STRUCTURAL AND FUNCTIONAL ARTERIAL CHANGES IN HEALTHY WOMEN
FOLLOWING PREGNANCY: MATERNAL VASCULAR ADAPTATIONS TO
PREGNANCY II STUDY
by
Mansi Mahir Desai
B.S. in Molecular Biochemistry and Biophysics, Illinois Institute of Technology, 2012
Submitted to the Graduate Faculty of
Graduate School of Public Health in partial fulfillment
of the requirements for the degree of
Master of Public Health
University of Pittsburgh
2015
UNIVERSITY OF PITTSBURGH
GRADUATE SCHOOL OF PUBLIC HEALTH
This essay is submitted
by
Mansi Mahir Desai
on
April 21, 2015
and approved by
Essay Advisor:
Emma Barinas-Mitchell, PhD
Assistant Professor
Department of Epidemiology
Graduate School of Public Health
University of Pittsburgh
__________________________________________
Essay Reader:
Marnie Bertolet, PhD
__________________________________________
Assistant Professor
Departments of Epidemiology, Biostatistics, and Clinical and Translational Science Institute
Graduate School of Public Health
University of Pittsburgh
Essay Reader:
Janet Catov, PhD
__________________________________________
Assistant Professor
Departments of Obstetrics, Gynecology and Reproductive Sciences, Epidemiology, and Clinical
and Translational Research
School of Medicine
University of Pittsburgh
ii
Copyright © by Mansi M. Desai
2015
iii
Emma Barinas-Mitchell, PhD
STRUCTURAL AND FUNCTIONAL ARTERIAL CHANGES IN HEALTHY WOMEN
FOLLOWING PREGNANCY: MATERNAL VASCULAR ADAPTATIONS TO
PREGNANCY II STUDY
Mansi M. Desai, MPH
University of Pittsburgh, 2015
ABSTRACT
Background: Parity is associated with increased maternal risk of cardiovascular disease (CVD)
in a J-shaped fashion. Vascular adaptation to changes during pregnancy may be an indicator of
future CVD risk. The literature indicates that pregnancy results in an increase in common carotid
artery inter-adventitial diameter (CCA IAD) and intima-media thickness (CCA IMT), markers of
vascular aging and future CVD risk. These changes were found to recede early postpartum,
although not consistently and not back to pre-pregnancy levels. This study aims to determine
whether vascular changes during pregnancy persist beyond the initial postpartum period.
Methods: This study is a follow-up to a prospective study that assessed vascular health in a
cohort of 43 healthy women during each trimester of their first pregnancy and 6-8 weeks
postpartum. Data on physical measures, biomarker assays, pregnancy outcomes, and health
history were collected 1-5 years following the index birth. B-mode ultrasound was used to
measure CCA IAD and CCA IMT. Paired t-tests and multivariable linear regression were used to
assess changes in vascular measures.
iv
Results: Sixteen participants with mean age 32.4 years completed the second postpartum visit.
The average time from index delivery was 2.7 years. Unadjusted mean CCA IAD slightly
decreased and CCA IMT increased at the second postpartum visit compared to the first
postpartum and first trimester visits; these changes were not statistically significant. Long-term
postpartum, larger CCA IAD was associated with higher blood pressure whereas thicker CCA
IMT was associated with less time since index birth, greater weight change, less time
breastfeeding, and lower insulin resistance (p<0.05).
Conclusions: Markers of vascular aging did not improve further out postpartum compared to the
early postpartum period. Persistence of vascular effects of pregnancy may suggest a possible
mechanism linking increased CVD risk with parity.
Public Health Significance: CVD is the leading cause of death in women. Increased
understanding of vascular adaptation during pregnancy may help explain differential future CVD
risk with parity and inform early detection of women at increased CVD risk long-term.
Prolonged breastfeeding and weight management during pregnancy may be potential lifestyle
modifications for women to decrease their future CVD risk.
v
TABLE OF CONTENTS
PREFACE .................................................................................................................................... IX
1.0
INTRODUCTION................................................................................................................ 1
2.0
MATERIALS AND METHODS ........................................................................................ 3
3.0
4.0
2.1
STUDY DESIGN AND POPULATION ................................................................ 3
2.2
ULTRASOUND ASSESSMENT OF THE COMMON CAROTID ARTERY .. 4
2.3
PREGNANCY HISTORY AND CVD RISK FACTORS .................................... 5
2.4
POWER CALCULATION ..................................................................................... 7
2.5
STATISTICAL ANALYSIS ................................................................................... 7
RESULTS ............................................................................................................................. 9
3.1
SUBJECT CHARACTERISTICS.......................................................................... 9
3.2
KEY VASCULAR MEASURES ............................................................................ 9
3.3
ADDITIONAL ANALYSES ................................................................................. 11
DISCUSSION ..................................................................................................................... 18
APPENDIX: SUPPLEMENTARY TABLES AND FIGURES ............................................. 24
BIBLIOGRAPHY ....................................................................................................................... 28
vi
LIST OF TABLES
Table 1. Characteristics of the study population at the second postpartum visit .......................... 12
Table 2. Unadjusted values for vascular measures and key time-varying covariates by visit ...... 13
Table 3. Vascular measures at second postpartum visit by categories of pregnancy-related
covariates ...................................................................................................................................... 14
Table 4. Adventitial diameter models adjusted for individual predictors at second postpartum
visit ............................................................................................................................................... 15
Table 5. Intima-media thickness models adjusted for individual predictors at second postpartum
visit ............................................................................................................................................... 16
Supplementary Table 1. Baseline demographic characteristics of MVP participants who attended
compared to those who did not attend second postpartum visit ................................................... 24
Supplementary Table 2. Correlations between major outcome variables and other covariates at
second postpartum visit................................................................................................................. 25
vii
LIST OF FIGURES
Figure 1. Flowchart of the study population ................................................................................... 3
Figure 2. Ultrasound Image of CCA ............................................................................................... 4
Figure 3. Changes in CCA inter-adventitial diameter by visit...................................................... 17
Figure 4. Changes in CCA intima-media thickness by visit ......................................................... 17
Supplementary Figure 1. Changes in CCA inter-adventitial diameter for MVP II participants
throughout and after pregnancy .................................................................................................... 26
Supplementary Figure 2. Changes in CCA inter-adventitial diameter for all MVP participants
throughout and after pregnancy .................................................................................................... 26
Supplementary Figure 3. Changes in CCA intima-media thickness for MVP II participants
throughout and after pregnancy .................................................................................................... 27
Supplementary Figure 4. Changes in CCA intima-media thickness for all MVP participants
throughout and after pregnancy .................................................................................................... 27
viii
PREFACE
Sixteen months ago, while discussing with my advisor the several options I could pursue
for my master’s internship, the Maternal Vascular Adaptations to Pregnancy II study was born.
