Medical University of South Carolina (MUSC) is an urban medical university in Charleston, SC that serves patients predominantly in the Charleston Tri-County area (80%) and 20% from surrounding regions in South Carolina. We perform 2000 deliveries each year. The ethnic composition of our obstetrical population is 57% African American, 36% Caucasian, and 6% Hispanic and 1% Asian. Franklin C. Fetter and Our Lady of Mercy Clinics primarily see Hispanic patients for prenatal care. These Hispanics have prenatal care coverage provided by a Roper Hospital, Roper St. Francis Healthcare, Charleston, SC grant and include approximately 150 deliveries per year at Roper Hospital. MUSC provides care to any woman presenting in labor who requires routine care or who is referred from outlying hospitals due to high-risk pregnancy for which delivery at a Regional Perinatal Center (RPC) is appropriate, regardless of ability to pay.
6. Enrollment and Follow-Up Subjects: The Study Coordinator will be responsible for assisting in screening, enrollment and follow-up. To maintain data quality, the Study Coordinator will review every form to check for basic problems such as missing data. Labor and delivery staff will alert the study coordinator or the Clinical PI's of the admission of any study subjects. Each month the Study Coordinators will generate a report on patient recruitment and retention to be reviewed jointly by the research team. A computer-generated calendar will serve as a reminder to contact recruited patients two days prior to their appointment. Each month the Study Coordinators will generate a report on patient recruitment and retention that will be reviewed by Dr. Wagner.
7. Follow-up Infant Visit: The Study Coordinator will follow up by phone several times from birth to 18 months in order to verify subject's contact information. The infant (accompanied by mother) follow-up visit will take place at MUSC's CTRC Outpatient Clinic around 18 months postpartum. The infant will have blood drawn for vitamin D and will have a dental assessment of the primary teeth, including digital photographic images.

With the assistance of Dr. Thomas Hulsey, epidemiologist and co-investigator of this application, sample size calculations were made. All outcome markers are ratio-scaled and normally distributed. Because of the possibility of premature delivery and patient attrition during this study, enrollment of additional subjects over that minimally needed, will provide a cost effective opportunity to conduct additional analyses examining changes in vitamin D associated with diet, seasonality, and maternal medical complications. As one of the major goals of this study is to explore maternal health disparities by ethnicity, the two supplemented groups (400 and 4,400 IU/day) will be balanced by ethnicity (equal numbers of Caucasian, African American, and Hispanic). This sampling distribution will be accomplished by stratified randomization. This sampling frame was selected to provide: (1) sufficient numbers to examine associations with covariates; (2) the volume of initial and follow up samples will be manageable by our laboratory; (3) over-samples to compensate for losses to follow up; and, (4) stabilizes the sophisticated statistical analysis techniques (repeated measures).

The sources of research material will be maternal interview, history and physicals at clinic visits, the questionnaires, the lab reports for blood and urine analyses, the dental periodontal exam results, the dental salivary occult blood results, the vaginal swab results, the placental pathology report, the medical records of each mother and her infant following delivery, and the photographic teeth results on the infants.

The material/data that is routinely found in a maternal health record during pregnancy (including information on past pregnancies and disease history) will be utilized in the study. There are additional materials and data from questionnaires, blood, urine, saliva, infant teeth, placental tissue and vaginal samples that will be obtained for research purposes only.

A master list will be stored with study files accessible for clinical intervention only. The PI and study staff will have access to the master list. All archived samples and samples provided to collaborating laboratories will be identified with study subject numbers only. The DNA samples collected will have no recontact.

The study subject's primary care provider(s) will make first contact on behalf of the study so there is no appearance of a breach in confidentiality. Once a subject has expressed interest in this research study, to obtain informed consent, one of the study coordinators will explain to each subject in lay language the purpose, benefits and risks of the investigation. Consent will be given following an informative discussion period and either by reading the Informed Consent to the subject or by allowing the subject to read the consent, then reviewing it with the subject. All study coordinators have taken the Research Coordinator course and have passed their Miami CITI. Every attempt will be made to conduct this study in full compliance for the protection of the subjects. For those women who are not fluent in English and whose first language is Spanish, a Spanish version of the consent form will be used (translated by BiLingo from IRB approved English consent and submitted as an amendment to IRB). Following the subject's verbal agreement to participate in the study, the subject will sign the informed consent; the study coordinator also will sign the consent. A copy of the signed, written informed consent will be given to the study subject. The original informed consents will be placed in locked research file in study coordinators' office, 20 Ehrhardt St, #4. A copy of the informed consent also will be placed in the subject's study binder (located in locked cabinets at same location). A study coordinator note will be placed in Epic identifying an MUSC woman as participating in a vitamin D and pregnancy RCT.

All of the investigators have conducted several clinical trials; they are well versed in the issues of patient/subject confidentiality and have completed the Miami CITI course. They are nationally and internationally known in their fields. All study coordinators have completed the Miami CITI and the Research Coordinator course at MUSC. The endeavors of the PI, co-investigators and study coordinators to maintain subject confidentiality will be ensured.

All maternal blood, urine, and vaginal samples as well as infant blood and placental tissue will be labeled with the patient's name and medical record number to ensure proper tracking. Each study subject (and 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 the web database. 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 offices of the study coordinators. The data will be entered in 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. All this information will be kept confidential, and when reported in scientific journals, the information will not have any identifying information regarding study subjects.

Should any untoward reaction occur, subjects would receive treatment at the Medical University of South Carolina. The study will cover the costs of laboratory tests; however, additional costs of treatment will be the responsibility of the subject as described in the informed consent.

This study meets the guidelines of clinicaltrials.gov as a clinical trial requiring a Data Safety and Monitoring Plan and a Data and Safety Monitoring Committee. Two of the members of the Data and Safety and Monitoring committee are scientists external to MUSC well known in the field of vitamin D metabolism and calcium metabolism. Two of the members are physician scientists external to MUSC, who are also well known in the field of calcium and vitamin D metabolism. A fifth member will be Dr. Tom Hulsey, epidemiologist at MUSC with considerable experience serving on the DSMC of other clinical trials. Dr. Hulsey was the chair of the DSMC of the two prior vitamin D supplementation trials conducted by this study team. He will maintain the database of the study, follow HIPAA guidelines, and conduct ongoing interim analyses to ensure that the risk: benefit analyses remain in favor of benefit to the subjects.

Creation of a Data and Safety Monitoring Board (DSMB): This grant application meets NIH policy and Guidelines on the inclusion of a Data and Safety Monitoring Plan for clinical trials mL and http://grants.nih.gov/grants/guide/notice-files/not98-084.html). MUSC as the Institute and Center (IC) of this grant will have a Data and Safety Monitoring Committee (DMSC) in place as outlined in this grant. Two of the members of the DSMC are scientists external to MUSC well known in the field of vitamin D and calcium metabolism. Two of the members are physician scientists external to MUSC who also are well known in the field of calcium and vitamin D metabolism. A fifth member will be Dr. Tom Hulsey, epidemiologist at MUSC with considerable experience serving on the DSMC of other clinical trials (current and completed). He will maintain the database of the study, follow 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 MUSC clinical lab is required to notify the PI of any critical lab values. The Study Coordinators will be responsible for checking laboratories of the women within 72 hours of their reporting and then entering the laboratory data into the computer. They will be provided with a set of normative laboratory values as a reference. The Clinical PI will review values during the weekly study meeting or will be notified if a value falls outside of the referent value range. All data will be verified independently by the Data Processing Center under the direction of Dr. Hulsey. The DSMC will be notified via telephone and fax 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 Kellogg Foundation on a quarterly basis and the IRB yearly. The Investigators will report all Serious Adverse Events by telephone to the IRB and the DSMC; in addition, the IRB, the DSMC, and Kellogg Foundation will receive a written 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.

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. The DSMC will serve to monitor for safety and efficacy. DSMB reports produced by Dr. Hulsey's team will include summary statistics: on mother recruitment (expected vs. actual), data form quality (completion and timeliness of forms); tracking of data editing; demographics of the randomized mothers; 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 (119).

First, differences in losses to follow-up and missed visits will be compared between the six comparison groups to detect any potential bias (400 IU group stratified by race: African American, Hispanic and Caucasian; 4000 IU group also stratified by race). The biggest problem with unbalanced losses will be the loss of statistical power. We will aggressively attempt to avoid any losses by monitoring adherence to visits. In the event that there are differential losses between ethnic groups or supplementation groups, we will conduct statistical tests to ascertain whether these are random or biased. Random losses will be reported but should not affect outcomes. Biased, or non-random, losses will be assessed on their potential impact on differences in outcomes. If necessary, we will conduct a propensity score analysis to control for any statistically significant differences in non-random losses. Following that, differences in compliance to supplementation will be compared as an 'intent-to-treat' design. Any significant deviation from protocol will be considered in subsequent analyses. In this section, we will report on: (1) the deviations from protocol; (2) outcomes comparisons using an efficacy design to test for true differences associated with subjects who adhered to protocol; and, (3) outcomes comparison using the intent to treat (effectiveness) design to test for actual differences that might be expected in a non-controlled intervention. This will help determine whether this type of supplementation could be expected to be effective if applied to the general population.

Next, vitamin D levels at study entrance (10-14 weeks' gestational age) will establish the baseline for each supplementation group. It is expected that at baseline there should not be any differences between groups. Because we do expect differences between ethnic groups (which accounts for the stratification), mean vitamin D at baseline will be compared by two-way analysis of variance (ethnicity by experimental group).

In step 3, using repeated measures analysis of variance, the changes in vitamin D will be described separately for each ethnic group within each of the supplemented groups. This will depict separately for each ethnic group the changes in vitamin D over time as a function of the level of supplementation. This method will reveal how soon after supplementation the changes in blood (from baseline) are detected, the linear change in blood vitamin D over time and, when and if a peak level (or plateau) was attained. Adjusted differences will be examined by use of multiple regression with and without the inclusion of other covariates. Regression coefficients provide the formula for the linear association between vitamin D in blood and time, separately for each ethnic group. In the event that there is a non-linear change over time, appropriate data transformations will occur as necessary. This step also will provide information on the magnitude of importance that covariates play in changes in vitamin D over time within each ethnic group. As an example, we may find that seasonality plays a significant role in changes in vitamin D for Caucasian women but the association is not as strong for African American women. Further, these associations may become similarly unimportant at the higher levels of supplementation.

The next series of analyses will compare the differences in vitamin D between supplemented groups over time between ethnic groups. This will be accomplished by use of a two-way (ethnicity and supplementation group) repeated measures analysis of variance. This technique will detect any significant differences between main effects (ethnic groups and supplementation groups) and interaction effects (ethnicity by supplementation) in changes in vitamin D over time. Multiple regression also will be used to examine the changes with and without the use of potential confounders.

In secondary analyses, multi-level mixed-effects models will be used to estimate the average individual monthly rate of change in 25(OH)D, compare this rate between dose groups, and explore the effects of covariates on the rate of change. These models include fixed effects for dose group, time, and the group-time interaction, and a random intercept effect, with additional covariate effects as required. Time will be considered a continuous variable, measured in months rather than assuming structured visit occurrences. An unstructured covariance matrix will be assumed. The multi-level modeling approach also will be used to evaluate the longitudinal association between 25(OH)D and the safety parameters calcium, iPTH (log-transformed), phosphorus, and urinary calcium, creatinine, and calcium: creatinine levels. The cumulative occurrence of pregnancy complications such as preterm labor and infection will be compared between dose group levels using logistic regression. All analyses will be performed using SAS (SAS Institute Inc., Cary NC).

