Supplements to Improve Vascular Inflammation After an Adverse Pregnancy Outcome
Phase: NA
Conditions: Pre-Eclampsia, Pre-Term, Intrauterine Growth Restriction, Hypertension in Pregnancy
Sponsor: University of South Carolina

Study Title: Dietary supplements to improve vascular inflammation after an adverse pregnancy 
outcome 
 
NCT04633551 
 
11/13/2020 
 
 
 

 
 
A. SPECIFIC AIMS  
 
1. Aim 1. Determine the effects of a 4-week course of anti-inflammatory 
supplementation (PolyResveratrol-SR®) on vascular function, subclinical 
atherosclerosis and CV biomarkers 
 
2. Aim 2. Define adherence and barriers to compliance to supplementation.  
 
 
B. BACKGROUND AND SIGNIFICANCE 
 
 
Adverse pregnancy outcomes (APOs) occur in up to 20% of all pregnancies and are associated 
with a lifetime of higher cardiovascular disease (CVD) risk1,2.  Aberrant and persistent 
systemic inflammation is a hallmark of both APOs and CVD.3 An inappropriate uterine 
innate immune response to invading fetal cytotrophoblasts is thought to underlie poor 
placentation and vascular-related APOs; inflammation of the uterine cavity evokes contractions 
and contributes to preterm birth.4 Levels of cytokines involved in endothelial inflammation, an 
instigating event in hypertensive and atherosclerotic processes, were higher in the 2nd trimester 
in women destined to have APOs.5 Women with a past APO have higher levels of systemic 
inflammation, including c-reactive protein and interleukin-6 (IL-6), in the years after delivery 
versus women with no APOs. A similar pro-inflammatory cytokine profile has been observed in 
patients with overt CVD. Intervening on systemic inflammation may be a viable strategy to 
interrupt the progression from APO to CVD.  
Dietary supplements, such as polyphenols, are anti-inflammatory and inhibit vascular 
inflammation with potentially few barriers to adherence. Given the hypothesized contribution 
of a persistent pro-inflammatory state to the excess CVD risk observed in women after 
APO, utilization of a polyphenol dietary supplement to specifically target inflammation 
holds promise as a low-burden intervention to prevent or delay APO-related CVD. To our 
knowledge, efficacy and tolerability of a dietary polyphenol supplement has not been 
investigated in women with a history of APO. To fill in this critical gap in knowledge, we propose 
to test the vascular function and CV biomarker, i.e., IL-6, IL-10, intracellular and vascular cell 
adhesion molecule-1 (ICAM-1 and VCAM-1), responses to a 4-week anti-inflammatory dietary 
supplementation in a clinical trial pilot study of 56 women, 28 assigned to the intervention and 
28 to the control group, in the first 3 years after an APO. 
C. PRELIMINARY STUDIES 
 
Other studies of endothelial dysfunction and APOs have consistently shown that women who go 
on to have APOs have evidence of endothelial dysfunction that is detectable in the 1st trimester 
of pregnancy5. In a recent paper, we demonstrated that there was no difference in evidence of 
pre-pregnancy endothelial dysfunction between women who did and did not go on to have a 
preterm or small for gestational age delivery ~3 years later (OR: 1.01, 95% CI: 0.74, 1.39). Our 
results suggest that the endothelial dysfunction that contributes to APOs (and eventual CVD) 
may not develop until pregnancy begins, highlighting the importance of the pregnancy period for 
influencing the CVD risk trajectory in women. In this investigation, only black race was 
associated with greater risk of preterm birth and/or small for gestational age delivery, even after 
adjustment for relevant covariates 
 

Higher BMI and central BP in women with a past 
APO. In early data from an ongoing study conducted in 
the PI’s lab, we found a trend towards higher central 
systolic BP in women with a past APO (Table, 
unpublished data). Findings suggest that differences in 
sensitive measures of vascular function are detectable 
soon after an APO but before overt CVD. 
 
 
 
D. RESEARCH DESIGN AND METHODS AND DATA 
ANALYSIS  
 
Research Design. We will invite 56 women with an APO within the past 3 yrs to participate. 
Women will be randomly assigned to the 4-wk intervention group (n=28) or a standard care 
group (n=28) using a computerized random number generator, stratified by race. Assuming up 
to 25% with poor adherence, we expect that 42 women (21 each arm) will complete the study. 
No data exists related to effects of supplementation on vascular function in our population, we 
assumed a medium effect size, i.e. Cohen’s d~0.50 and accepting a β=0.80, to generate sample 
sizes. Generation of effect sizes is a key outcome of this study. We will conduct a vascular 
assessment and blood draw at two visits: before the intervention begins and within 3 days of 
completing the intervention. We will conduct visits during the early follicular phase of menstrual 
cycle to account for fluctuating hormones. Vascular testing will be performed in the supine 
position and ≥4 hours after a light meal. Surveys for Aim 2 will be completed within the first 2 
weeks of study enrollment, and interviews for Aim 2 will be conducted within 1 week of Visit 2, 
in-person or via telephone.  
 