What seemed like an over-ambitious goal and a long journey could not be accomplished without
the help of many people. I am very thankful to my advisor and mentor Emma Barinas-Mitchell
who stood by me every step of the way. You have set an example of excellence as a researcher,
mentor, and role model. I greatly appreciate all the help from Nancy Niemczyk; without your
previous work, this study would not have existed. I am thankful to my mentor Marnie Bertolet
for her statistical expertise and support at all times. I would like to thank my essay readers and
co-investigators; your constant feedback and encouragement has been absolutely invaluable.
I would like to thank all the staff of the Ultrasound Research Laboratory, Health Studies
Research Clinic, and Heinz Nutrition Laboratory for their help, support, and for being so flexible
with the needs of this study. I am very grateful to all the study participants, who despite the
unpredictable weather and young babies, made it to the study visits.
I would especially like to thank my amazing family for the love, support, and constant
encouragement. In particular, I would like to thank my husband, parents, in-laws and sister. You
are the sunshine of my life, and I could not have done this without you.
ix
Finally, I would like to dedicate this work to my husband, Mahir, for his remarkable
patience, unwavering support, and motivation throughout my graduate program. Thank you for
always being there for me.
x
1.0 INTRODUCTION
Maternal cardiovascular disease risk rises with increased parity in a J-shaped fashion with
2 births representing the nadir of risk
16,24,25
, however the mechanism for this increased risk is
unknown. Some of the increased risk may be attributed to behavioral and socioeconomic risk
factors related to increased parity.16 In addition, pregnancy may unmask an underlying
cardiovascular disease (CVD) susceptibility in some women due to increased stress on the
cardiovascular system, with each pregnancy potentially adding to increased cumulative risk.16
The cardiovascular demands of a normal pregnancy are substantial and vascular remodeling is
required to handle the increased circulating fluid volume.
During a healthy pregnancy, uterine vasculature remodels in response to a stimuli of
hemodynamic, hormonal, and metabolic changes.27 Hemodynamic changes starting early in
pregnancy include an increase in heart rate, cardiac output and stroke volume; sodium and water
retention, which leads to increased blood volume; and decreased blood pressure and systemic
vascular resistance.17,18 Hormonal changes during pregnancy include an increase in estrogen,
progesterone, testosterone and maternal cortisol concentrations.19,20 Metabolic changes during
pregnancy include increased insulin21, triglyceride22, lipid22, and C-reactive protein23
concentrations. Although not well-understood, results from small studies suggest that during
pregnancy, systemic arteries adapt to these changes in a similar fashion to the uterine
vasculature12,14,17. How the systemic vasculature adapts to these changes during pregnancy may
be an indicator of future CVD risk. Furthermore, many pregnancy complications have a vascular
component.38-41 Better understanding of the effects of pregnancy on the systemic arteries may
lead to early detection of women for pregnancy complications and CVD risk, and help explain
the differences in cardiovascular risk found in women of different parity.12
1
A small number of studies13-15,26 have examined changes in systemic arteries during the
course of normal pregnancy, and found that pregnancy results in increased common carotid
artery (CCA) intima-media thickness (IMT) and inter-adventitial diameter (IAD), markers of
vascular aging30,33 and future CVD risk26,36,37. These changes were found to recede postpartum,
although not consistently and not back to pre-pregnancy levels.13-15,26 Many of these studies have
been limited by lack of serial vasculature26 and biomarker13-15,26 measures throughout normal
pregnancy and later postpartum, and use of less well-established techniques to assess changes in
the vasculature14.The Maternal Vascular Adaptations to Healthy Pregnancy (MVP) study
prospectively assessed vascular health in a cohort of 43 healthy women during each trimester of
their first pregnancy and 6-8 weeks postpartum.12 Consistent with existing literature, the MVP
study found that in uncomplicated first pregnancies, some vascular changes resolved (increased
CCA IAD) and others persisted (increased CCA IMT) 6-8 weeks postpartum.12 This persistence
may be because 6-8 weeks postpartum does not represent adequate time to return to baseline. It
remains to be explored if this persistence of vascular effects of pregnancy indicates long-term
cardiovascular disease risk.12 If the increased CCA IMT identified in the MVP study persists
long-term, it could be a mechanism by which parity contributes to greater CVD risk.
Thus, the aim of this study was to bring back MVP participants for a second postpartum
visit to determine whether the vascular change that occurs during pregnancy, increased CCA
IMT, persists beyond the initial postpartum period. We hypothesized that: 1) CCA IAD would
decrease long-term postpartum as compared to 6-8 weeks postpartum and 2) CCA IMT would
decrease long-term postpartum as compared to 6-8 weeks postpartum. Our secondary hypotheses
were that: 1) CCA IAD would decrease long-term postpartum as compared to first trimester
2
(baseline) and 2) CCA IMT would decrease long-term postpartum as compared to first trimester
(baseline).
2.0 MATERIALS AND METHODS
2.1 Study Design and Population
The Maternal Vascular Adaptations to Pregnancy II (MVP II) study is a follow-up study
to the MVP study. MVP study visits were scheduled at 12-14 weeks, 24-26 weeks and 36-38
weeks of pregnancy, and then at 6-8 week postpartum. The MVP II study visit took place
between 1 and 5 years after the index delivery. This is the second postpartum visit for the MVP
study participants.
44 women enrolled in MVP Study
1 dropout
43 eligible MVP women
4 lost to follow-up
14 moved out of Pittsburgh
4 pregnant
2 had pregnancy outcomes
within last 4 months
19 eligible for MVP II study visit
3 withdrawals
MVP II study (n=16)
Figure 1. Flowchart of the study population
3
Inclusion criteria for this study included: 1) prior participation in the MVP study; 2) at
least 4 months since last pregnancy outcome; and 3) at least 1 to 5 years since index delivery.