Participant attrition and missing data:

Because the primary endpoint of the study is change in 25(OH)D from baseline to delivery, the primary analysis will be restricted to participants who have remained in the study until delivery and provided a blood sample within 6 weeks prior to delivery, at delivery, or at the post-delivery visit (completers-only analysis). Typically, multiple imputations would be used to impute missing values in support of the favored intention-to-treat analytic approach. Because the multiple imputation model for this analysis would have required variables also measured in the final blood sample, however, it cannot be used to impute cases with a missing final blood sample. Thus, to assess the primary findings' robustness to assumptions about the missing data, we will perform a sensitivity analysis. Missing data will be imputed under the following conditions: cases with missing endpoints that experienced no change in both groups; experienced the group-specific median change observed in completers; experienced no change in the 400 IU group and minimal change in the 4000 IU group. In the secondary analyses using multi-level mixed-effects models for longitudinal modeling, all available data points will be used, as it is not necessary to delete cases with missed timepoints when using this approach.

Arms: arm 1: Subjects randomized into this arm will receive supplementation with 4000 IU/day vitamin D3 in gummy vitamin form, plus the standard prenatal vitamin (containing 400 IU vitamin D3. arm 2: Supplementation with placebo gummy vitamin form, plus the standard prenatal vitamin (containing 400 IU vitamin D3)
Interventions: intervention 1: Vitamin D3 4000 IU in gummy form intervention 2: Placebo gummy vitamin
Intervention descriptions: intervention 1: Subjects randomized to the Vitamin D3 4000 IU gummy vitamin arm, the investigational drug, will consume 4 gummies/day beginning at 10-14 weeks of your pregnancy. All subjects will consume a prenatal vitamin (either chewable or pill form) also beginning at 10-14 weeks of your pregnancy. intervention 2: Subjects randomized to receive placebo, also in gummy form but manufactured without Vitamin D3, will consume 4 gummies/day beginning at 10-14 weeks of pregnancy. All subjects will take a prenatal vitamin (either chewable or pill form) also beginning at 10-14 weeks of pregnancy.

Protocol documents:
Prot_000.pdf: 
 
 
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PI Name:  Carol L. Wagner, M.D. 
 
 
Study Title:  Preventing Health Disparities during Pregnancy through Vitamin D Supplementation  
 
TABLE OF CONTENTS  
 
Specific Aims  
 
 
 
 
 
 
 
2 
Background and Significance  
 
 
 
 
 
2 
Preliminary Studies 
 
 
 
 
 
 
 
6 
Research Design and Methods 
 
 
 
 
 
8 
Protection of Human Subjects  
 
 
 
 
 
13 
References 
 
 
 
 
 
 
 
 
19 
 
NCT#01932788  
 
 
6/17/2016 
  
 
 
 
 
 
 
 
 
 
 
Medical University of South Carolina 
Protocol 


 
 
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A. SPECIFIC AIMS 
 
This project, comprised of discrete studies, expert evaluation and translation for public health campaign, 
promotes racial health equity for pregnant women. A strong racial disparity in the US surrounds vitamin D 
status: African American women have 20-fold and Hispanic women 2.4-fold greater risk of deficiency than 
Caucasian women. In South Carolina, greater than 70% of African American, 33% of Hispanic and 12% of 
Caucasian pregnant women meet the Institute of Medicine’s (IOM’s) 2010 definition of vitamin D deficiency 
(1-3). Such disparity on the basis of race/ethnicity represents a serious public health issue. Yet, some—
including the IOM—argue that vitamin D deficiency minimally affects maternal and fetal health and that no 
vitamin D supplementation study has shown improved pregnancy outcome (3, 4). Results of our two recently 
completed vitamin D trials involving 510 pregnant women throughout pregnancy suggest otherwise (5-7). In 
the NICHD trial, women supplemented with 400 IU vitamin D/day (the amount in most prenatal vitamins) vs. 
2000 or 4000 IU/day not only had worse vitamin D status throughout pregnancy, but combined higher risk of 
comorbidities of pregnancy (gestational diabetes, hypertensive disorders, infection, preterm labor/birth and 
periodontal inflammation) (7). In trial 2, women at two South Carolina community health centers were 
supplemented with vitamin D and again, higher vitamin D status was associated with lower comorbidities of 
pregnancy risk (6, 8).  
A novel finding from the NICHD trial was that serum levels of active, hormonal vitamin D (1,25(OH)2D) 
during pregnancy were optimized at twice the level normally observed in non-pregnant women, levels that 
would be considered toxic in nonpregnant individuals. At no other time in life does this relationship exist. Is 
the hormone at such levels driving some vital process during pregnancy? If so, what is that process? There are 
hints as to why 1,25(OH)2D is elevated in pregnant women. During pregnancy, the maternal immune system 
undergoes drastic changes for fetal protection. For example, if a solid organ composed of 50% mismatched 
cell markers (antigens) was transplanted into a new host, it would be rejected within hours; however, a fetus 
that is 50% mismatched with paternal antigens is protected from immune destruction through a process 
known as immune privilege. During pregnancy the body increases immune suppressive cells, reduces highly 
activated natural killer and cytotoxic T-cells with capacity to destroy the fetus and still has the capacity to 
fight off foreign pathogens. While the mechanisms that orchestrate this complex balance are unknown, we 
hypothesize that due to vitamin D’s ability to regulate immune cells, vitamin D is responsible for many 
immune alterations associated with fetal immune privilege. We further hypothesize that correction of vitamin 
D deficiency during pregnancy will protect against immune-mediated disorders such as recurrent miscarriage, 
preterm birth, bacterial infections, periodontal inflammation and gestational diabetes. This premise,tested 
and confirmed by this project, will lead to public policy changes and equality in vitamin D status during 
pregnancy. To test the validity of these hypotheses, the goals of this project are as follows: 
The central goal of this project is to realize racial equity for all pregnant women and their developing babies 
in the US. To achieve this goal, there are 3 specific objectives:  
1) Determine how vitamin D deficiency leads to immune imbalance and subsequent disparities in pregnancy 
health outcomes;  
2) Determine how vitamin D supplementation prevents such imbalance and disparities; and  
3) Translate such findings into public health policy.  
By comparing women of diverse racial/ethnic backgrounds who receive the current vitamin D IOM standard 
of 400 IU/day compared to 4000 IU/day [daily dose shown to achieve optimal production of active vitamin D 
hormone (1,25(OH)2D)], the clear effects of vitamin D will be realized. Furthermore, these effects of sufficient 
vitamin D during pregnancy transcend the racial differences of the women.  
 
B. BACKGROUND AND SIGNIFICANCE  
 
Despite its discovery a hundred years ago, vitamin D has emerged as one of the most controversial nutrients 
and prohormones of the 21st century. Its role in calcium metabolism and bone health is undisputed but its role in 
immune function and long-term health is debated. There are clear indicators from in vitro and animal in vivo 
studies that point to vitamin D’s indisputable role in both innate and adaptive immunity; however, the 


 
 
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translation of these findings to clinical practice, including the care of the pregnant woman, has not occurred. 
Until recently, there has been a paucity of data from randomized controlled trials to establish clear cut beneficial 
effects of vitamin D supplementation during pregnancy. 
What is vitamin D? Vitamin D is a preprohormone that is made by most living plants and terrestrial animals. 
In the true sense of the word, vitamin D is not a “vitamin” because the main source of vitamin D is that which we 
synthesize ourselves—in our skin—with less than 10% coming from dietary sources. Vitamin D comes in two 
major forms—vitamin D2 or ergocalciferol and vitamin D3 or cholecalciferol. While certain plants are capable of 
making both forms of vitamin D, the major form made by plants is vitamin D2 following ultraviolet B exposure of 
the provitamin D2 ergosterol (9, 10). In comparison, humans can metabolize both vitamin D2 and D3, but can only 
synthesize de novo vitamin D3. The de novo synthesis of vitamin D3 in humans and other animals begins in the 
skin with the parent compound 7-dehydrocholesterol or provitamin D3. Following exposure to ultraviolet B 
radiation in the range of 290-315 nm, 7-dehydrocholesterol becomes previtamin D3. Through a subsequent 
thermal reaction in the skin, previtamin D3 is isomerized into vitamin D3. It is important to note that unlike other 
hormones in the body whose main substrate is cholesterol, vitamin D synthesis requires the 7-
dehydrocholesterol precursor and sunlight at a specific wavelength and angle. Without this reaction, humans 
are dependent on dietary intake of vitamin D, which may be in the form of either vitamin D2 or D3. A western 
diet, however, provides less than 200 IU (5 µg) vitamin D/day, a point that was reestablished in our two recently 
completed vitamin D clinical trial during pregnancy (5).  
Following its synthesis, vitamin D binds to vitamin D binding protein (VDBP) and finds its ways into the 
circulation. Dietary and endogenous vitamin D appear to act similarly with half-life between 12-24 hours, the 
length of time depending on how quickly the liver converts vitamin D to 25-hydroxy-vitamin D (also known as 
calcidiol). Vitamin D is measured in international units (IU) or micrograms with a known conversion of 40 IU 
equal to 1 microgram.  
While there appears to be a differential conversion rate of the two forms of vitamin D to 25(OH)D (11), the 
conversion of either form is dependent on a functional liver and the activity of 25-hydroxylase. Thus, those with 
impaired liver function will have diminished conversion of vitamin D to 25(OH)D. Following its synthesis, 25(OH) 
then enters the circulation where it is tightly bound to VDBP. Only a small amount of 25(OH)D is unbound or 
“free.” The half-life of 25(OH)D is 2-3 weeks, making it a much better indicator of the body’s vitamin D status 
than vitamin D. Of note, 25(OH)D can be expressed as ng/mL or nmol/L. The conversion from ng/mL to nmol/L is 
2.5; thus, a 25(OH)D concentration of 20 ng/mL equals 50 nmol/L. 
Once 25(OH)D is formed in the liver, it enters the circulation. Best known is the processing of 25(OH)D by 
the kidney where 25(OH)D complexed with VDBP and megalin is taken up by the epithelial cells of the proximal 
tubules and converted to the active hormonal form of vitamin D—di-hydroxy-vitamin D (1,25(OH)2D or 
calcitriol)—by the action of the mitochondrial enzyme 1-α-hydroxylase.  
1,25(OH)2D’s endocrine effects include the following classic triad of action: (1) increase intestinal calcium 
(as Ca2+ ions) absorption through the actions of calbindin; (2) increase urinary calcium reabsorption; and (3) 
regulation of parathyroid hormone in a negative feedback loop that allows calcium to be absorbed from the 
gastrointestinal tract, reabsorbed from urine, and metabolized from bone in order to maintain calcium 
homeostasis within the body. Because calcium is essential to all tissues and organs, particularly the heart, 
skeletal muscle and brain, the body will scavenge calcium if necessary from the skeleton. Adequate vitamin D 
must be on hand to provide enough substrate to form 25(OH)D, which in turn, is converted to 1,25(OH)2D, 
whose half-life is 8 hours. In individuals with vitamin D deficiency, only trace amounts of vitamin D will be found 
in the body because whatever comes into the circulation is quickly converted to 25(OH)D and then to 
1,25(OH)2D to maintain calcium homeostasis. 
For decades, it was thought that only the kidney has the capacity to metabolize 25(OH)D; however, 
extrarenal metabolism has been demonstrated in every organ system in the body (12-15). During pregnancy, the 
placenta is probably the most prominent site for extra-renal activation of vitamin D (16). It appears that the 
extrarenal function of vitamin D has more to do with immune function than with calcium metabolism and 
homeostasis. Support for this premise first came from the observations of Mellanby and others at the turn of 
the 20th century: during that time, Mellanby in his study of rachitic children and dogs noted an increased risk of 
respiratory infections in those afflicted (17, 18). Additional reports came from those working with tuberculosis 


 
 
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patients and the beneficial effect of being in sunlight and outdoors in the treatment of the condition (19). Weick 
(20) in 1967 and Rehman (21) in 1994 independently observed that children with rickets appeared ill, with 
decreased energy and activity, and were more susceptible to respiratory illnesses. Despite these observations, it 
was concluded that the condition of vitamin D deficiency led to weakness and malnutrition and was not a direct 
effect of vitamin D on the immune system. The mechanism of action of these processes and health 
derangements would not be understood until the advent of molecular biology. 
Vitamin D appears to affect immune function in two ways: (1) upregulation of the innate immune system; 
and (2) downregulation of the adaptive immune system. Focusing on the innate immune system first, a major 
mechanism of action of vitamin D is via an endogenous antimicrobial peptide called cathelicidin (LL-37), which is 
generated in response to microbial invasion through activation of toll-2 receptors (TLR) on monocytes and 
macrophages (22-24). Not surprisingly, the vitamin D receptor (VDRE) is contained in the promoter region of the 
gene for LL-37. VDRE are found only in the LL-37 gene promoters of primates, suggesting that the ability of 
vitamin D to promote LL-37 antibacterial action is a relatively recent event in evolution. Both 1,25(OH)2D and 
25(OH)D have the ability to induce the expression of cathelicidin in monocyte/macrophage and epidermal 
lineage in cells that simultaneously have the 25(OH)D hydroxylase (25).  
 
