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Sex hormones and atherosclerosis in postmenopausal women
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Sex hormones and atherosclerosis in postmenopausal women
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Content
SEX HORMONES AND ATHEROSCLEROSIS IN POSTMENOPAUSAL
WOMEN
by
Roksana Karim
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(EPIDEMIOLOGY)
May 2007
Copyright 2007 Roksana Karim
ii
ACKNOWLEDGEMENTS
I would like to express my utmost gratitude to Dr. Wendy Mack, my chair, for
mentoring me and making this dissertation possible. I am also indebt to Drs. Howard
Hodis, Frank Stanczyk, Leigh Pearce and Eileen Crimmins for their guidance and
support in preparing this Dissertation. My special thanks to Dr.Stanley Azen for his
invaluable advice throughout the graduate program. I would also like to thank Dr.
Rogerio Lobo for his suggestions and comments in the preparation of the
manuscripts. Finally, I am grateful to my family for their endless support in making
my dream come true.
iii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS .........................................................................................ii
LIST OF TABLES .....................................................................................................vii
LIST OF FIGURES ....................................................................................................ix
ABSTRACT.................................................................................................................x
CHAPTER I. INTRODUCTION AND BACKGROUND ON SEX
HORMONES AND ATHEROSCLEROSIS IN POSTMENOPAUSAL WOMEN ...1
1.1 Introduction........................................................................................................1
1.2 Background on atherosclerosis ..........................................................................8
1.2.1 Definition and clinical features of atherosclerosis......................................8
1.2.2 Pathophysiology of atherosclerosis...........................................................10
1.2.3 Descriptive statistics of CVD in women...................................................12
1.2.4 Effect of Menopause on CVD/atherosclerosis..........................................14
1.2.5 Risk factors of CVD/atherosclerosis.........................................................15
1.2.5.1 Traditional risk factors .......................................................................16
1.2.5.2 Predisposing risk factors ....................................................................18
1.2.5.3 Some novel risk factors......................................................................19
1.2.5.4 Biomarkers of oxidative stress and inflammation..............................20
1.3 Background on sex steroid hormones in women .............................................22
1.3.1 Regulation of sex hormone production, transport and metabolism ..........22
1.3.2 Biosynthesis, metabolism and cardiovascular functions of sex
hormones............................................................................................................24
1.3.2.1 Estrogens............................................................................................24
1.3.2.2 Progesterone.......................................................................................28
1.3.2.3 Androgens ..........................................................................................29
Chapter I References..............................................................................................32
CHAPTER II. LITERATURE REVIEW OF CVD/ATHEROSCLEROSIS
AND SEX HORMONES IN POSTMENOPAUSAL WOMEN ...............................39
2.1 Exogenous hormone therapy and CVD/ atherosclerosis in
postmenopausal women .........................................................................................39
2.1.1 Observational epidemiologic studies ........................................................39
2.1.1.1 CVD or CHD in relation to HT..........................................................39
2.1.1.2 Carotid atherosclerosis in relation to HT ...........................................46
2.1.1.3 Critique of observational studies........................................................47
iv
2.1.2 Clinical Trials............................................................................................48
2.1.2.1 Secondary prevention trials................................................................48
2.1.2.2 Primary prevention trials....................................................................55
2.1.2.3 Summary and comments on clinical trials .........................................58
2.1.3 Discussion on the discrepancy between observational studies and
randomized trials................................................................................................60
2.1.4 Impact of HT on CVD risk factors............................................................67
2.1.5 Impact of HT on circulating levels of sex hormones ................................70
2.2 Endogenous sex hormones, CVD and atherosclerosis in postmenopausal
women....................................................................................................................72
2.2.1 Cardiovascular disease & mortality outcomes..........................................72
2.2.2 Studies with coronary atherosclerosis outcomes.......................................78
2.2.3 Studies with carotid atherosclerosis outcome ...........................................82
2.2.4 Critique and discussion of the studies on sex hormones and
CVD/atherosclerosis in postmenopausal women...............................................84
2.2.5 Endogenous sex hormones and risk factors for CVD/ atherosclerosis
in postmenopausal women .................................................................................88
2.2.5.1 Serum estrogen concentrations and CVD risk factors .......................89
2.2.5.2 Serum androgens and risk factors ......................................................90
2.2.5.3 Serum SHBG concentrations and risk factors...................................91
2.2.5.4 Serum sex hormone concentrations and obesity ................................92
2.2.5.5 Conclusion of chapter II: Issues to be discussed in future research...95
Chapter II References.............................................................................................99
CHAPTER III. Relationship Between Serum Levels of Sex Hormones and
Progression of Subclinical Atherosclerosis in Postmenopausal Women.................112
Chapter III Abstract .............................................................................................113