The participants were excluded if they: 1) were currently pregnant; 2) moved out of Pittsburgh
area; or 3) were lost to follow-up. Thus, 19 women remained eligible for the MVP II study of
which 3 withdrew from the study, leaving 16 women in the final evaluation (Figure 1). MVP II
participants thus included a total of 16 healthy women, age 23-37 years, currently non-smoking,
and with up to 2 additional pregnancies since their index delivery. All participants signed an
informed consent document approved by the University of Pittsburgh Institutional Review
Board. The visit included questionnaires to access pregnancy-related factors, physical
measurements, and B-mode carotid ultrasound scans, performed by the research staff. Study
visits were conducted between September 2014 and March 2015.
2.2 Ultrasound Assessment of the Common Carotid Artery
IAD
IMT
Figure 2. Ultrasound Image of CCA
4
Bilateral images of the CCA were obtained by a certified vascular sonographer via B-mode
ultrasound using an Acuson Cypress portable ultrasound machine (Siemens Medical Solutions,
Malvern, PA) equipped with a 7L3 linear transducer. Images were taken from the near and far
walls of the distal CCA (1 cm proximal to the carotid bulb) and all images were read centrally at
the Ultrasound Research Laboratory (University of Pittsburgh, Pittsburgh, PA). CCA IMT
measures were electronically traced between the lumen-intima interface and the media-adventitia
interface across the 1 cm segment using a semi-automated reading software (AMS system
developed in Sweden by Dr. Thomas Gustavsson).1 The mean of the near and far wall IMT
measurements from the left and right CCA comprises the average CCA IMT. The CCA IAD was
measured from the same 1 cm CCA segment as the average distance between the adventitialmedial interface on the near wall and the medial-adventitial interface on the far wall (Figure 2).
Reproducibility of CCA IMT measures was good to excellent with an intra-class correlation
coefficient within sonographer of 0.85 and within reader of > 0.96. These scanning and reading
protocols have been used in numerous studies.2-4
2.3 Pregnancy History and CVD Risk Factors
Study Forms. At the second postpartum study visit, participants completed a self-administered
interval health history form with information on smoking, diabetes, health conditions,
medications, breastfeeding, number of pregnancies, and contraceptive use. Participants reported
whether they were currently breastfeeding and the time (in months) they breastfed each of their
babies. Cumulative breastfeeding was calculated as a sum of total time (in months) of
breastfeeding by each participant. Participants then completed a self-administered pregnancy
outcomes form for each subsequent pregnancy since the last MVP visit including information
5
such as outcome of pregnancy, date of outcome, gestational age, birth weight and length,
complications, and duration of breastfeeding.
Physical Measures. Research staff palpated the right radial pulse for 30 seconds and then
measured blood pressure in that arm using a mercury sphygmomanometer after the participants
rested for 5 minutes in a quiet room.
Three measurements were taken according to a
standardized protocol, and the average of the last two measures was used for the analysis.
Research staff then weighed the participants on a standard balance scale twice, and the average
of the readings was used for the analysis. Lastly, the research staff took two measurements each
of the waist and the hip circumference with a tape measure according to a standardized protocol,
and the average of each was used to calculate the waist-to-hip ratio.
Data Management Tool. Data on physical measures and from study forms were managed using
REDCap (Research Electronic Data Capture) hosted at the University of Pittsburgh.11 REDCap
is a secure, web-based application designed to support data capture for research studies.11
Biochemical Assays. A 30 ml venous blood sample was collected from each participant at the
study visit. After collection, the samples were kept at room temperature for ~0.5 h before being
centrifuged at 4ºC for 15 minutes at 1500 g. These fasting serum samples were delivered on dry
ice to the Heinz Nutrition Laboratory (University of Pittsburgh Graduate School of Public
Health, Pittsburgh, PA) where assays were performed. Standard laboratory procedures were
used to determine the blood glucose,5 total cholesterol,6 high density lipoprotein (HDL-c),7 low
density lipoprotein (LDL-c),8 and triglyceride9 levels. Insulin was measured using standard radioimmune assay (Linco Research, St. Charles, MO). HOMA-IR, a measure of insulin resistance,
was calculated as (glucose x insulin)/405 for the purposes of analysis.10 High-sensitivity C-
6
reactive protein (hsCRP) was measured with an enzyme-linked immunoassay (Alpha Diagnostics
International, Inc. San Antonio, TX).
2.4 Power calculation
A statistical power analyses was performed to estimate the smallest detectable difference
in vascular outcomes between the first and second postpartum visits, based on results from the
MVP study (n=43). The mean standard deviation of differences in CCA IAD ranged from 0.16 to
0.67 mm and the mean standard deviation of differences in CCA IMT ranged from 0.02 to 0.09
mm between the various time-points of the MVP study12. Using a two-sided two-sample paired ttest for 16 participants with an alpha of 0.05 and power of 80%, the smallest detectable
difference in CCA IAD was calculated to be in the range of 0.112 to 0.469 mm and the smallest
detectable difference in CCA IMT was calculated to be in the range of 0.014 to 0.063 mm. Since
the number of participants in our study is very small, the detectable effect sizes for statistical
significance for each of the vascular outcomes are quite large. Thus, this study can be seen as a
pilot study to determine effect sizes for sample calculations for future studies.
2.5 Statistical Analysis
For descriptive purposes, data are presented as mean (standard deviation) for normally
distributed continuous variables, median [Q1, Q3] for continuous variables not normally
distributed, and as n (%) for categorical variables. For parametric analyses, variables were
transformed as needed to meet model assumptions. Paired t-tests of mean differences were used
separately for CCA IAD and CCA IMT to 1) estimate mean differences for CCA IAD and CCA
IMT between first (6-8 weeks) postpartum visit and second (1-5 years) postpartum visit; and 2)
estimate mean differences for CCA IAD and CCA IMT between first trimester visit and second
postpartum visit. Ordinary least squares regression was used to assess the carotid artery outcome
7
measures at the second postpartum visit by categories of important CVD risk factors and
pregnancy-related covariates. Correlations between the outcome measures and normally
distributed covariates at the second postpartum visit were assessed using Pearson correlation test.