Significant support for the role of vitamin D in immune processes and function came in 2006 when Liu et al 
published their landmark study in Science (22). Serum samples taken from African American subjects with low 
25(OH)D were inefficient in supporting cathelicidin mRNA induction; however, with  the addition of 25(OH)D to 
those samples with low 25(OH)D levels this pattern was reversed. Thus, in this series of experiments, the 
addition of 25(OH)D3 restored the ability of sera from individuals with low 25(OH)D concentrations to support 
TLR2/1L-mediated induction of cathelicidin mRNA. A related study by Fabri, et al (26), showed that IFN-ɣ-
mediated antimicrobial activity of human macrophages, especially important in HIV and tuberculosis patients, is 
dependent on vitamin D. Both study findings have implications for the pregnant woman and her developing 
fetus, but our understanding of such processes following maternal exposure to a pathogen or maternal infection 
remains scant. There is every reason to suggest that such processes are fully functional in the pregnant woman.  
 
Vitamin D’s role as a modulator of the immune system encompasses the adaptive immune system as well. 
1,25(OH)2D not only has the ability to affect processes within macrophages and monocytes, but also in T and B 
lymphocytes as well. The vitamin D receptor (VDR) is found on activated (but not resting) human T- and B-
lymphocytes. Whereas 1,25(OH)2D appears to activate the bacteriocidal process within macrophages and 
monocytes, it has different effects, that include suppression of T-cell proliferation and modulation of T-cell 
phenotype —with anti-inflammatory properties (27). By binding to the VDR on T cells, 1,25(OH)2D acts to: (1) 
inhibit the proliferation of uncommitted TH (helper) cells and (2) promote the proliferation of 
immunosuppressive regulatory T cells, or TregS, with notable accumulation of these cells at sites of inflammation 
(25). It appears that 1,25(OH)2D suppresses certain B cell functions such as proliferation and immunoglobulin 
production and retards the differentiation of B-lymphocyte precursors to mature plasma cells in vitro. These in 
vitro findings explain the significant association between vitamin D deficiency and autoimmune diseases, such as 
systemic lupus erythematosus (28), multiple sclerosis (29-38), rheumatoid arthritis (34, 39, 40), diabetes—both 
types 1 (34, 41-45) and 2 (46-48), and certain cancers, such as colon (49-52), breast (53-58), and prostate (58-
62). Additionally, the role of vitamin D in immune function intensifies the need to establish vitamin D sufficiency 
during pregnancy.  
It is clear that vitamin D deficiency during pregnancy is common throughout the world yet what effect does 
deficiency have on the mother and her developing fetus? There is a strong relationship between maternal and 
fetal (cord blood) circulating 25(OH)D levels (63-66) such that maternal vitamin D deficiency is mirrored by 
neonatal vitamin D deficiency. With severe maternal vitamin D deficiency, the fetus rarely may develop rickets in 
utero with manifestation at birth (67). Such readily observable fetal and neonatal skeletal effects of profound 
vitamin D deficiency are easily understood in terms of cause and effect, but the more subtle effects of deficiency 
on the developing immune system, for example, and subsequent infection risk or immune dysfunction are more 
difficult to understand (22, 68-71).  
Vitamin D status during pregnancy appears to play a role in fetal skeletal development, tooth enamel 
formation, and general fetal growth and development (72, 73). Mannion et al. (74), comparing growth 
parameters in newborn infants with the maternal intakes of milk and vitamin D during pregnancy, found an 


 
 
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association between vitamin D intake during pregnancy and birth weight. They reported with every additional 40 
IU (1 µg) of maternal vitamin D intake, there was an associated 11-g increase in birth weight. Pawley and Bishop 
(75) in their study of 108 pregnant women and their offspring found a significant association between umbilical 
cord 25(OH)D concentrations and head circumference at 3 and 6 months’ postnatal age that persisted after 
adjusting for confounding factors. Maghbooli et al. found significantly wider posterior fontanelle diameter in 
neonates of mothers with vitamin D deficiency (as defined by a 25(OH)D level <34.9 nmol/L or ~14 ng/mL) 
compared to neonates whose mothers were not deficient (76). Beyond growth, recent reports of neonates 
followed prospectively for acute viral infections and bronchiolitis from respiratory syncytial virus (RSV) support 
the premise that these states of deficiency do impact on the health of the young infant and suggest a greater 
role of vitamin D beyond bone health (77-79). 
McGrath and others continue to investigate whether there are lasting effects of fetal and early infancy 
vitamin D deficiency on later adult disease processes such as anatomical changes of the brain, schizophrenia, 
multiple sclerosis, certain cancers, cardiovascular disease, and various other autoimmune diseases such as 
diabetes and lupus (80-95). Because vitamin D status has not been a consistent concern during pregnancy, long-
term data are sparse. The few studies that have been conducted have focused more on discernible neonatal 
effects of vitamin D during pregnancy, rather than the long-latency and later health effects. Reports of neonatal 
seizures due to severe hypocalcemia or rickets in utero that is manifested at birth  from severe maternal and 
thus fetal vitamin D deficiency are rare and do not further our understanding of potential epigenetic effects of 
vitamin D (96, 97). During pregnancy, supplementation with the current standard amount of vitamin D in 
prenatal vitamins—400 IU (10 µg) vitamin D/day—has minimal effect on circulating 25(OH)D concentrations in 
the mother and her infant at term (5, 98). It is also known that infants of women who were deficient throughout 
pregnancy will reach a state of deficiency more quickly and with greater severity than infants of women replete 
during pregnancy (67).  
 
While there are numerous epidemiological studies that bear evidence of the association between vitamin D 
deficiency and altered health, definitive proof in terms of randomized controlled trials is often lacking. For 
example, higher circulating 25(OH)D levels have been linked with improved glucose handling and beta-cell 
function (99), and a reduction in risk for a growing list of long latency diseases that include cardiovascular 
disease (60, 100-104), multiple sclerosis (30, 35, 37), rheumatoid arthritis (39), systemic lupus erythematosus 
(28), type 1 and 2 diabetes (39), and various cancers (49, 55, 58, 105-110), but critics counter that while such 
findings are intriguing, they do not provide definitive evidence of causality or a mechanism of action that come 
from randomized controlled trials, and may lead to what is referred to as “circular epidemiology” (111). While 
we await the results of numerous clinical trials now underway to determine if there is a discernible effect of 
vitamin D in altering risk for various disease states to understand the role of vitamin D in health, it is important 
not to discount the mounting evidence from laboratory studies and prospective observation trials (77-79) that 
vitamin D—as a preprohormone—is essential in maintaining the immune system with profound implications (22, 
112-114).  
 
Significance: The central tenet of this project is enhanced understanding of the underlying mechanisms 
of action of vitamin D deficiency during pregnancy, which result in poor maternal and fetal health, and health 
disparities in pregnancy outcomes. By focusing on immune imbalance associated with vitamin D deficiency—first 
utilizing a well-developed mouse model of preterm birth and preeclampsia and then studying pregnant women 
themselves looking at adverse maternal and fetal health effects and markers of deficiency—we expect to show 
improvement in various pregnancy outcomes. During this project, we will complete the following activities:  
1) Define more fully the health disparity during pregnancy between African American, Hispanic and 
Caucasian women on the basis of vitamin D status;  
2) Demonstrate that vitamin D deficiency impairs placental function, leading to abnormal immune 
responses and adverse pregnancy outcomes;  
(3) Demonstrate that vitamin D supplementation will enhance placental biology and protect against 
adverse pregnancy outcomes;  
(4) Define the immune mechanisms by which vitamin D acts during pregnancy to enhance immune 
suppression and decrease inflammation for optimal health outcomes of mother and fetus through measurement 
and characterization of: 


 
 
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(a) immune T-cell modulatory changes during pregnancy (% change ratio of CD4+ and CD25+ 
over total lymphocyte population where there would be an increase in the ratio as a function of vitamin 
D repletion;  
(b) inflammatory cytokine changes as a function of vitamin D status;  
(c) tooth developmental changes as a function of vitamin D status;  
(5) Validation of immune mechanisms of action of vitamin D through a mouse model developed by Dr. 
Martin Hewison at UCLA;  
(6) Examine the metabolism and processing of vitamin D supplementation to better understand and 
prevent deficiency; 
(7) Dissemination of findings at several levels through: a. the National Association of Community Health 
Centers; b. vitamin D think tank meetings with all involved partners; c. public health forum held regionally 
utilizing vitamin D grassroots organization/health center networks at community, state, and federal levels.  
The first 6 activities will generate supportive data for supplementing pregnant women with vitamin D at 
doses that enhance immune balance and regulation. With greater knowledge about vitamin D gained from this 
project, we will educate health care providers, policy makers, and the pregnant women themselves about why 
vitamin D is important and how to prevent deficiency (Activity 7). In Year 4, a “think tank”-type of vitamin D 
meeting of scientists, philosophers, politicians, health care providers, and educators will be hosted at MUSC. 
Also, a vitamin D public forum specifically about vitamin D during pregnancy will be launched. This will allow the 
creation of partners at the local, state and federal levels in changing the course of pregnancy outcomes in 
America. Such networking and interactions will allow utilization of existing infrastructure to sustain such change 
with the expectation that public health policy changes will be created that address the established need of 
pregnant women for vitamin D repletion.  
SubObjective of Activity 7: Vitamin D Status and Health Implications for Women during Pregnancy: An 
MUSC Research-to-Practice Implementation and Assessment: Year 3-5 of Kellogg Foundation Grant  
 
To accomplish Activity #7 (Objective #3), the following Objectives are to be met:  
(1) 
Vitamin D status assessments by trimester of every woman presenting to MUSC for obstetrical 
care 
(2) 
Determine the effect of vitamin D status on the pregnancy outcomes of women and their infants 
who receive health care through the MUSC health care system 
(3) 
Assess the impact of real-time vitamin D status on the prescription of vitamin D 
supplementation during pregnancy by MUSC health care providers 
 
 
C. PRELIMINARY STUDIES___________________________________________________________________ 
 
Summary from two papers (one in press) (see appendix for manuscript): 
 
Hollis BW, Johnson D, Hulsey TC, Ebeling M, Wagner CL. Vitamin D supplementation during pregnancy: double-
blind, randomized clinical trial of safety and effectiveness. Journal of bone and mineral research : the official 
journal of the American Society for Bone and Mineral Research. 2011;26(10):2341-57. Epub 2011/06/28. doi: 
10.1002/jbmr.463. PubMed PMID: 21706518; PubMed Central PMCID: PMC3183324. 
 
Wagner C, McNeil R, Hamilton S, Winkler J, Rodriguez Cook C, Warner G, et al. A Randomized Vitamin D 
Supplementation Trial in Two Community Health Center Networks in South Carolina. Am J Obstet Gynecol. 
2013;in press. 
 
