Preventing Health Disparities During Pregnancy Through Vitamin D Supplementation
Preventing Health Disparities During Pregnancy Through Vitamin D Supplementation
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.
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.
Inclusion Criteria:

 -  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.

 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.