Based on the literature, MVP study trends, and correlations and significance in our analyses, we
picked the variables with the strongest significant associations with each vascular measure to be
included in base models for the vascular measures. The base model for CCA IAD included
current age and systolic blood pressure whereas the base model for CCA IMT included weight
change from pre-pregnancy to second postpartum visit. The relationship between vascular
measures CCA IAD and CCA IMT, time since index delivery (months), and other key predictors
was evaluated by multivariable linear regression analysis. A stepwise regression approach was
used to find the ‘best’ set of predictors for each vascular measure (p-value for removal = 0.2).
Key predictors, including weight change, waist-to-hip ratio, triglycerides, hsCRP, systolic blood
pressure, fasting glucose, insulin, HDL-c, LDL-c, total cholesterol, time since last visit, smoking,
breastfeeding, use of hormonal birth control, and parity, were included simultaneously to the
base model for each vascular measure. Lower akaike information criterion, lower mean-squared
error of prediction, and higher overall model significance were used as criteria to compare the
performance of these multivariable models. The full models for each vascular measure were then
adjusted for the other measure since both CCA IAD and CCA IMT are highly correlated
measures that affect each other. Sensitivity analyses were performed 1) excluding women with
pregnancy complications; and 2) including only women with all visits. All statistical analyses
were performed using SAS statistical software release 9.3 (SAS Institute, Cary, NC) and STATA
statistical software release 13 (StateCorp LP., College Station, TX). Two-tailed p-values of
<0.05 were considered statistically significant.
8
3.0 RESULTS
3.1 Subject Characteristics
Of the 16 participants who completed the second postpartum visit, 8 completed all study
visits and 3 had complications during their MVP pregnancy. Participants who returned for the
second postpartum visit were predominantly white, married or living as married, well-educated,
and employed. No significant differences were seen between participants who returned for the
second postpartum visit (n=16) and those who did not (n=27) (Supplementary Table 1).
Important characteristics of the study population (n=16) at the second postpartum visit are shown
in Table 1. These participants had an average age of 32.4 ± 4.4 years and an average body-mass
index of 25.6 ± 3.1 kg/m2. Since the first postpartum visit 50% of the participants had additional
pregnancies since their first birth (6 participants had 1 and 2 participants had 2 additional
pregnancies), 44% were currently breastfeeding, 31% were currently using hormonal birth
control, and 56% were overweight or obese at the time of the visit. The mean time since the
index delivery was 32.6 ± 9.5 months.
3.2 Key vascular measures
Table 2 shows the mean (SD) for the vascular measures and key time-varying covariates
at the first trimester, first (6-8 weeks) postpartum, and second postpartum visits. Heart rate and
triglyceride and the HDL-c concentrations significantly changed between the first trimester and
the second postpartum visit. No covariates changed significantly between the first postpartum
and the second postpartum visit. Mean CCA IAD slightly decreased from 6.45 ± 0.28 mm at first
trimester and 6.42 ± 0.32 mm at 6-8 weeks postpartum to 6.38 ± 0.42 mm at the second
postpartum visit (Table 2). No statistically significant differences were seen in the CCA IAD
values between the second postpartum and the first postpartum or first trimester visit. These
9
values changed but remained non-significant after adjustment for current age and systolic blood
pressure (Figure 3). Mean CCA IMT increased from 0.55 ± 0.04 mm at first trimester and 0.56 ±
0.05 mm at 6-8 weeks postpartum to 0.58 ± 0.05 mm at the second postpartum visit (Table 2),
although there were no statistically significant differences in CCA IMT values between the
second postpartum and the first postpartum or first trimester visit. These values changed but
remained non-significant after adjustment for weight change (Figure 4).
The beta-coefficients (p-values) for pregnancy-related categorical covariates were
calculated using ordinary least squares linear regression (Table 3). Unadjusted CCA IAD was
higher with lower parity. Adjusting for current age and systolic blood pressure, CCA IAD was
higher with no complications during index pregnancy and less time breastfeeding, although
statistically significant differences were not observed for any of these categories. Unadjusted
CCA IMT did not statistically differ by categories of pregnancy-related covariates. Although not
statistically significant, after adjusting for weight change, CCA IMT was thinner with current use
of hormonal birth control.
We explored whether individual CVD risk factors or pregnancy-related factors may
explain CCA IAD and CCA IMT at the second postpartum visit. Pearson correlations between
the major outcome measures and other covariates at the second postpartum visit are reported in
Supplementary Table 2 (Appendix). Systolic and diastolic blood pressures were significantly
positively associated with CCA IAD at the second postpartum visit. Similar associations were
seen when adjusted for current age. CCA IMT was significantly positively associated with CCA
IAD when adjusted for current age and systolic blood pressure (Supplementary Table 2). Higher
systolic blood pressure was significantly associated with CCA IAD in all multivariable models,
whereas current age, time since index birth, and weight change were not (Table 4). Neither
10
cumulative breastfeeding nor parity were associated with CCA IAD. When CCA IMT was
included in the full model for CCA IAD, higher CCA IAD remained significantly associated
with higher systolic blood pressure and the results did not change significantly (data not shown).
Weight change was significantly positively associated with CCA IMT at the second
postpartum visit. Significant negative associations with CCA IMT were observed for time since
index birth and time since first postpartum visit, when adjusted for weight change
(Supplementary Table 2). CCA IAD was borderline significantly (p = 0.08) positively associated
with CCA IMT (Supplementary Table 2). In the multivariable models, both weight change and
time since first birth significantly predicted CCA IMT at the second postpartum visit (Table 5).
When current breastfeeding and log HOMA were added to the model, they were significantly
associated with CCA IMT adjusting for log hsCRP. When CCA IAD was included to the full
model for CCA IMT, higher CCA IMT remained significantly associated with only weight gain
and lesser time since first birth (data not shown). Results from sensitivity analyses including only
those women without pregnancy complications and only those women who completed all the
study visits were consistent with the main results (data not shown).
3.3 Additional Analyses
Changes in CCA IAD throughout pregnancy and postpartum for MVP II (n=16)
participants and all MVP (n=43) participants are shown in Supplementary Figures 1 and 2
(Appendix). Second and third trimester CCA IAD values are significantly different when
compared to the second postpartum visit. Changes in CCA IMT throughout pregnancy and
postpartum for MVP II (n=16) participants and all MVP (n=43) participants are shown in
Supplementary Figures 3 and 4 (Appendix). No significant differences are seen in CCA IMT
values for all visits compared to the second postpartum visit.