 
In our recently completed NICHD vitamin D supplementation trial involving a diverse group of pregnant 
women less than 16 weeks of gestation, 4000 IU (100 µg) vitamin D3/day was superior to 400- or 2000 IU/day in 
achieving circulating 25(OH)D of at least 40 ng/mL (100 nmol/L), the point at which 1,25(OH)2D begins to be 
optimized (see Figure 2) (5). While 4000 IU/day was superior to 2000 IU/day in achieving the Institute of 
Medicine’s threshold for sufficiency of a 25(OH)D concentration of ≥20 ng/mL (50 nmol/L), it was not statistically 


 
 
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significant—both 2000 and 4000 IU/day will achieve this level in pregnant women. If the goal, however, is to 
reach the point of 1,25(OH)2D optimization of 40 ng/mL (100 nmol/L), then there is a clear advantage of taking 
4000 IU/day. While there was restriction of randomization in the NICHD trial necessary for initial IRB approval, 
such a restriction was not in place for the second pregnancy Thrasher Research Fund Trial (described below). 
Even in the latter trial where women were randomized to either 2000 IU (50 µg) or 4000 IU (100 µg) vitamin 
D3/day, there were no safety issues surrounding vitamin D supplementation and there was a trend where 
specific health complications of pregnancy such as preterm birth, preterm labor, hypertensive orders of 
pregnancy, gestational diabetes, and infection were higher in the 400 IU- compared with the 4000 IU-group, but 
did not reach statistical significance (115). When analyzed together as comorbidities of pregnancy and 
controlling for race, there were statistically significant differences between the 400 IU-, the 2000 IU-, and the 
4000 IU groups with fewer events in the 4000 IU group (p=0.03) (115).  
 
Consistent with the NICHD trial results, another recently completed study funded by the Thrasher Research 
Fund where women were randomized to either 2000 (50 µg)- or 4000 IU (100 µg) vitamin D3/day showed that 
the while the mean change from maternal 25(OH)D baseline was +12.2±13.2 ng/mL in the 2000 IU group and 
+15.2±12.9 ng/mL in the 4000 IU group (p=0.16), the overall 25(OH)D rate of increase differed significantly 
between the two dose groups (p=0.036) with a higher rate in the 4000 IU group. Mean infant 25(OH)D level 
(ng/mL) was 22.1±10.3 in the 2000 IU group and 27.0±13.3 in the 4000 IU group (p=0.024). Secondary analysis 
indicated that preterm birth and labor were inversely related to pre-delivery vitamin D status (preterm birth 
OR=0.50 per 10 ng/mL, p=0.002; preterm labor or birth OR=0.72 per 10 ng/mL, p=0.012), an effect that persisted 
even after controlling for race. There were no adverse events associated with vitamin D supplementation (116).  
Analysis of the two concurrent vitamin D pregnancy NICHD (n=350) and Thrasher Research Fund (n=157) 
trials was undertaken to assess the health characteristics and outcomes of this larger, combined group using a 
common data dictionary (8). As noted above, in the NICHD trial, women were randomized to 400 IU (10 µg), 
2000 IU (50 µg) or 4000 IU (100 µg)/day, stratified by race. In Thrasher trial, participants were randomized to 
2000 (50 µg) or 4000 IU (100 µg)/day, also stratified by race. All participants received study drugs from the same 
manufacturing lot. Studies administered identical questionnaires to produce comparable 
sociodemographic/clinical characteristics. Outcome measures included: (1) maternal baseline and delivery 
25(OH)D; (2) neonatal 25(OH)D; (3) comorbidities of pregnancy: gestational diabetes, hypertensive disorders of 
pregnancy, infection—any, bacterial vaginosis (BV), and preterm birth without preeclampsia. In the combined 
cohort, there were 110 in the control group, 197 in 2000 IU group, and 189 in the 4000 IU group. The treatment 
groups differed on the basis of parity (p=0.022); insurance status (Medicaid status more common in the 2000 IU 
group p=0.006); and education (higher education level in the 400 IU group, p=0.012). No differences were noted 
between groups in baseline 25(OH)D (p=0.24), but differences between groups were noted in vitamin D status 
within one month of delivery (p<0.0001) and in cord blood (p=0.0001), with improved status in the 4000-IU 
group overall. No differences were noted between groups in terms of independent or combined comorbidities 
of pregnancy. There was a trend where preterm birth without preeclampsia was lower with increasing 25(OH)D 
concentration (Odds Ratio (OR) 0.83 (Confidence Interval (CI) 0.68-1.01); p=0.057).The risk of combined 
comorbidities of pregnancy adjusted for race/ethnicity and study was significantly lower with increasing 
25(OH)D concentration (Odds Ratio per 10 ng/mL increase in 25(OH)D: 0.84; 95% CI 0.74-0.96; p=0.0095) (8). 
The debate about what constitutes frank deficiency, insufficiency, and sufficiency continues (3, 117). The 
answer varies depending on the question—is the outcome bone integrity or immune function? There could be a 
different cut point for each category. Most would agree—including the Institute of Medicine in their most recent 
statement –that levels below 20 ng/mL (100 nmol/L) represent deficiency in the nonpregnant individual. There 
is less consensus with respect to pregnancy: based on our recent randomized controlled trial with pregnant 
women, it is clear that optimization of 1,25(OH)2D does not occur until total circulating 25(OH)D levels have 
reached 40 ng/mL (100 nmol/L) (5).  
The significance of these trials is that vitamin D status is improved with 4000 IU vitamin D3 taken daily to 
achieve: (1) optimization of 1,25(OH)2D production, and (2) improved cord blood 25(OH)D concentration. Having 
a higher 25(OH)D concentration was associated with improved health outcomes in both studies, but whether 
improved vitamin D status is a marker of some other parameter or a direct effect of vitamin D supplementation 
remains unclear at this time. Studies specifically designed and powered to answer the question of whether or 


 
 
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not vitamin D supplementation leads to improved health outcomes—lower risk of preterm birth, preeclampsia, 
and infection remain to be done. We can conservatively say that while we do not understand completely the 
role of 1,25(OH)2D during pregnancy, achieving optimal production can be done safely with 4000 IU vitamin 
D3/day, which appears to be the amount of vitamin D that would be conservatively generated by the body with 
adequate sunlight exposure over the duration of gestation. 
 
D. RESEARCH DESIGN AND METHODS (including data analysis)  
 
Study Design: This is a randomized, placebo-controlled clinical trial of 450 pregnant women who will be enrolled 
between 10-14 weeks of gestation and followed until delivery. Following written, informed consent, each 
mother will be randomized to receive either placebo or 4000 IU/day vitamin D3 plus the standard prenatal 
vitamin (containing 400 IU vitamin D3). She will be followed monthly for a total of nine study visits. If, after 3 
months of vitamin D supplementation, a mother is still frankly vitamin D deficient (less than 15 ng/ml), she will 
receive open label active vitamin D gummies for the remainder of her pregnancy. These visits will include the 
initial recruitment of mother at 10-14 weeks of pregnancy, monthly study visits, delivery study visit, and 18-
month post-partum follow-up visit for mother and child. The primary outcome variable is maternal and neonatal 
health status as a function of maternal and infant vitamin D status. Secondary outcome variables to be analyzed, 
as a function of maternal vitamin D status during pregnancy will include: T-lymphocyte profile, immune function 
indicators, neonatal growth, inflammatory cytokine profile, methylation patterns of DNA of both mother and her 
neonate,  
(1) Subject Selection Criteria and Recruitment: Any mother (18-45 years of age) who presents to her 
obstetrician or midwife at the Medical University of SC (MUSC), Charleston, SC obstetrical facilities within the first 
14 weeks after her last menstrual period (LMP) with confirmation of a singleton pregnancy will be eligible for 
enrollment in the study. Mothers of diverse ethnic backgrounds (African-American, Asian, Caucasian and Hispanic) 
will be actively recruited. A database will be generated weekly of undelivered patients for the labor and delivery 
staff continue to alert the staff of impending admissions.  
(2) Exclusion Criteria: Mothers with pre-existing calcium, uncontrolled thyroid disease, parathyroid 
conditions, or who require chronic diuretic or cardiac medication therapy including calcium channel blockers will 
not be eligible for enrollment into the study. Mothers with pre-existing sickle cell disease (not trait only), 
sarcoidosis, Crohn’s disease, or ulcerative colitis may not participate in the study. In addition, because of the 
potentially confounding effect of multiple fetuses, mothers with multiple gestations will not be eligible for 
participation in the study. A sub-group of approximately 100 subjects with known diabetes, hypertension, HIV, or 
morbid obesity (body mass index > 49) will participate in the study.  
(3) Subject Enrollment: The potential study subject’s primary care provider will make first contact on 
behalf of the study and refer interested mothers to study personnel for further information and invitation. Upon 
enrollment into the study, expectant mothers at approximately 10-14 weeks’ gestation will be randomized to 
receive one of two treatment regimens of vitamin D3. They will be randomized to 1 of 2 groups: (1) Group A: 400 
IU vitamin D3/day—Standard dose treatment of placebo (0 IU vitamin D3) plus prenatal vitamin (400 IU/day); or 
(2) Group B: 4,400 IU/day (4,000 IU/4 gummies/day + 400 IU/day in prenatal). To attain equal group numbers 
(n=150) and balanced numbers by racial/ethnic group (African-American, Caucasian and Hispanic, which represent 
the predominant groups delivering at MUSC), a stratified block randomization will be used. 
(4) Race/Ethnicity Defined: Each mother will be asked to define the racial/ethnic group to which she 
belongs: (a) African-American, (b) Caucasian (including Asian and American Indian), and (c) Hispanic. Because few 
mothers of American Indian (0.08% of all deliveries) and Asian (1% of all deliveries) descent deliver at MUSC, there 
is insufficient power to include those groups individually in this study. In addition, the mother, by self-report, will 
be asked to define the race/ethnicity of the father of the baby. 
(5) Study Site Population: The Medical University of South Carolina (MUSC) is an urban medical 
university in Charleston, SC that serves patients predominantly in the Charleston Tri-County area (80%) and 20% 
from surrounding regions in South Carolina. We perform 2000 deliveries each year. The ethnic composition of our 
obstetrical population is 57% African American, 36% Caucasian, and 6% Hispanic and 1% Asian. MUSC provides 
care to any woman presenting in labor who requires routine care or who is referred from outlying hospitals due 


 
 