Anti-inflammatory supplementation intervention. Participants randomized to the 
supplementation intervention will receive PolyResveratrol-SR® (100 mg curcumin phytosome, 
100 mg quercetin phytosome, 100 mg green tea phytosome, 100 mg trans-resveratrol, 100 mg 
trans-pterostilbene; Thorne Research). Participants will be asked to take 2 doses/day (1g total). 
Participants randomized to supplementation will receive a daily email reminding them to take 
the supplement. Covariates. Medical history, age, and sociodemographics will be obtained via 
self-report. We will use validated surveys to determine physical activity, sedentary behavior, and 
smoking history. Height and weight will be measured. 
 
 
Aim 1 Outcomes. The outcomes will be measured in-person. All outcomes are listed and 
described below. Dr. Lane-Cordova has extensive experience collecting the measurements 
described below. 
  
1. Blood pressure. Resting brachial systolic BP (SBP) and diastolic BP (DBP) will be 
measured using an automated oscillometric cuff (HEM-907 XL; Omron, Shimane, Japan) 
after 10 min of supine rest in a quiet, dimly lit room. Brachial BP will be obtained in 
duplicate with 1 min between the first and second reading. If the two values are not 
within 5 mmHg of each other, another measurement was taken until two consecutive 
values within 5 mmHg of each other are obtained. These two values will be averaged 
and used for analysis. 
2. Arterial stiffness. Participants will remain supine in a quiet room. The aortic waveform 
will be reconstructed from radial artery pressure waveforms obtained from a 10-s epoch 
 
APO+ 
n=17 
APO- 
n=36 
Age (yrs) 
33 
33 
Race (% AA)* 
28 
17 
aSBP (mmHg)* 
102 
97 
bSBP(mmHg) 
113 
109 
BMI (kg/m2)* 
29.2 
26.0 
Table Legend. AA: African American; aSBP: aortic systolic 
BP; bSBP: brachial systolic BP 

using applanation tonometry (Millar Instruments, Houston, TX) and calibrated with 
brachial MAP and diastolic BP with a generalized validated transfer function6 
(SphygmoCor; AtCor Medical, Sydney, Australia) From this central waveform, we will 
derive central BP, augmentation index (AIx), and end-systolic pressure. An in-device 
quality rating of ≥80 % will be required for all recordings used in analysis. We will 
measure central (aortic) pulse wave velocities in the supine position using gold-standard 
techniques7. We will measure distances from the suprasternal notch to the femoral artery 
and from the carotid artery to the suprasternal notch with a tape measure, recorded to 
the nearest mm. The distance from the carotid site to the suprasternal notch will be 
subtracted from the distance between the suprasternal notch and femoral artery to 
account for the divergent directions of pulse wave propagation. We will use the same 
high-fidelity strain-gauge transducer to obtain pressure waveforms at the right common 
carotid artery and then at the right femoral artery for central pulse wave velocity. Pulse 
wave velocity will be calculated as distance/time, using the distances between 
measurement points and the measured time delay between 10 proximal and distal 
waveforms (SphygmaCor; AtCor Medical). ECG will be recorded simultaneously, and the 
peak of the R wave will be our timing marker. BP influences pulse wave velocity, so we 
will also adjust pulse wave velocity for aortic MAP.  
3. Resting forearm blood flow. Forearm blood flow (FBF) will be measured using strain 
gauge plethysmography (EC-4; Hokanson, Inc., Bellevue, WA). A standard BP cuff will 
be positioned around the upper arm. An appropriately sized strain gauge will be placed 
around the widest part of the forearm, and a pediatric cuff placed around the wrist to 
occlude hand blood flow. Before measuring FBF, the wrist cuff will be inflated to 250 mm 
Hg of pressure for 1 minute. FBF will be determined by inflating the upper cuff to 50 mm 
Hg (to oppose venous backflow) for 7 s and then deflating it for 8 s. An average of six of 
these 15-s plethysmographic cycles will be used to determine FBF, expressed as 
milliliters per minute per 100 mL of forearm tissue and as flow per unit pressure, e.g. 
conductance, using the equation: conductance = (FBF/MAP) x1000.  
4. Vascular endothelial function.  Reactive hyperemia, e.g. resistance vessel endothelial 
function, will be measured immediately after FBF. Arm blood flow will be occluded by 
inflating the upper arm BP cuff to 250 mm Hg for 5 min. One minute prior to the release 
of the upper arm cuff, the wrist cuff will also be inflated to 250 mm Hg. The upper arm 
cuff pressure will be rapidly released and changes in forearm volume will be recorded 
via the strain gauge, in 15-s cycles as described above, for 3 min (13 readings)8. The 
highest reading will represent peak blood flow. All 13 measurements will be plotted 
against time, and the area under this curve will represent total reactive hyperemia8. We 
chose to measure reactive hyperemia rather than flow-mediated dilation due to the high 
intra- and interobserver variability in flow-mediated dilation measurements9. 
5. Carotid intima-media thickness. The right common carotid artery will be imaged with 
ultrasound (Logiq GE S8) using a 12-MHz linear array probe. Intima-medial thickness 
will be defined as the distance between the leading edge of the lumen–intima border and 
the leading edge of the media–adventitia border on the far wall of the artery10. We will 
average a 5mm segment obtained approximately 20 mm proximal to the carotid 
bifurcation for analysis. 
6. Blood analyses. We will collect 30 mL(2 Tb) of blood into an EDTA containing tube and 
serum tube using venipuncture and a butterfly needle. Blood will be centrifuged at 1300 
g for 10 min, aliquoted into 0.25 mL cryotubes, and stored at -80ºC until analysis.  We 
will determine levels of circulating IL-6, IL-10, ICAM-1, and VCAM-1 using commercially 
available ELISA kits (R&D Systems).  We will bank unusedl blood for future, as yet 
undetermined analyses.  Participants will be informed of this intent before providing 
blood.  Blood will be labelled and linked to clinical data and demographics using a 