11
Table 1. Characteristics of the study population at second postpartum visit (n=16)
Characteristics
Descriptive
Current age, years
32.44 ± 4.43
Total parity
1
≥2
Time since index birth, months
50.0% (8)
50.0% (8)
32.55 ± 9.47
Time since last pregnancy, months
19.05 ± 13.46
Current body-mass index, kg/m2
25.61 ± 3.12
Complications during index pregnancy
18.7% (3)
Body-mass index, kg/m2
18.5 – 24.9 (normal)
25.0 – 29.9 (overweight)
≥ 30.0 (obese)
Waist-to-hip ratio
43.8% (7)
50.0% (8)
6.2% (1)
0.81 ± 0.09
Smoking habits
Never smoker
Ever smoker
Currently breastfeeding
62.5% (10)
37.5% (6)
43.7% (7)
Cumulative breastfeeding, months
19.78 ± 11.24
Cumulative breastfeeding
0-6 months
≥ 6 months
Currently using hormonal birth control
18.7% (3)
81.3% (13)
31.2% (5)
Continuous variables presented as mean ± SD and categorical variables presented as % (n)
12
Table 2. Unadjusted values for vascular measures and key time-varying covariates by visit
Trimester 1 visit
(n=8)
Postpartum I
(n = 16)
Postpartum II
(n = 16)
6.38 (0.42)
p-value
(Trimester 1
vs.
Postpartum
II)
0.38
p-value
(Postpartum
I vs.
Postpartum
II)
0.45
CCA inter-adventitial diameter (mm)
6.45 (0.28)
6.42 (0.32)
CCA intima-media thickness (mm)
0.551 (0.04)
0.561 (0.05)
0.579 (0.05)
0.47
0.17
Weight (kg)
68.90 (8.86)
70.85 (7.75)
69.79 (7.43)
0.69
0.22
Weight change (kg)
-0.10 (1.83)
3.36 [2.23,5.45]
2.97 (4.58)
0.69
0.22
Systolic blood pressure (mm Hg)
104.69 (6.09)
107.06 (9.66)
103.38 (9.04)
0.69
0.14
Diastolic blood pressure (mm Hg)
64.94 (5.28)
69.50 (6.04)
67.38 (7.49)
0.30
0.10
Heart rate (bpm)
79.38 (11.92)
69.00 (9.22)
64.69 (8.35)
0.08
Cardiac output (L/min)
5.39 (1.04)
4.47 (0.55)
4.66 (0.59)
0.01*
0.07
Total cholesterol (mg/L)
195.43 (33.57)
199.75 (30.99)
192.00 (28.62)
0.38
0.22
LDL-c (mg/dl)
105.60 (23.84)
121.05 (28.07)
117.81 (23.34)
0.15
0.59
Triglycerides (mg/dl)
122.14 (41.68)
84.0 [64.0,121.0]
85.19 (37.13)
0.01*
0.26
HDL-c (mg/dl)
65.34 (8.53)
59.33 (9.75)
57.26 (11.51)
0.45
Glucose (mg/dl)
78.0 [75.0,86.0]
84.56 (6.98)
87.56 (7.38)
0.02*
0.23
Insulin (µU/ml)
9.94 (3.82)
9.77 (3.09)
10.59 (3.13)
0.23#
0.40
HOMA-IR
2.00 (0.95)
2.06 (0.75)
2.31 (0.79)
0.61
0.32
hsCRP (mg/L)
3.9 [2.16,6.82]
1.42 [0.93,3.71]
1.03 [0.58,1.44]
0.13#
0.21#
0.24
0.13
Continuous normal variables presented as mean (SD); non-normal variables presented as median [Q1, Q3]
2-sided p-values for paired t-test (of mean differences) are reported for normal differences; *p<0.05 is considered significant
#
2-sided p-values for sign rank test (of medians) are reported for non-normal differences and non-symmetric populations; p<0.05 is considered significant
13
Table 3. Vascular measures at second postpartum visit by categories of pregnancy-related covariates (n=16)
Characteristics
Complications during index pregnancy
No
Yes
Total parity
1
2+
Body-mass index, kg/m2
18.5 – 24.9 (normal)
25.0 – 30.0+(overweight or obese)
Smoking habits
Never smoker
Ever smoker
Currently breastfeeding
No
Yes
Cumulative breastfeeding
0-6 months
≥ 6 months
Current use of hormonal birth control
No
Yes
Inter-adventitial diameter
Intima-media thickness
Unadjusted
Adjusted for
current age and
SBP
Unadjusted
Adjusted for
weight change
Reference
-0.37 (0.18)
Reference
-0.34 (0.06)
Reference
-0.05 (0.14)
Reference
-0.03 (0.36)
Reference
-0.35 (0.09)
Reference
-0.12 (0.46)
Reference
0.01 (0.73)
Reference
0.01 (0.65)
Reference
-0.12 (0.60)
Reference
0.02 (0.88)
Reference
0.02 (0.45)
Reference
-0.001 (0.96)
Reference
-0.08 (0.72)
Reference
-0.11 (0.50)
Reference
0.01 (0.64)
Reference
-0.01 (0.60)
Reference
-0.33 (0.11)
Reference
-0.19 (0.23)
Reference
-0.04 (0.16)
Reference
-0.01 (0.74)
Reference
-0.34 (0.22)
Reference
-0.33 (0.09)
Reference
-0.03 (0.46)
Reference
-0.01 (0.73)
Reference
-0.15 (0.54)
Reference
-0.09 (0.60)
Reference
-0.04 (0.23)
Reference
-0.04 (0.10)
Categorical variable descriptives presented as % (n)
Beta coefficient (p-value) reported for each model using ordinary least squares linear regression; No statistical significance observed (p>0.05)
14
Table 4. Adventitial diameter models adjusted for individual predictors at second postpartum visit (n=16)
Model 1
(base model)
3.87 (<0.01)
4.01 (<0.01)
3.75 (<0.01)
3.80 (0.01)
Model 5
(full model)
3.45 (<0.01)
-0.04 (0.06)
-0.03 (0.09)
-0.03 (0.13)
-0.03 (0.09)
-0.03 (0.13)
0.04 (<0.01)
0.04 (<0.01)
0.04 (<0.01)
0.04 (<0.01)
0.04 (<0.01)
Time since first birth (mo)
-
-0.01 (0.30)
-0.01 (0.27)
-0.01 (0.14)
-0.02 (0.10)
Weight change (kg)
-
-
0.02 (0.30)
0.02 (0.34)
0.02 (0.29)
Cumulative breastfeeding (mo)
-
-
-
-0.01 (0.37)
-
Log hsCRP (mg/L)
-
-
-
0.16 (0.13)
0.19 (0.09)
Parity (1 vs. ≥2)
-
-
-
-
-0.22 (0.25)
Overall model p-value
0.003*
0.007*
0.012*
0.024*
0.019*
Adjusted correlation coefficient
(R2)
0.5241
0.5301
0.5373
0.5720
0.6252
Intercept
Current age (years)
Systolic blood pressure (mm Hg)
Model 2
Model 3
Values presented as beta-coefficients (p-value); * overall p-values < 0.05 are considered significant.