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to high-risk pregnancy for which delivery at a Regional Perinatal Center (RPC) is appropriate, regardless of ability 
to pay.  
(6) Enrollment and Follow-Up Subjects: The Study Coordinator will be responsible for assisting in 
screening, enrollment and follow-up. To maintain data quality, the Study Coordinator will review every form to 
check for basic problems such as missing data. Labor and delivery staff will alert the study coordinator or the 
Clinical PI’s of the admission of any study subjects. Each month the Study Coordinators will generate a report on 
patient recruitment and retention to be reviewed jointly by the research team. A computer-generated calendar 
will serve as a reminder to contact recruited patients two days prior to their appointment. Each month the Study 
Coordinators will generate a report on patient recruitment and retention that will be reviewed by Dr. Wagner. 
(7) 
Follow-up Infant Visit: The Study Coordinator will follow up by phone several times from 
birth to 18 months in order to verify subject’s contact information. The infant (accompanied by mother) follow-
up visit will take place at MUSC’s CTRC Outpatient Clinic around 18 months postpartum. The infant will have blood 
drawn for vitamin D and will have a dental assessment of the primary teeth, including digital photographic images.  
(8) 
Sample Size: With the assistance of Dr. Thomas Hulsey, epidemiologist and co-
investigator of this application, sample size calculations were made. All outcome markers are ratio-scaled and 
normally distributed. Because of the possibility of premature delivery and patient attrition during this study, 
enrollment of additional subjects over that minimally needed, will provide a cost effective opportunity to conduct 
additional analyses examining changes in vitamin D associated with diet, seasonality, and maternal medical 
complications. As one of the major goals of this study is to explore maternal health disparities by ethnicity, the 
two supplemented groups (400 and 4,400 IU/day) will be balanced by ethnicity (equal numbers of Caucasian, 
African American, and Hispanic). This sampling distribution will be accomplished by stratified randomization. This 
sampling frame was selected to provide: (1) sufficient numbers to examine associations with covariates; (2) the 
volume of initial and follow up samples will be manageable by our laboratory; (3) over-samples to compensate for 
losses to follow up; and, (4) stabilizes the sophisticated statistical analysis techniques (repeated measures). 
(9) 
Subject Sampling: Since this study will enroll equal numbers from each ethnic group, the 
actual enrollment will be approximately 150 in each group. The sampling procedure is designed to achieve an 
ethnically balanced study population with over-sampling to accommodate lost to follow-up and missed visits. The 
random assignment will be conducted as a stratified (by ethnicity) blocked (by supplementation group) design. 
The randomization schedule will be developed using ProcPlan in SAS statistical software. Dr. Hulsey has extensive 
experience in developing stratified blocked randomization allocation plans for clinical trials.  
(10) 
Multi-Vitamin and Vitamin D Supplementation.  
(a) Preparation of Vitamin D Tablets: The vitamin D3 gummies will be manufactured by 
Vitafusion (Church and Dwight, Vancouver, Washington) and provided to the PI for this study at no cost in 
bottles with child-proof caps. Two different gummies will be produced: all will be identical in appearance due to 
the blinding of the study. Gummies will contain no vitamin D (placebo) or 1000 IU vitamin D3. All subjects will be 
instructed to chew 4 gummies/day. All subjects will receive prenatal vitamins (pills or chewable, subject 
preference) once daily containing an additional 400 IU of vitamin D3 (one/day). Thus, a total vitamin D intake by 
group will be 400 or 4400 IU/day. In a large study of this type, we believe that oral intake by gummy is a more 
reliable means to administer vitamin D to the subjects, many of whom are experiencing pregnancy nausea or 
dislike swallowing large capsules to achieve even the 400 IU vitamin D3/day. 
(b) Maternal Supplementation: At each visit, mothers enrolled in the Study will be given an 
additional 40-day supply of prenatal vitamins with 400 IU/vitamin D3 per tablet and an additional 40-day supply 
of vitamin D3 gummies (placebo or 1000 IU/gummy). The subjects and the investigators will not be aware to 
which vitamin D group the subjects have been assigned. Each mother will be instructed to take one prenatal 
vitamin along with four vitamin D3 gummies daily. At the completion of each month at the time of the prenatal 
clinic visit, each mother enrolled in the study will receive another 40-day supply of the prenatal vitamins and the 
specified vitamin D3 gummy strength to which she is assigned. Mother will be instructed to bring the vitamin D 
and prenatal containers with her to each follow-up visit. Pill counts will be performed monthly and compliance 
noted. 
(11) 
Maternal Sociodemographic Questionnaire: Upon enrollment in the study, each mother 
will be asked to complete a sociodemographic questionnaire. This questionnaire will ascertain certain 


 
 
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sociodemographic information: maternal age, race of herself and her parents and grandparents, educational level, 
occupation of herself and the father of her infant, the number of people in her household including herself, and 
maternal insurance status. A Hollingshead index from 1-7 will be derived for each mother based on the educational 
status and occupation of the mother (and father if he is involved and living in the same residence as the mother).  
(12) 
Pregnancy Intake Survey and Monthly Maternal Health/Medication Questionnaire: 
Upon enrollment, and then at each monthly obstetrical visit, the mother will be asked to complete a health 
assessment questionnaire to ascertain her use of medications (checklist) and over-the-counter preparations that 
may influence vitamin D/calcium homeostasis. She will be specifically asked questions regarding her use of 
cigarettes and alcohol, and overall health status. In addition, if mother has required hospitalization, this will be 
recorded and a copy of the hospital record obtained after she has signed a release of medical information form 
to obtain the medical information. In addition, e.g., if mother required tocolysis, antibiotics or IV rehydration, this 
will be recorded on the questionnaire. 
(13) 
Maternal Dietary History: Each mother will complete a standardized Food Frequency 
Questionnaire (FFQ) at the 2nd visit to ascertain her generalized eating pattern, with specific calculation of calcium 
and vitamin D intake. A Diet and Lifestyle Questionnaire will also be distributed. Dr. Sarah Rothenberg will use 
data obtained from the FFQ and Diet and Lifestyle Questionnaire to determine fish/shellfish consumption as it 
pertains to methylmercury intake and the interaction between vitamin D and methylmercury. This questionnaire 
is advantageous to collecting data over food diaries or 3-day food records for the following reasons: (a) The 
questionnaire does not rely on memory as much as other validated methods; (b) Its readability is designed for 
lower literacy populations; and (c) It accurately estimates individual and population-wide average daily intakes of 
all macro- and micronutrients. Individual and collective data can be gathered for statistical analysis to assess 
dietary intakes for this study population. This tool has been validated and reproduced against other dietary intake 
tools. Each completed FFQ form will be sent to the processing center (Berkeley, California) in batches of 50-100. 
Food models also will be provided to assist subjects in identifying most commonly consumed portion sizes rather 
than assuming each subject can accurately discern serving sizes on her own.  
(14) 
Maternal Physical Activity Assessment Questionnaire: Each mother will be asked to 
complete a Physical Activity Assessment Questionnaire. This questionnaire utilizes a well-established physical 
activity instrument with known validity and reliability. Because physical activity can affect calcium metabolism 
and because a woman’s activity level may change considerably during her pregnancy, it will be important to 
reassess maternal activity level throughout the entire period of gestation. Therefore, each mother will complete 
this questionnaire monthly.  
(15) 
Sunlight Exposure: Two body sites will be scored at each monthly visit with a Smart Probe 
(measures skin color at 450 nm wavelength) to assess sunlight exposure. 
(16) 
Maternal Physical Examination: As part of MUSC’s OB clinic, each mother undergoes a 
physical examination at scheduled clinic visits, according to standard of care for the OB clinic. Some of these results 
of the physical exam will be recorded directly on the case report forms. 
(17) 
Maternal Dental Examination:  Women with 1 or more natural teeth and not having a 
health condition that requires antibiotic prophylaxis before periodontal probing are eligible for the periodontal 
examination.  
(a) 
The commonly used full mouth periodontal examination (FMPE) may be done up to 
three times during the pregnancy (baseline, 28 weeks and 36 weeks). The FMPE protocol will be used for 
classification of periodontal disease. The major outcome measure of change in the periodontal measures will be 
correlated with change in serum circulating 25(OH)D concentration from baseline to 28 and 36 weeks. The FMPE 
will be done by trained personnel (Registered Dental Hygienist or Dentist) in a dental chair setting. Data are 
either recorded on paper or direct data entry will be done. The research instruments and questionnaires have all 
been previously used in the research environment. 
(b) 
Gingival inflammation is measured using a salivary occult blood test (SOBT). The 
chairside SOBT is done monthly by rinsing the mouth with water and then spitting into a small paper cup. The 
outcome for SOBT will be analyzed with respect to the periodontal measures, and in particular, the percent of 
sites with BOP. Trained study personnel will perform the SOBT. Data will be recorded on visit questionnaire. This 
research instrument has been previously used in the research environment.  


 
 
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(c) 
Oral bacteria will be measured using a GenProbe for Chlamydia trachomatis at delivery 
or visit 7 (36 weeks). A swab is used to make the oral collection by gently swabbing the inside of the cheeks and 
oral cavity. The dichotomous outcome for Chlamydia (positive or negative) will be analyzed with respect to the 
periodontal measures, BOP, and birth outcomes. Trained study personnel will perform the oral bacterial test. 
Data will be recorded on the visit questionnaire. This research instrument has been previously used in the 
research environment. 
(18) 
Labor and Delivery Questionnaire: Characteristics of each mother’s labor and delivery 
will be recorded on a data form. Prior complications of pregnancy will be listed on the monthly pregnancy 
assessment questionnaire. Such complications at the time of delivery will be listed here according to ACOG 
definitions: e.g., preeclampsia; HELLP syndrome; gestational diabetes; hypertension; preterm labor; premature 
rupture of membranes (PROM); deep venous thrombosis or a coagulopathy; maternal infection such as varicella 
or herpes (primary or secondary); placental abnormalities such as previa or abruption. The following information 
about labor will be included: (a) length of labor, (b) use of pitocin augmentation, (c) time of rupture of membranes, 
and (d) complications of labor: fetal distress or decelerations, failure to progress, shoulder dystocia, breech 
presentation or abnormal fetal lie. Should the infant be delivered at an outlying hospital, mother will complete a 
release of medical information form so that medical records relating to the labor and delivery and the infant’s 
health status/physical examination will be forwarded to the Clinical PI. 
(19) 
Maternal Body Mass Index (BMI) Measurement: Pre-pregnancy height and weight of 
each mother will be recorded at the first outpatient visit to determine BMI (weight [kg]/height2 [m2]). During 
subsequent outpatient visits, only the mother’s weight will be recorded.  
(20) 
Maternal Baseline and Monthly Calcium and C-Reactive Protein Serum Studies: Whole 
blood (3.5 mL) will be obtained from each mother at the time of her scheduled obstetrical visit monthly as well as 
at delivery and placed in an SST tube. The blood will be sent to the Clinical Chemistry Laboratory. The following 
blood studies will be measured: total Ca and C-reactive protein. These results will be reported directly to Dr. 
Wagner, then entered into the Study’s Database. 
(21) 
Maternal Baseline and Monthly Vitamin D and Calcium Homeostatic Parameters: A  
14 mL sample of whole blood will be drawn from the mother at the scheduled obstetrical visit monthly as well as 
at delivery and placed in an SST tube to be processed in the CTRC Clinical Laboratory for serum separation. The 
serum will be placed in a –80oC freezer and sent to Dr. Bruce Hollis’ laboratory on dry ice. Each serum sample will 
be analyzed for maternal circulating levels of total 25(OH)D, 1,25(OH)2D, iPTH (5),  batched for serum cytokine 
analysis, and batched for measurement of megalin and cubulin.  25(OH)D will be run on all subjects after 3 months 
supplementation.  Mothers who are still frankly vitamin D deficient (less than 15 ng/ml) will be placed on open 
label vitamin D gummies for the remainder of their pregnancy. 
(22) 
Monthly Maternal Urine Sample: A random urine sample will be obtained from the 
mother at each obstetrical visit to be sent to the Clinical Chemistry Laboratory for urinary Ca and creatinine 
measurement and derivation of the urinary Ca: creatinine ratio. This ratio will be used as an indicator of 
hypercalciuria that precedes hypercalcemia. Results of these urine studies will be reported directly to Dr. Wagner. 
An additional aliquot of the urine sample will be frozen for future cytokine analysis. 
(23) 
Cord Blood Vitamin D and Calcium Measurements. At the time of delivery, 10-mL of 
whole blood will be drawn from the umbilical vein then sent to the CTRC Clinical Laboratory facility for processing. 
One red bullet (0.5 cc aliquot) will be sent to the Clinical Chemistry laboratory for measurement of total calcium 
and creatinine. The remaining blood will be processed in the CTRC Clinical Laboratory immediately for serum 
separation and placed in a –80oC freezer then transported on dry ice to Dr. Hollis’ laboratory for later 
determination of each neonate’s circulating levels (at the time of delivery) of intact iPTH, total 25(OH)D, and 
1,25(OH)2D (5). Additional frozen serum will be batched for analysis of cytokines. Every effort will be made to 
collect cord blood at the time of delivery. An additional 15 mL plasma sample will be obtained from the cord for 
immune privilege flow cytometry studies. 
(24) 
Immune Privilege Flow Cytometry Studies:  Maternal plasma (14 mL) at first visit 
(baseline), fourth visit (24 weeks gestation) and seventh visit (36 weeks gestation), as well as 15 mL cord plasma 
will be analyzed by Dr. Jennifer Mulligan’s laboratory for circulating immune cells by flow cytometric analysis. 
Plasma cytokines will be measured by ELISA per manufacturer’s instructions. Expression of genes related to 


 
 