unique identifier; samples will be de-identified for storage at the PHRC and for future 
analyses. 
7. Covariates: Medical and Pregnancy History and Demographics. We will ask questions 
about demographic, medical, and pregnancy history.  Answering questions will not 
provide identifiable information, Self-reported activity: We will use validated surveys to 
quantify physical activity and sedentary behavior,12,13 Diet History. We will use a 
validated survey to assess diet composition in the previous 6 months.14 
 
Data Analyses. We will calculate the 25th and 75th percentile for each biomarker and vascular 
function test and use these values as cut-offs for “low” and “high” levels, respectively. We will 
test for differences in biomarker levels between women by APO status and race using a t-test or 
Mann-Whitney U test, depending on the distribution of the variables. We will use separate 
multiple linear regression models to determine associations of each exposure (continuous levels 
of biomarkers; quartiles; and high or low levels of these biomarkers) with continuous measures 
of our outcomes (individual indices of vascular function, e.g. BP, arterial stiffness, augmentation 
index, FBF, endothelial function, and intima-media thickness) in all 56 women. We will use 
logistic regression to test for associations between continuous and categorical levels of 
biomarkers with categorical outcomes: the likelihood of having hypertension or prehypertension, 
high central arterial stiffness, low endothelial function, and high carotid intima-media thickness 
in all 56 women.  We will test for unadjusted associations and then adjust all regression 
analyses for key co-variables: maternal age, race, and smoking. We will test for associations 
after furthering adjusting for years of education, body mass index (BMI), parity, and physical 
activity, in the index pregnancy in exploratory analyses.  We will test for an interactive effect of 
APO status and race on these associations using a multiplicative interaction term in separate 
analyses and conduct all analyses stratified by APO status and/or race if the interaction is 
significant.  We will conduct a sensitivity analysis by repeating our statistical tests only in the 
primiparous women as prior pregnancy is associated with lasting changes in vascular function 
and may influence our outcome and exposure variables.   
Aim 2 Outcomes:  
1.  Acceptability of Dietary Supplementation. We will administer surveys to all participants 
to understand their perceptions of dietary supplementation, including potential benefits 
and consequences associated with supplementation. We will determine willingness to 
use supplements, barriers to supplementation, and who, i.e., physician or partner, 
influences their decision to use dietary supplements.  
2. Adherence and Barriers to Adherence. We will count pills to determine adherence to 
supplementation regime. We will assess each intervention participant’s perceptions of 
supplementation with a brief interview at the final visit. Interviews will capture 
participant’s perceptions of the role of supplementation for CVD prevention, and any side 
effects of supplementation. We will ask participants to recommend ways to encourage 
participation and ask questions regarding their perceptions of the incentive structure and 
contact with the study team. We will contact participants who drop out of the study to 
identify reasons for drop out.  
 