15
Model 4
Table 5. Intima-media thickness models adjusted for individual predictors at second postpartum visit (n=16)
Model 1
(base model)
Model 2
Model 3
Model 4
(full model)
Intercept
0.56 (<0.01)
0.64 (<0.01)
0.50 (<0.01)
0.76 (<0.01)
Weight change (kg)
0.01 (0.04)
0.01 (0.02)
0.004 (0.06)
0.01 (0.02)
Time since first birth (mo)
-
-0.002 (0.04)
-0.002 (0.06)
-0.003 (0.03)
Current weight (kg)
-
-
0.002 (0.22)
-
Cumulative breastfeeding (mo)
-
-
-
-0.002 (0.05)
Log HOMA
-
-
-
-0.07 (0.04)
Log hsCRP (mg/L)
-
-
-
0.02 (0.08)
Overall model p-value
0.043*
0.017*
0.024*
0.019*
Adjusted correlation coefficient (R2)
0.2081
0.3845
0.4147
0.5434
Values presented as beta-coefficients (p-value); * overall p-values < 0.05 are considered significant.
16
6
6.2
6.4
6.6
6.8
7
7.2
Mean adjusted CCA IAD by visit
Trimester 1
Postpartum I
Postpartum II
Figure 3. Changes in CCA inter-adventitial diameter by visit (n=16)
*All comparisons non-significant at p>0.05, Adjusted for current age and systolic blood pressure
.54
.56
.58
.6
Mean adjusted CCA IMT by visit
Trimester 1
Postpartum I
Postpartum II
Figure 4. Changes in CCA intima-media thickness by visit (n=16)
*All comparisons non-significant at p>0.05, Adjusted for weight change.
17
4.0 DISCUSSION
This study evaluated the course of vascular remodeling that occurs long-term postpartum
after a healthy pregnancy. We found that the vascular changes in the CCA seen during the MVP
study, namely an increase in CCA IMT and the return to baseline of the CCA IAD, both
persisted long-term. CCA IAD was similar in the initial and long-term postpartum visits.
Although not statistically significant, CCA IMT appeared to thicken during the postpartum
period. Contrary to our hypothesis, markers of vascular aging did not improve further out
postpartum compared to the early postpartum period. Long-term postpartum, larger CCA IAD
was associated with higher blood pressure, and thicker CCA IMT was associated with less time
since index birth, greater weight gain, less time breastfeeding, and lower insulin resistance.
Pregnancy is a time of dramatic hemodynamic changes that affect the vasculature during
pregnancy and may also have long-term effects.18,29,31 Acute changes including increased cardiac
output lead to arterial dilation.12,29 This increase in diameter may lead to intimal medial
thickening as a way of normalizing shear and tensile strength on the blood vessel.17,18,29 This is
consistent with MVP study findings of increased CCA IMT during pregnancy and early
postpartum.12 However, in our study CCA IMT did not return to baseline long-term postpartum
as hypothesized, which may be partially explained by subsequent pregnancies. In our sample,
50% of the women had additional pregnancies following the index birth. More acute vascular
remodeling from subsequent pregnancies following the index birth, some of them being as close
as 5 months to the second postpartum visit date, may mask the vessel walls’ return to baseline
and may explain the observed increased wall thickness at the second postpartum visit.
18
As in non-pregnant adults33, we found that inter-adventitial diameter was significantly
associated with higher systolic blood pressure, a risk factor for CVD. This might represent an
adaptive response to the greater tensile strength on the common carotid artery. We found that
unadjusted CCA IAD was higher (borderline statistically significant) for women with 1 child
compared to women with more than 2 children. This may be due to the fact that women with
more than 2 children were currently breastfeeding which may have a protective effect on the
vascular measure of CCA IAD as previously reported in the literature34, although in our study
current breastfeeding status was not statistically associated with CCA IAD. Since CCA IAD is a
result of acute changes during pregnancy, we ran the multivariable models for CCA IAD
including time since last pregnancy instead of time since MVP birth (data not shown). CCA IAD
decreased with more time since last birth as expected suggesting that the vasculature of these
women is still remodeling long-term postpartum.
Although CCA IMT did not decrease as hypothesized, our multivariable models suggest
that CCA IMT was thicker with less time since index birth adjusting for weight change, duration
of breastfeeding, insulin resistance, and C-reactive protein. Since CCA IMT may change more
gradually after pregnancy compared to CCA IAD and time since last birth was not associated
with CCA IMT, we chose not to run these models with time since last birth. The association
between CCA IMT and less time since index birth is consistent with slower changes in IMT and
potential cumulative effects of subsequent pregnancies. It would be expected that CCA IMT
would be thicker closer to the pregnancy in order to normalize stresses on the arterial wall and as
an adaptation to changes in arterial diameter. We found that certain pregnancy-related factors,
like weight gain, persist long-term postpartum and may account for the thicker CCA IMT and
19
may potentially accumulate with each pregnancy. The increased weight may have served as a
surrogate for the increased blood volume12 due to subsequent pregnancies.
We also found that lower breastfeeding is associated with thicker CCA IMT. This is
consistent with results by Schwarz et al.34 who found that mothers who do not breastfeed have
vascular characteristics associated with a greater CVD risk. To the best of our knowledge, our
study is the first to establish an association between duration of recent breastfeeding by women
and their current CCA IMT values. Our results add to the existing literature that lactation may
help resolve the vascular adaptations and fat accumulation associated with pregnancy.16
However, our findings are limited by the measure of lactation used in the study since we did not
include a measure of exclusive breastfeeding. Our study also suggests that metabolic changes did
not explain the postpartum thickening of CCA IMT. Contrary to previous literature35, we found
that lower insulin resistance was significantly associated with thicker CCA IMT adjusting for
hsCRP. This finding is counterintuitive and other studies need to explore this further.