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vitamin D3 activation and metabolism will be measured by custom PCR Array (SA Biosciences) for the following 
genes: VDR (vitamin D receptor), CYP2R1 (vitamin D 25-hydroxylase), CYP27B1 (1-alpha-hydroxylase), CYP24A1 
(24-hydroxylase), and GC (vitamin D binding protein). The plasma immunoregulatory products to be examined 
include IL-10, TGF-beta, TNF-alpha, IFN-gamma, IL-2, IL-18 and IL-12. 
(25) 
 Vaginal Self-Swabs: At each visit, subjects will be asked to perform 3 vaginal self-swabs:  
(a) First swab that the study coordinator will use to prepare a wet mount slide for 
identification of bacterial vaginosis and gram staining (these slides will be air dried and saved for batch analysis 
by the pathologists in Clinical Chemistry);  
(b) Second swab will be a GenProbe swab for identification of Trichomonas vaginalis, 
Neisseria gonorrheae and Chlamydia trachomatis; 
(c) Third swab will be a GenProbe swab for evaluation of vaginal cytokines.  
When study subjects come into hospital for delivery, we will also obtain these three vaginal self-swabs. Vaginal 
cytokines that are of interest include: tumor necrosis factor-α, interleukin (IL)-1-α and 1-β, IL-2, IL-3, IL-4, IL-5, 
IL-6, IL-7, IL-8, IL-10, IL-12, IL13, IL-15, IL-17, IL-18, monocyte chemotactic protein (MCP), TGF-β, macrophage 
inflammatory protein (MIP), alpha-defensins, leukotriene B, s-tumor necrosis factor-R1, E-selectin, necrosis 
factor and cAMP element response binding. These cytokines can be batch-analyzed using a custom Luminex 
multiplex ELISA assay.   
(26) 
Placental Pathology Placentas will be obtained at time of delivery for research purposes. 
Each placenta will be placed in the Labor and Delivery refrigerator until transport to Pathology, Dr. Michael 
Caplan’s lab refrigerator. Dr. Caplan will record gross placental findings, membranes, fetal surface, maternal 
surface, and placental parenchyma, using methodology already in place in Dr. Caplan’s pathology laboratory.  
(27) 
 DNA and mRNA Expression:  At visit 2, each study subject will have an additional 6 mL 
of blood drawn in a plasma tube for analysis of DNA and RNA by Dr. Inderjit Singh’s lab. Specifically, vitamin D 
binding protein DNA and vitamin D receptor will be analyzed per subject as per established protocol. mRNA 
expression of CYP24A1, CYP27B1, IL-4, IFNg, IL-6, IL-17A, IL-23, FoxP3, and 24,25(OH)2D also will be measured 
using methodology already in place in Dr. Singh’s laboratory and previously reported (118).  
(28) 
Methylation: Cord blood, buccal mucosal swabs, and placental tissue samples will be 
used to determine the variation in infant DNA methylation as a function of maternal vitamin D status, maternal 
pregnancy complications, and infant and maternal characteristics. The methylation will be probed at both the 
whole genome level and at the specific locus level. To evaluate the methylation status of the entire genome we 
will utilize a liquid chromatography-mass spectroscopy based method, which determines the proportion of 
methylated cytosine to total cytosine in the entire genome. We will use locus-specific methylation probes to 
determine the methylation of the promoter regions for cortisol receptor, vitamin D receptor, and other steroid 
receptor sites. Locus-specific methylation will be determined by bisulfite sequencing to determine the 
methylation of individual cytosine base pairs at the nucleotide level. 
(29) 
Metabolism and Processing of Vitamin D:  Dr. Sarah Rothenberg will examine the effect 
of dietary methylmercury intake on the metabolism and processing of vitamin D supplementation during 
pregnancy. A recent publication reported the ability of methylmercury to inhibit the bioactive form of vitamin D 
to upregulate the vitamin D receptor, thereby potentially negatively altering the innate immune system (119). 
To examine this pathway in this cohort, at visits 2, 7 and delivery, each study subject will have an additional 6 mL 
(1.2 teaspoons) of whole blood drawn in a royal blue vacutainer. Additionally, data obtained from both the FFQ 
and Diet and Lifestyle Questionnaire will determine fish/shellfish consumption as it pertains to methylmercury 
intake. The methylmercury levels will be determined and compared with the vitamin D status and vitamin D 
receptor gene expression of the subjects.  
(30) 
Child Dental Visit   At 18 months of age, the dentist or hygienist will ask routine dental 
utilization questions, examine and make digital images (take photographs) of the child’s teeth. Likewise, an oral 
bacterial collection may be done by a gentle swabbing of the mouth. 
 
Activity #7 (Objective #3):  Through the joint efforts of this Kellogg-sponsored project, the Medical 
University of South Carolina and GrassrootsHealth.net, activity #7 represents a significant public health 
breakthrough. GrassrootsHealth.net is a nonprofit organization located in San Diego and affiliated with the 


 
 
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University of California in San Diego, whose sole purpose is to improve the health of individuals throughout the 
world through education about vitamin D and the adverse effects on health during times of deficiency. Through 
the assistance of GrassrootsHealth.net and the University of California’s San Diego’s CME office, we developed 
educational media and CME credits for physicians and other health care professionals to teach them about 
vitamin D, its role in immune function and overall health, with the latest scientific information 
(GrassrootsHealth.net received funding for this during year 4 of the Kellogg project). 
 
We launched “Protect Our Children Now” campaign on April 7, 2015. Protect Our Children NOW! is a community 
outreach and clinical implementation project to reduce the incidence of preterm births quickly, easily, and safely 
by addressing the vitamin D deficiency epidemic through the engagement of pregnant women in a value‐
changing program of Good Health vs. ‘Treating Illness.’ The purpose is to implement vitamin D testing and 
supplementation of pregnant women as standard of care. Specifically, it is to help them achieve a serum level of 
at least 40 ng/mL to aid in the prevention of many comorbidities of pregnancy. 
 
The Protect Our Children NOW! project involves educating prenatal care practitioners by providing free CME 
opportunities about vitamin D, and the importance of testing serum levels. These CME courses were and are 
offered to MUSC physicians sponsored by the University of California San Diego and GrassrootsHealth. In 
addition to the CME credits, each physician and health care provider receives 'D*certification' for this project. 
 
Once again, these goals can only be reached when the Obstetrical community embraces the importance of 
vitamin D supplementation during pregnancy as an important factor in affecting the outcome of the health and 
well‐being of the mother and the developing fetus during pregnancy. To this end, MUSC has partnered with 
GrassrootsHealth to implement universal vitamin D screening of all pregnant women who present to MUSC for 
their obstetrical care. 
 
The Obstetrics Department at MUSC embraced the mounting evidence that vitamin D deficiency is not beneficial 
to our pregnant women and began universal screening in September 2015 of all pregnant women presenting for 
obstetrical care at MUSC. Specifically, all pregnant women who present for obstetrical care within the MUSC 
system have a baseline 25(OH)D concentration measured with appropriate counseling and supplementation to 
achieve a total circulating 25(OH)D concentration of at least 40 ng/mL by second trimester with repeat measure 
now enacted at the 24-28 weeks’ gestation obstetrical visit to coincide with the planned glucose tolerance test. 
This change in practice began on September 1, 2015, after the Obstetrics Department at MUSC unanimously 
agreed to implement this change. Without surveillance data, however, there is no way to assess the 
effectiveness of this program and its impact on pregnancy outcomes at MUSC. To address this deficit, we have 
implemented the following: 
 
Methods for Activity #7 (Objective #3): 
Using the MUSC Perinatal Database and the associated laboratory data, the vitamin D status of all women who 
present for obstetrical care at MUSC and who deliver their infants at MUSC will be assessed. We will request a 
waiver of consent and HIPAA as it is not practical to consent these women and their babies after they have been 
discharged, and the data will be queried in a manner that will reduce the risk of loss of confidentiality by utilizing 
deidentified data after the laboratory data are merged with the PINS database by Myla Ebeling.  The data to be 
abstracted from PINS includes complications of the pregnancy, complications of the delivery and abnormal 
diagnoses of the offspring child. The data will be used to address specific research questions as noted above. 
Grassroots Health will analyze the deidentified data as an independent entity that is already involved in the 
Kellogg Vitamin D Pregnancy Project.   
 
 
E. PROTECTION OF HUMAN SUBJECTS________________________________________________________ 
 
1. RISKS TO THE SUBJECTS  


 
 
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This is a randomized, double-blind, placebo-controlled trial of vitamin D supplementation during pregnancy, 
funded by the Kellogg Foundation. Dr. Carol Wagner is the PI of the study. For the parent protocol, 300 women 
must complete the study (through delivery) for the necessary statistical power (100 Hispanic, 100 African-
American, and 100 Caucasian); half of each ethnicity will be randomized to 400 IU vitamin D/day and half to 
4400 IU vitamin D/day. These women will be between 18-45 years of age, in good general health; exclusion 
criteria will be: mothers with pre-existing parathyroid disease or uncontrolled thyroid disease, mothers with 
multiple fetuses, and mothers with sickle cell disease, sarcoidosis, Crohn’s disease, or ulcerative colitis. There 
will be a subgroup of pregnant women (projected 100 total) of all ethnicities who will have known diseases such 
as diabetes, hypertension, HIV or a body mass index > 49; these women will also be randomized to either 400 IU 
vitamin D/day or 4400 IU vitamin D/day, but in a separate randomization scheme, not dependent on completed 
subjects yielding a statistical power difference.  
 
The targeted/planned enrollment table allows for a 30% dropout rate; information for our two previous large 
randomized clinical trials of vitamin D and pregnancy supports this rate. The targeted/planned enrollment table 
does NOT include the subgroup of pregnant women of all ethnicities who will have known diseases. 
 
Targeted/Planned Enrollment Table 
 
Total Planned Enrollment 450 
 
TARGETED/PLANNED ENROLLMENT: Number of Subjects 
Ethnic Category 
Sex/Gender 
Females 
Males 
Total 
Hispanic or Latino 
   150 
    0 
      150 
Not Hispanic or Latino 
   300 
    0 
      300 
Ethnic Category: Total of All Subjects* 
450 
Racial Categories 
 
American Indian/Alaska Native 
0 
0 
0 
Asian 
5 
0 
5 
Native Hawaiian or Other Pacific Islander 
0 
0 
0 
Black or African American 
150 
0 
150 
White 
295 
0 
295 
Racial Categories: Total of All Subjects* 
450 
0 
450 
 
*The “Ethnic Category: Total of All Subjects” must be equal to the”Racial Categories: Total of All Subjects”.  
 
Pregnant women, 18-45 years of age will be recruited, of Hispanic, African-American and Caucasian ethnicities 
(may have a few Asians; will not exclude American Indian and Native Hawaiian if they are interested). All 
subjects will be receiving their prenatal care in the Charleston, SC area. All women will be less than 14 weeks of 
gestation at enrollment. This is a vitamin D supplementation study in pregnant women; so therefore, all women 
must be pregnant. The research question to be addressed is relevant only to women (pregnancy). A blood 
sample from the child of the pregnant women will be collected at birth, and the child will have a visit (with 
mother) at around 18 months of age to examine the first 4 baby teeth that have erupted and also to obtain a 
sample of the child’s blood at that age for measurement of vitamin D. 
 
Activity #7 (Objective #3) will not consent any additional subjects, because we are requesting a waiver of 
consent and HIPAA for deidentified record review of women who delivered babies at MUSC since September 
2015. 
 
b. Sources of Materials 


 
 
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The sources of research material will be maternal interview, history and physicals at clinic visits, the 
questionnaires, the lab reports for blood and urine analyses, the dental periodontal exam results, the dental 
salivary occult blood results, the vaginal swab results, the placental pathology report, the medical records of 
each mother and her infant following delivery, and the photographic teeth results on the infants. 
 