Statistical analyses: Acceptability survey data will be collected using a Likert Scale, tallied and 
averaged. We will determine the proportion of participants with adequate adherence (≥80%) to 
supplementation. If ≥80% of women have adequate adherence, we will conclude acceptable 
adherence is feasible with the existing incentive structure. Analysis of all post-intervention 
interview data will use an inductive approach and systematically guided by the constant 

comparison technique. Qualitative data analysis software (QRS NVivo 10) will be used to code 
data and organize the analysis.  
 
 
E. PROTECTION OF HUMAN SUBJECTS 
 
1.  TARGET POPULATION: 
Participants and incentives. We will invite women who had a vascular APO 6 months-3 years 
ago15 and were 18 years or older at the time of the APO, to participate. Exclusion criteria: 
currently pregnant or breastfeeding, current smoking, active cancer, regular use of NSAIDs, 
steroidal medications, statins, or other anti-inflammatory supplements, HIV/AIDS, uncontrolled 
high blood pressure, unwilling or unable to use a dietary supplement, known sensitivity to 
resveratrol, curcumin, green tea, or quercetin. We will provide a cash incentive of $50 after 
study Visit 1 and Visit 2. If enrollment numbers are below target at 6-months, we will open 
enrollment to women with a history of gestational diabetes (GDM) as GDM is also associated 
with inflammation and excess CVD risk in women. We chose the time interval of 6 months to 3 
years after delivery because it takes about 6 months to return a pre-pregnancy physiological 
state, but vascular dysfunction has been documented for at least 3 years after an APO15.  
 
2.  RECRUITMENT PLANS:  
 
We will target recruitment channels including: 1) promotions in publications with low or no 
advertising cost (i.e., Craig’s List); 2) electronic and social media recruitment blurbs (i.e., 
Facebook, Twitter, Instagram, website, email newsletter, electronic billboard, etc.); 3) posting 
flyers around USC campus area and local Columbia businesses and health centers; 4) word of 
mouth, 5) ads in local papers and place flyers in public places. 
 
Additionally, patients will be recruited from Prisma USC Obstetrics and Gynecology Center (2 
Medical Park, Columbia, SC). Prisma staff may identify potentially eligible participants and direct 
them to the lab website or to talk to research staff for more information about the study.   
 
All recruitment material will list study eligibility criteria and requirements and include a phone 
number and email address where participants can reach staff if they have any questions 
regarding study participation. Potential participants will be screened over the phone by a 
research assistant and scheduled for a visit if they meet requirements. 
 
3. EXISTING DATA/SAMPLES:  
Not applicable 
 
4.  CONSENT/ASSENT: 
Participants fill complete a written, informed consent form prior to participation in the study. 
 
5.  POTENTIAL RISKS: 
The risks of the proposed studies are minimal. The main risks are (1) minor discomfort or 
bruising during phlebotomy and plethysmography; (2) unforeseen side effects of 
supplementation; and (3) loss of confidentiality. We will minimize risks 1 and 3 with standardized 
training of all study staff and by using HIPAA-compliant databases. Participants will be seated 
during the blood draw, and juice and snacks provided if needed.  A maximum of three needle 
sticks will be permitted. Given that the supplement for the study is readily available over the 
counter and we’re screening for known sensitivity, we do not anticipate severe side effects. We 

will inform participants of the supplement ingredients during screening and in the Informed 
Consent document and exclude individuals with known sensitivity to the ingredients. 
 
6.  POTENTIAL BENEFITS: 
Participants will not experience any benefits from participating in this study.  
 
7.  CONFIDENTIALITY 
All of participants data will be deidentified. Only study team members will have access to 
participant information and study data.  All data will be uploaded from these secure sites by 
study personnel and saved on the department server.  These data are stored behind an 
encrypted firewall, and automatically backed up. All data will be stored at least 7 years after the 
completion of the study.   
 
8. COMPENSATION: 
Participants will be compensated $50 at both study visits. We will pay for parking at our lab. 
 
9.  WITHDRAWAL: 
Participants will be informed they can leave the research at any time without penalty. If a 
participant decides to withdraw, no more information will be collected.  
 
Any data collected during participation in this research study prior to the date the participant 
chooses to withdraw consent may continue to be used by the investigators for the purposes 
described above.   
 
Choosing not to be in the study will not result in any penalty or loss of benefit to which 
participants are entitled. Specifically, the choice not to be in this study will not negatively affect a 
participant’s right to any present of future medical treatment or his/her present or future 
employment (for employees at USC or its affiliates).  
 
 
F. REFERENCES/LITERATURE CITATIONS 
 
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consequences of adverse pregnancy outcomes. J Amer Coll Cardiol. 2019. 
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Higashi Y, Sasaki S, Nakagawa K, Matsuura H, Kajiyama G and Oshima T. A 
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