Our findings complement prior studies that have demonstrated that the vascular
remodeling of pregnancy persists in healthy women.13,15,26,28 In contrast, Akhter et al.14 found
that the CCA intima to media (I/M) ratio decreased (healthier arteries) by one year postpartum. It
is difficult to compare our results to the study by Akhter et al. since they used I/M ratio, a
vascular measure which is not widely accepted in the field of CVD and vascular imaging.14
Mersich et al.13 followed the participants only to 12 weeks postpartum, Visontai et al.15 followed
their participants up to 6 months postpartum, and Clapp et al.28 followed their participants to 1
year postpartum. Our study is the first study to follow the participants throughout their course of
pregnancy and up to 4 years after pregnancy (with a median follow-up of 2.5 years after first
birth).
20
Our finding that CCA IMT increased long-term postpartum could potentially help explain
the increase in CVD risk that occurs in women of higher parity.16,24,25 Greater CCA IMT is an
established risk factor for CVD because thickened arteries are less capable of effectively
responding to changes in blood pressure29 and are more prone to development of
atherosclerosis.28,30 While several studies have identified increased CVD risk with parity16,24,25, it
is unknown if the increase in CVD risk is caused by persistence of acute vascular changes of
pregnancy or by accumulation of CVD risk factors that occurs with subsequent pregnancies.12
Our study might suggest that an acute pregnancy change and its persistence well after pregnancy,
thickened CCA IMT, may contribute to higher future maternal CVD risk. Triglyceride levels
significantly changed between first trimester and second postpartum visit which reflects the wellestablished changes that occur very early in pregnancy from the non-pregnancy values.42 The
significant change in HDL-c levels between first trimester and second postpartum visit need to
be explored further. However, lipid concentrations did not appear to be related to long-term
postpartum vascular measures.
The findings of this study need to be considered in the context of its strengths and
limitations. The strengths of our study include the use of a highly valid and reproducible measure
of carotid structure and the detailed data collection of various pregnancy-related covariates like
time of total breastfeeding, use of hormonal birth control, and data on each subsequent
pregnancy and health history since the first pregnancy. This follow-up study adds a one to five
year follow-up to the MVP study, allowing for a more complete understanding of vascular
adaptations during normal pregnancy and further out postpartum than previously published
studies. To the best of our knowledge, this is the first study that has prospectively assessed
vascular adaptations in systemic arteries during, and for a long period of time after pregnancy.
21
Persistence of vascular effects of pregnancy may indicate long-term CVD risk and might suggest
a possible mechanism behind increased CVD risk with increased parity.
However, several limitations should also be acknowledged. A key limitation is the loss to
follow-up of participants and the resulting small sample size (n=16) for the follow-up visit. The
small sample size may be a major reason for statistically insignificant changes in CCA IMT
during the second postpartum visit compared to the first postpartum and first trimester visits.
This might also be the reason for not observing statistically significant differences in the vascular
outcomes by categories of covariates (Table 3) and for the lack of statistically significant
differences between visits for CVD risk factors and other important pregnancy-related covariates
(Table 2). The small sample size may have limited extensive exploration of the effects of
potentially confounding variables such as subsequent pregnancies and pregnancy complications.
Although the largely white, well-educated women in this cohort may not be representative of the
general population reducing the generalizability of the study results, this study has utility as
providing a basis against which systemic arterial remodeling in other population-based cohorts
can be assessed. Some of the pregnancy and health-related data is collected by self-report from
the participants, and may introduce some bias in the study. The first trimester visit (12-14 weeks
of pregnancy) is assumed to be a baseline for our secondary hypotheses. Since significant
hemodynamic changes of pregnancy begin as early as week 5 of gestation17, the first trimester
values might not represent a true pre-pregnancy baseline. Because of relatively small size of the
cohort, this study should be considered as a pilot study, and suggests a need for larger studies
with a population-based sample of participants. We think that the effect sizes observed in this
study might be clinically relevant, and this needs to be explored using larger studies.
22
To understand the trajectory of CVD risk after pregnancy, more studies are needed to
measure CVD risk factors among participants before, during, and beyond pregnancy. Larger
cohort studies are needed to establish whether changes in the vasculature after pregnancy
account for higher CVD risk in women of higher parity and may help identify women at
increased risk later in life. Studies can use the data presented here as a basis for assessing
differences in vascular adaptation among women of different socio-economic status and different
races, overweight or obese population, and women experiencing pregnancy complications. This
study provides baseline data for future prospective studies to help determine when arterial
changes in pregnancies with complications deviates from the normal pattern. Understanding
normal vascular adaptation to changes in pregnancy may allow for better understanding of the
physiology of pregnancy complications, pathophysiology of the disease, as well as possible
means for early prediction of women who are at risk for the pregnancy complications. This study
provides a better understanding of vascular adaptions during and following pregnancy that may
help explain differences in future CVD risk with parity and may aid in early detection of women
who may be at increased CVD risk long-term. This study also suggests that prolonged
breastfeeding and maintaining weight during pregnancy may be potential lifestyle modifications
that may decrease a woman’s CVD risk.
In conclusion, these data suggest that markers of vascular aging, CCA IAD and CCA
IMT, did not improve further out postpartum as compared to early postpartum. Persistence of
vascular effects of pregnancy may indicate long-term CVD risk and suggest a possible
mechanism behind increased CVD risk with increased parity. Lactation and weight management
may play a significant role in reducing future maternal CVD risk.