The material/data that is routinely found in a maternal health record during pregnancy (including information on 
past pregnancies and disease history) will be utilized in the study. There are additional materials and data from 
questionnaires, blood, urine, saliva, infant teeth, placental tissue and vaginal samples that will be obtained for 
research purposes only. 
 
A master list will be stored with study files accessible for clinical intervention only. The PI and study staff will 
have access to the master list. All archived samples and samples provided to collaborating laboratories will be 
identified with study subject numbers only. The DNA samples collected will have no recontact. 
 
c. Potential Risks  
 
Potential Risks of Phlebotomy and Blood Removal: The total amount of blood to be drawn at each maternal visit 
is as follows: visits 1 and 4, 31.5 mL; visits 2 and 8 (delivery), 22.5 mL; visits 3, 5, and 6, 17.5 mL; and visit 7, 37.5 
mL. The total amount of blood drawn over the study will not exceed 200 mL. The risks of maternal phlebotomy 
include a slight pain when the needle is inserted into the vein. Momentary discomfort may occur. Pressure will 
be applied to the arm following the procedure to minimize the risk of bruising. There is a slight chance of 
inflammation of the vein and/or blood clot formation, but this is extremely rare. Fainting could occur, but this is 
rare. The same risks apply to the blood draw for the child. Every attempt will be made to minimize discomfort, 
including application of a topical anesthetic (EMLA cream) to the child’s arm prior to venipuncture. These 
procedures were in place for our two previous vitamin D supplementation trials during pregnancy (HR #10727 
and HR #16476). 
 
Potential Risks of Dental Procedures: The risks of the dental procedures include temporary discomfort 
associated with periodontal probing and temporary discomfort of a dry mouth after providing a saliva specimen. 
Because of the transient bacteremia with periodontal probing, the few women who are usually premedicated 
with antibiotics before routine dental treatments will be excluded from the periodontal probing. 
 
Potential Risk of Obtaining Umbilical Cord Blood: The total amount of blood to be collected from the umbilical 
cord is 30 mL. Since this procedure will be performed after delivery, there is no risk to either the mother or the 
infant during this procedure. 
 
Potential Risks of Vitamin D Supplementation: There is a possibility that the treatment (dose of vitamin D) that 
each subject receives may prove to be less effective or to have more side-effects than the other study treatment 
(other dose of vitamin D) or other available treatments. The risks of maternal high dose 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. In the more than 540 women in the 
two prior vitamin D pregnancy supplementation trials (5, 6) and in the more than 340 mothers and infants who 
participated in the recently completed vitamin D lactation supplementation trial conducted at MUSC and the 
University of Rochester, this has not happened previously at this dose. The dose of vitamin D (4400 IU/day) used 
in this study is just over the limits established by the Institute of Medicine in 2010 (3). The PI and co-investigator 
do have an IND from the FDA (IND 66,346) for the administration of this dose of vitamin D and a higher dose in 
pregnant and lactating women; this protocol has been added to the IND. If, after 3 months on vitamin D 
supplementation, a woman is still frankly vitamin D deficient (less than 15 ng/ml), she will receive open label 
vitamin D gummies as requested by the FDA (IND 66,346). In two previous large randomized controlled trials of 
vitamin D supplementation during pregnancy and one large randomized controlled trial of vitamin D 


 
 
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supplementation during lactation, there was no difference in serum calcium or urinary calcium/creatinine ratios 
between the treatment groups (please see attached papers). 
 
Potential Risks to the Fetus: During pregnancy, the developing fetus’ vitamin D levels are about 70% of maternal 
serum vitamin D levels (5, 6). In the more than 540 women who participated in our two previous vitamin D 
supplementation studies, there was no identified increased risk to the developing fetus as per our two Data 
Safety and Monitoring Committees. Yet, some unknown risk of high dose maternal vitamin D supplementation 
on the fetus could be possible. By monthly monitoring of maternal serum vitamin D levels, we will ensure that 
the fetal levels remain in the normal range. 
 
Potential Risks of Vaginal Swabs: Vaginal swabs obtained by the subjects do not carry a risk greater than 
insertion of a tampon. 
 
Potential Risk of Breach of Confidentiality: As with any research study, there is a small risk that medical 
information, laboratory reports and data could become available to individuals outside the study. Every effort 
will be made to ensure that this does not happen. 
 
Potential of Unknown Risks: The experimental trial may have unknown side effects that previously have not 
been reported (unexpected and unanticipated adverse events or effects). 
 
The only alternative to this treatment regimen with vitamin D is the standard prenatal vitamin prescription (with 
400 IU vitamin D3 per tablet) taken daily throughout pregnancy. This regimen may not be advantageous if a 
woman is deficient in vitamin D. 
 
 
2. ADEQUACY OF PROTECTION AGAINST RISKS  
 
a. Recruitment and Informed Consent 
 
The study subject’s primary care provider(s) will make first contact on behalf of the study so there is no 
appearance of a breach in confidentiality. Once a subject has expressed interest in this research study, to obtain 
informed consent, one of the study coordinators will explain to each subject in lay language the purpose, 
benefits and risks of the investigation. Consent will be given following an informative discussion period and 
either by reading the Informed Consent to the subject or by allowing the subject to read the consent, then 
reviewing it with the subject. All study coordinators have taken the Research Coordinator course and have 
passed their Miami CITI. Every attempt will be made to conduct this study in full compliance for the protection 
of the subjects. For those women who are not fluent in English and whose first language is Spanish, a Spanish 
version of the consent form will be used (translated by BiLingo from IRB approved English consent and 
submitted as an amendment to IRB). Following the subject’s verbal agreement to participate in the study, the 
subject will sign the informed consent; the study coordinator also will sign the consent. A copy of the signed, 
written informed consent will be given to the study subject. The original informed consents will be placed in 
locked research file in study coordinators’ office, 20 Ehrhardt St, #4. A copy of the informed consent also will be 
placed in the subject’s study binder (located in locked cabinets at same location). A study coordinator note will 
be placed in Epic identifying an MUSC woman as participating in a vitamin D and pregnancy RCT. 
 
b. Protection against Risk 
 
All of the investigators have conducted several clinical trials; they are well versed in the issues of patient/subject 
confidentiality and have completed the Miami CITI course. They are nationally and internationally known in their 
fields. All study coordinators have completed the Miami CITI and the Research Coordinator course at MUSC. The 
endeavors of the PI, co-investigators and study coordinators to maintain subject confidentiality will be ensured. 


 
 
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All maternal blood, urine, and vaginal samples as well as infant blood and placental tissue will be labeled with 
the patient’s name and medical record number to ensure proper tracking. Each study subject (and 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 the web database. 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 offices of the study coordinators. The data will be entered in 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. All this information will be kept confidential, and when reported in scientific 
journals, the information will not have any identifying information regarding study subjects. 
 
Should any untoward reaction occur, subjects would receive treatment at the Medical University of South 
Carolina. The study will cover the costs of laboratory tests; however, additional costs of treatment will be the 
responsibility of the subject as described in the informed consent. 
 
See main section #5 Data Safety and Monitoring Plan that follows for full details; this is a clinical trial. This study 
meets the guidelines of clinicaltrials.gov as a clinical trial requiring a Data Safety and Monitoring Plan and a Data 
and Safety Monitoring Committee. Two of the members of the Data and Safety and Monitoring committee are 
scientists external to MUSC well known in the field of vitamin D metabolism and calcium metabolism. Two of the 
members are physician scientists external to MUSC, who are also well known in the field of calcium and vitamin 
D metabolism. A fifth member will be Dr. Tom Hulsey, epidemiologist at MUSC with considerable experience 
serving on the DSMC of other clinical trials. Dr. Hulsey was the chair of the DSMC of the two prior vitamin D 
supplementation trials conducted by this study team. He will maintain the database of the study, follow HIPAA 
guidelines, and conduct ongoing interim analyses to ensure that the risk: benefit analyses remain in favor of 
benefit to the subjects.  
 
 
3. POTENTIAL BENEFITS OF THE PROPOSED RESEARCH TO THE SUBJECTS AND OTHERS  
 
Pregnant women enrolled in the study may avoid vitamin D deficiency during their pregnancy. The developing 
fetus may have more normal calcium and vitamin D levels, better bone mineralization, and improved immune 
function and brain development. Another potential benefit of this study is that data will be generated that will 
translate into recommendations for optimal vitamin D status during pregnancy. Increasing numbers of 
Americans are becoming aware of the risks of hypovitaminosis D, and have begun taking supplemental vitamin 
D. Without monitoring, the potential for overdosing—although small, exists. There needs to be additional 
controlled trials that will serve as a guide for practitioners and health care policy makers when recommending 
the DRI for vitamin D during pregnancy in all ethnicities.  
 
The risks to the subjects in participating in this study (phlebotomy, hypercalciuria, hypercalcemia, confidentiality 
issues) are reasonable for the following reasons: The issue of adequate vitamin D levels in pregnant women and 
in women in general is a recent public health problem that reflects the vitamin D-poor content of modern diets 
and markedly decreased sunlight exposure (a relatively new phenomenon in our evolution) that follows the 
recommendation of the American Cancer Society and the American Dermatological Society. Thus, the issue of 
maternal supplementation with vitamin D becomes relevant for thousands of women and their developing 
fetuses. Hypovitaminosis D during pregnancy is a preventable disorder whose occurrence is widely pervasive in 
the African American community (affects >75% of reproductive age African Americans) with the majority of their 
neonates being hypovitaminotic in utero and at birth. Hypovitaminosis D is also prevalent in the Hispanics 
(affects approximately 60% of women of reproductive age). Secondly, the maximal dose that women will take 
for 10-40 weeks gestation is 5-fold lower than the per kg dose given to preterm and term infants routinely in 
total parenteral nutrition and formulas. Compared to the Masaii tribe in East Africa, who receive 5-9 hours of 


 
 
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sunlight per day and achieve vitamin D levels well above that achieved taking 4000 IU vitamin D3, day, this 
supplementation regimen is aimed at reversing the vitamin D deficiency epidemic in the U.S. and other areas of 
the world. We now know that vitamin D deficiency has far more clinical implications than integrity of the 
skeleton, including impaired immunity and increased risk of various disease states, including diabetes, neoplasia, 
and multiple sclerosis. Through vitamin D supplementation during pregnancy, we believe that participation will 
beneficially improve overall health status of mother and her developing fetus. Because of the close monitoring 
of the pregnant women and their developing fetuses through pregnancy, we will have minimized the risks of 
participation in this study. Should a woman develop hypervitaminosis D, hypercalciuria or hypercalcemia, there 
are appropriate safeguards built into the study to minimize the risk to the mother and her infant. 
 
4. IMPORTANCE OF THE KNOWLEDGE TO BE GAINED  
 
The issue of maternal supplementation with vitamin D becomes relevant for thousands of women and their 
unborn fetuses due to recent public health information showing that the majority of pregnant women have 
inadequate vitamin D levels and decreased sunlight exposure. The potential co-morbidities of pregnancy caused 
by vitamin D insufficiency or deficiency are increasing and occur with more frequency in African-American and 
Hispanic ethnicities. As shown in our previous vitamin D supplementation pregnancy trials, hypovitaminosis D is 
a preventable disorder with adequate supplementation; we strongly believe that the DRI for pregnant women, 
especially darkly pigmented individuals is woefully inadequate. Optimal vitamin D supplementation becomes an 
issue for the majority of pregnant women in this country and is necessary to decrease health disparities due to 
ethnicity. 
 
The results of our previous vitamin D supplementation clinical trials in pregnancy did not show any detrimental 
effects (hypercalcemia or hypercalciuria) of daily intake of 4000 IU of vitamin D (5, 6). While in the NICHD 
vitamin D supplementation trial, there was a trend toward reduction in co-morbidities of pregnancy (5), in the 
Thrasher Research Fund trial involving community-based women living at or near the poverty level, there was 
evidence of reduced preterm labor, preterm birth, and infection as a function of maternal total circulating 
25(OH)D levels, effects that persisted even after controlling for race (6). Yet, in both studies where the health 
status of the mother was not the focus of the trial nor was immune function, these are post-hoc analyses and 
can only suggest correlation. In the present RCT, we hope to show statistical differences in the co-morbidities, 
and show improved immune function with improved vitamin D status. 
 
Based on the data of vitamin D supplementation in normal adult men and women of diverse ethnic 
backgrounds, our previous vitamin D pregnancy and lactation trials, and the current practice of safely 
supplementing with 5-8 fold higher IU/kg doses in infants and young children, there is little risk of 
hypervitaminosis D occurring in pregnant subjects enrolled in this study. Rather, it is quite likely that the woman 
and her fetus (later, infant) will achieve more satisfactory vitamin D and calcium homeostasis that will yield 
improved immune function and reduction in co-morbidities of pregnancy. Should a woman develop 
hypervitaminosis D, hypercalciuria or hypercalcemia, there are appropriate safeguards built into the study to 
minimize the risk to mother and her infant. Thus, the risks to the subjects are reasonable in relation to the 
importance that reasonably may be expected to result. 
   
 
5. SUBJECT SAFETY AND MINIMIZING RISKS (Data and Safety Monitoring Plan) 
 
 
DATA AND SAFETY MONITORING PLAN: The operation of the DSMB is crucial to the safety of the subjects who will 
be enrolled in the study. Following the recommendations of the DSMB will be an essential and fundamental part 
of this Research Protocol. 
 
A. Creation of a Data and Safety Monitoring Board (DSMB): This grant application meets NIH policy and 
Guidelines on the inclusion of a Data and Safety Monitoring Plan for clinical trials mL and 


 
 
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http://grants.nih.gov/grants/guide/notice-files/not98-084.html). MUSC as the Institute and Center (IC) of this 
grant will have a Data and Safety Monitoring Committee (DMSC) in place as outlined in this grant. Two of the 
members of the DSMC are scientists external to MUSC well known in the field of vitamin D and calcium 
metabolism. Two of the members are physician scientists external to MUSC who also are well known in the field 
of calcium and vitamin D metabolism. A fifth member will be Dr. Tom Hulsey, epidemiologist at MUSC with 
considerable experience serving on the DSMC of other clinical trials (current and completed). He will maintain the 
database of the study, follow 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 MUSC clinical lab is required to notify the PI of any critical lab values. The Study 
Coordinators will be responsible for checking laboratories of the women within 72 hours of their reporting and 
then entering the laboratory data into the computer. They will be provided with a set of normative laboratory 
values as a reference. The Clinical PI will review values during the weekly study meeting or will be notified if a 
value falls outside of the referent value range. All data will be verified independently by the Data Processing Center 
under the direction of Dr. Hulsey. The DSMC will be notified via telephone and fax 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 semiannually by the DSMC, whose report will be forwarded to the Kellogg Foundation on a quarterly 
basis and the IRB yearly. The Investigators will report all Serious Adverse Events by telephone to the IRB and the 
DSMC; in addition, the IRB, the DSMC, and Kellogg Foundation will receive a written 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. The DSMC will serve to monitor 
for safety and efficacy. DSMB reports produced by Dr. Hulsey’s team will include summary statistics: on mother 
recruitment (expected vs. actual), data form quality (completion and timeliness of forms); tracking of data editing; 
demographics of the randomized mothers; 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 (119). 
 
 
 
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Johnson DD, Wagner CL, Hulsey TC, McNeil RB, Ebeling M, Hollis BW. Vitamin D Deficiency and 
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20640974  
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Supplementation Trials during Pregnancy: Health Characteristics and Outcomes. Pediatr Res. 2012;#750466 
(abstract). 


 
 
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to recommend an intake of vitamin D that is effective. The American journal of clinical nutrition. 2007;85:649 - 50. 
118. 
Paintlia AS, Paintlia MK, Hollis BW, Singh AK, Singh I. Interference with RhoA-ROCK Signaling 
Mechanism in Autoreactive CD4(+) T Cells Enhances the Bioavailability of 1,25-Dihydroxyvitamin D(3) in 
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 G. CONSULTANTS__________________________________________________________________________ 
Where applicable, attach electronic versions of appropriate letters from all individuals confirming their roles in 
the project. Go to the application under “additional uploads” to attach this information. 
   n/a 
 
H. FACILITES AVAILABLE____________________________________________________________________ 
Describe the facilities available for this project including laboratories, clinical resources, etc. 
Study subjects will be seen in obstetrical clinics at MUSC; interested pregnant women not being followed at any 
of these clinics can consent for the study and be seen in the CTRC at MUSC.  Research laboratories will include 
several in the Children’s Research Institute – Dr. Bruce Hollis/Dr. Carol Wagner, Dr. Jennifer Mulligan and Dr. 
Inderjit Singh as well as several in Obstetrics and Gynecology – Dr. Louis Guillette and Dr. David Soper/Dr. 
Gweneth Lazenby, and laboratories of Dr. Michael Caplan and Dr. Scott Argraves at MUSC. The laboratory of Dr. 
Sarah Rothenberg is located in the Department of Environmental Health Sciences, University of South Carolina. 
 
I. INVESTIGATOR BROCHURE ____________________________________________________ 
If applicable, attach the electronic version of the investigator brochure. Go to the application under “additional 
uploads” to attach this information.  
n/a 
 
J. APPENDIX_______________________________________________________________________________ 
Attach any additional information pertinent to the application, such as surveys or questionnaires, diaries or logs, 
etc. Go to the application under “additional uploads” to attach this information.  
 
 
 
Relevant Papers: 
Hollis BW, Johnson D, Hulsey TC, Ebeling M, Wagner CL. Vitamin D supplementation during pregnancy: double-
blind, randomized clinical trial of safety and effectiveness. Journal of bone and mineral research : the official 
journal of the American Society for Bone and Mineral Research. 2011;26(10):2341-57. Epub 2011/06/28. doi: 
10.1002/jbmr.463. PubMed PMID: 21706518; PubMed Central PMCID: PMC3183324. 
 
Wagner C, McNeil R, Hamilton S, Winkler J, Rodriguez Cook C, Warner G, et al. A Randomized Vitamin D 
Supplementation Trial in Two Community Health Center Networks in South Carolina. Am J Obstet Gynecol. 
2013;in press. 
 
 
 
 
 
 


 
 
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SAP_001.pdf: 
Preventing Health Disparities during Pregnancy through Vitamin D Supplementation 
NCT#01932788 
 
 
11/6/2013 
 
Statistical Analysis: The primary analysis in this investigation will compare levels of maternal 
25(OH)D across the two supplementation groups as ‘Intention-to-Treat’ and will employ Mantel-
Haenszel mean scores; however, a ‘Per Protocol’ (oversampling to consider attrition) analysis 
also will be done. This two-step analysis provides information regarding the efficacy of these 
dosage levels in increasing vitamin D and the effectiveness of oral supplementation to increase 
vitamin D. Whether the losses are random or are biased with respect to ethnicity, or 
supplementation arm will be tested. Any differential losses will be reported and examined with 
the use of propensity scores. 
 
Because of the unique nature of the contributions of potential confounders, two approaches 
to the analysis will be employed. The first and primary analysis will use a Mantel-Haenszel 
estimate of the mean treatment difference, computed from stratum-specific treatment differences, 
which will utilize a weighted average of treatment differences across ethnicity. Mean differences 
will be calculated for the experimental dosage relative to the 400 IU group. Bonferroni 
adjustments will be made for the Type I per comparison error rates to correct for multiple testing. 
The treatment differences, estimated variances, and confidence intervals for the differences will 
be estimated for maternal serum 25(OH)D. This mode of analysis is quite effective in clinical trials 
as it is less dependent on normal theory assumptions and associated assumptions such as 
homogeneity of variance. Parametric models also will be fit, but the primary analysis will rest on 
Mantel-Haenszel mean scores techniques readily done within SAS. Parametric dose response 
curves also will be generated to describe the relationship between 25 (OH)D and dosage. 
  
 
The proportion of participants who completed the daily supplement regimen will be estimated, 
with a similar “per protocol” style analysis restricted to those individuals. Finally, techniques of 
multiple linear regression will be employed to describe the interactive effects of ethnicity, as well 
as the effects of other covariates, such as seasonality, mother’s age and fecundity, on baseline 
serum 25(OH)D. These models will be assessed for violations of the model assumptions, and 
corrected for such violations as appropriate.  
 
  
The second statistical approach measures the effects of the confounders and takes the more 
traditional approach of assuming normal distribution for Vitamin D, and will be performed in 
parallel fashion to compare treatment effects in 400 IU and 4400 IU vitamin D groups. The primary 
research aim is to determine if supplementation is associated with an increase in vitamin D 
concentrations. As discussed below, it will be necessary to identify and control for other factors 
(confounders) that may also be associated with changes in vitamin D levels. 
 
      Paired analyses according to the study plan will be conducted by employing McNemar chi-
square for categorical variables, paired t-tests for normally distributed variables, repeated 
measures ANOVA for longitudinal analyses, and multiple linear regression for multi-variable 
analyses to control for confounding. Correlation will be used to assess the linear association 
between outcomes and those factors expected of an accumulated dose-response relationship 
(e.g., sunlight exposure), and individual regression tests to identify the strength of the association 
between outcomes and each characteristic. The resulting coefficients then will be translated into 


a formula for the linear association between each factor and outcomes. In the event that there are 
non-linear distributions, appropriate data transformations will occur as necessary.  
 
Both groups will be compared for differences in any factors at baseline, and will include: 
information contained in the maternal sociodemographic, maternal pregnancy and delivery, and  
maternal calcium, creatinine, vitamin D3, 25(OH)D, and urinary calcium creatinine ratio. While the 
stratified blocked randomization technique should minimize the potential for baseline differences, 
this first step will confirm whether there should be adjustment for any initial differences in follow-
up analyses. Next, differences between baseline and follow-up will be examined for those 
variables that may have changed over the follow-up period (health assessment; physical activity; 
and blood and urine outcome measures) to identify those factors that may serve as confounders 
or effect modifiers for the vitamin D assessments. The potential influence of identified confounders 
or interaction terms will have to be considered for any change in vitamin D status. 
 
      Changes in vitamin D outcomes will be compared between treatment arms with the additional 
information from sunlight exposure, pigmentation/melanin index, maternal physical activity 
assessment, maternal dietary history, health, and medication questionnaires. This detail will allow 
the separation of changes in vitamin D parameters as a function of confounding factors from 
supplementation. The study is powered to explore the degree to which changes in vitamin D 
parameters are a function of supplementation and ethnicity. To assess whether there are 
differences in the slopes or linear dissimilarities in the outcomes over time, repeated measures 
techniques will be used to compare longitudinal changes in outcomes between groups. This 
technique also provides a graphical depiction of the slopes over time and can identify if there are 
linear or plateau responses to differences in vitamin D supplementation concentration; it also will 
allow determination of data transformation techniques required for subsequent analyses if the 
slopes over time are non-linear. 
 
 
The final step in the second statistical approach will determine whether potential group 
differences in the various outcomes are a function of any of the potential confounders—with 
particular attention to skin pigmentation and sunlight exposure. This step will identify changes in 
outcomes associated with population factors independent of the group (supplementation) 
assignment. The extent of more complex statistical analyses will be predicated on the number of 
individuals comprising specific strata, and thus, restricted by a reduction in the statistical power. 
Such comparisons will be reported as information for future studies. To clarify, using sunlight 
exposure as a representative example, the independent effect of vitamin D supplementation may 
be found to have been modified by the amount of sunlight exposure. As such, the changes in 
outcomes attributable to each factor, independently, and then when considered in combination 
will be partitioned. While the major goal of this study is to examine the various outcomes as an 
independent function of vitamin D supplementation—thereby controlling for all other group 
differences, the changes in outcomes that are associated with these group differences will be 
examined and reported in detail. In this way, the changes in vitamin D that are associated with 
each factor independently will be partitioned.