23
APPENDIX: SUPPLEMENTARY TABLES AND FIGURES
Supplementary Table 1. Baseline demographic characteristics of MVP participants who
attended second postpartum visit (n=16) compared to those who did not attend second
postpartum visit (n=27)
Characteristics
Attended second
postpartum visit
(n=16)
Did not attend
second postpartum
visit (n=27)
p-value
Age (baseline), years
Ethnicity:
Hispanic
Non-hispanic
Race:
African American
Asian
Native American
Marital Status:
Never married
Married or living as married
Divorced
Educational level:
Some college (at least 1 year)
College (bachelor’s degree)
Graduate/Professional degree
Employment:
Full-time
Part-time
Unemployed
Household income:
< $25k
$25k - $49,999
$50k - $74,999
$75k - $99,999
$100k - $200k
30.0 [28.0,32.0]
27.5 ± 4.6
0.11#
0.37*
6.2% (1)
93.8% (15)
0.0% (0)
100.0% (27)
0.0% (0)
0.0% (0)
6.2% (1)
3.7% (1)
7.4% (2)
3.7% (1)
0.70*
1.00*
6.2% (1)
93.8% (15)
0.0% (0)
11.1% (3)
85.2% (23)
3.7% (1)
0.75*
12.5% (2)
56.3% (9)
31.2% (5)
11.1% (3)
44.4% (12)
44.4% (12)
81.3% (13)
6.2% (1)
12.5% (2)
55.6% (15)
33.3% (9)
11.1% (3)
6.2% (1)
18.8% (3)
18.7% (3)
37.6% (6)
18.7% (3)
14.8% (4)
44.4% (12)
18.5% (5)
7.4% (2)
14.8% (4)
0.11*
0.12*
Continuous normal variables presented as mean ± SD, continuous non-normal variables presented as median [Q1,
Q3] and categorical variables presented as % (n).
*2-sided p-value for Fisher’s exact test are reported for categorical variables with expected frequency<5 in atleast
one cell.
#2-sided p-value for Kruskal-Wallis test are reported for non-normal continuous variables.
No statistical significance seen between the two groups (p>0.05).
24
Supplementary Table 2. Correlations between major outcome variables and other covariates at second postpartum visit (n=16)
Inter-adventitial diameter
Unadjusted
Current age (years)
Current body-mass index (kg/m2)
Weight (kg)
Weight change (kg)
Waist-to-hip ratio
Systolic blood pressure (mm Hg)
Diastolic blood pressure (mm Hg)
Heart rate (bpm)
Total cholesterol (mg/L)
LDL-c (mg/dl)
Triglycerides (mg/dl)
HDL-c (mg/dl)
Glucose (mg/dl)
Insulin (µU/ml)
HOMA-IR
Log-hsCRP
Time since index birth (mo)
Time since last birth (mo)
Time since 6-8 week postpartum visit (mo)
CCA inter-adventitial diameter (mm)
CCA intima-media thickness (mm)
-0.26 (0.32)##
-0.07 (0.78)
0.24 (0.37)
0.11 (0.69)
-0.30 (0.26)
0.67 (<0.01*)
0.56 (0.02*)
0.15 (0.59)
-0.01 (0.96)
-0.03 (0.92)
-0.02 (0.93)
0.03 (0.91)
0.17 (0.52)
-0.23 (0.38)
-0.20 (0.47)
-0.28 (0.29)
-0.22 (0.40)
0.25 (0.35)##
-0.18 (0.49)
0.45 (0.08)
Adjusted for current
age and systolic blood
pressure
0.01 (0.98)
0.01 (0.96)
0.28 (0.34)
-0.27 (0.35)
0.75 (<0.01*)@
0.66 (<0.01*)@
-0.29 (0.31)
-0.21 (0.48)
-0.14 (0.63)
-0.08 (0.80)
-0.14 (0.64)
0.17 (0.57)
-0.17 (0.56)
-0.16 (0.59)
0.12 (0.69)
-0.30 (0.30)
0.003 (0.99)##
-0.18 (0.54)
0.55 (0.04*)
Intima-media thickness
Unadjusted
Adjusted for
weight change
-0.13 (0.64)##
0.13 (0.63)
0.48 (0.06)
0.51 (0.04*)
-0.41 (0.11)
0.05 (0.84)
-0.13 (0.63)
-0.30 (0.26)
-0.41 (0.12)
-0.28 (0.28)
-0.37 (0.16)
-0.20 (0.45)
-0.38 (0.15)
-0.30 (0.26)
-0.39 (0.13)
-0.08 (0.76)
-0.44 (0.08)
0.07 (0.80)##
-0.41 (0.12)
0.45 (0.08)
-
0.02 (0.96)##
-0.05 (0.86)
0.37 (0.17)
-0.21 (0.45)
0.17 (0.55)
0.20 (0.47)
-0.22 (0.43)
-0.28 (0.31)
-0.14 (0.61)
-0.30 (0.27)
-0.19 (0.50)
-0.39 (0.15)
-0.38 (0.16)
-0.47 (0.07)
-0.13 (0.66)
-0.53 (0.04*)
-0.07 (0.80)##
-0.53 (0.04*)
0.47 (0.08)
-
Correlation coefficients presented as rho (p-value); *p-value<0.05 is considered significant
##Spearman correlation coefficients reported for covariates that are not normally distributed; @Adjusted for only current age
25
6
6.2
6.4
6.6
6.8
7
7.2
Mean adjusted CCA IAD by visit
T1
T2*
T3*
PP I
PP II
Supplementary Figure 1. Changes in CCA inter-adventitial diameter for MVP II participants
throughout and after pregnancy (n=16)
T = Trimester; PP = Postpartum
All comparisons non-significant EXCEPT *Trimester 2 vs postpartum II (p=0.01) and *Trimester 3 vs postpartum II
(p=0.02). Adjusted for current age and systolic blood pressure.
6
6.2
6.4
6.6
6.8
7
7.2
Mean adjusted CCA IAD by visit
T1
T2*
T3*
PP I
PP II
Supplementary Figure 2. Changes in CCA inter-adventitial diameter for all MVP participants
throughout and after pregnancy (n=43)
T = Trimester; PP = Postpartum
All comparisons non-significant EXCEPT *Trimester 2 vs postpartum II (p=0.01) and *Trimester 3 vs postpartum II
(p=0.02). Adjusted for current age and systolic blood pressure.
26
.54
.56
.58
.6
Mean adjusted CCA IMT by visit
T1
T2
T3
PP I
PP II
Supplementary Figure 3. Changes in CCA intima-media thickness for MVP II participants
throughout and after pregnancy (n=16)
T = Trimester; PP = Postpartum
All comparisons are non-significant. Adjusted for weight change.
.54
.56
.58
.6
Mean adjusted CCA IMT by visit
T1
T2
T3
PP I
PP II
Supplementary Figure 4. Changes in CCA intima-media thickness for all MVP participants
throughout and after pregnancy (n=43)
T = Trimester; PP = Postpartum
All comparisons are non-significant. Adjusted for weight change.
27
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