Introduction..........................................................................................................114
Methods................................................................................................................116
Carotid Ultrasonography and Image Analysis .................................................117
Laboratory Measurements................................................................................117
Statistical analysis ............................................................................................119
Results..................................................................................................................121
Discussion ............................................................................................................129
Chapter III References .........................................................................................137
CHAPTER IV. Circulating Levels of Sex Hormones, Markers of
Inflammation, and Proinflammatory Factors in Postmenopausal Women ..............140
Chapter IV Abstract .............................................................................................141
Introduction..........................................................................................................142
Methods................................................................................................................145
Laboratory Measurements................................................................................145
v
Statistical analysis ............................................................................................148
Results..................................................................................................................150
Discussion ............................................................................................................155
Chapter IV References .........................................................................................162
CHAPTER V. Sex Hormone Levels and Subclinical Atherosclerosis
Progression in Postmenopausal Women ..................................................................168
5.1. Specific Aims................................................................................................168
5.1.1. Overall Objectives..................................................................................168
5.1.2. Specific aims ..........................................................................................170
5.2. Background and significance ........................................................................170
5.2.1. Sex hormones in postmenopausal women .............................................170
5.2.2. Coronary Heart Disease in Postmenopausal Women.............................171
5.2.3. Significance of Studying Atherosclerosis Progression ..........................173
5.2.4. Circulating sex hormone levels and CHD..............................................175
5.2.5. Measurement Error in Sex Hormone Determination: ............................177
5.2.5.1. Within subject variability of sex hormones in postmenopausal
women..........................................................................................................177
5.2.5.2. Sensitivity of sex hormone assay methods .....................................178
5.2.6. Circulating Sex Hormone Levels and CVD Risk Factors......................179
5.2.8. Importance of the proposed work ..........................................................180
5.3. Previous Work/Preliminary Studies..............................................................181
5.3.1. Randomized Controlled Trials in Postmenopausal Women ..................181
5.3.1.1. Estrogen in the Prevention of Atherosclerosis Trial (EPAT)
(NIH R01 AG-18798, PI: Howard N. Hodis, M.D).....................................182
5.3.1.2. Women's Estrogen-progestin Lipid-Lowering Hormone
Atherosclerosis Regression Trial (WELL-HART)(U01-HL-49298,
PI : Howard N. Hodis, M.D)........................................................................184
5.3.1.3. The Vitamin E Atherosclerosis Prevention Study (VEAPS)
(R01- NIA AG13860, PI: Dr. Howard N. Hodis, M.D) ..............................185
5.3.1.4. The B Vitamin Atherosclerosis Intervention Trial (BVAIT)
(R01-NIH AG RO1 17160, PI: Dr. Howard N. Hodis, M.D)......................186
5.3.1.5. Women’s Isoflavone Soy Health (WISH) Trial
(NIH U01-AT001653, PI: Dr. Howard N. Hodis, M.D)..............................186
5.3.2 Ultrasound measures of carotid IMT ......................................................187
5.3.3 Determination of Sex Steroid Hormones ................................................188
5.3.4 Preliminary Data on Sex Hormones, Subclinical Atherosclerosis,
and CVD Risk Factors .....................................................................................190
5.4. Research Design and Methods......................................................................199
5.4.1. Design of the Proposed Study................................................................199
5.4.2. Study subjects ........................................................................................200
5.4.3. Study Protocols ......................................................................................201
vi
5.4.4. Ancillary Data Collection ......................................................................201
5.4.5. Laboratory measurements ......................................................................202
5.4.6. Storage of blood samples .......................................................................203
5.4.7. Primary End point Measurement- Carotid Artery Intima-Media
Thickness .........................................................................................................204
5.4.8. Determination of Serum Sex Hormone levels .......................................206
5.4.9. Data co-ordination and external oversight of the randomized trials......208
5.4.10. Statistical Analysis...............................................................................209
5.4.11. Power Considerations ..........................................................................214
5.4.12. Timeline ...............................................................................................215
5.5. Exempt Human Subjects Research ...............................................................215
5.6. Inclusion of Women......................................................................................216
5.7. Inclusion of minorities .................................................................................216
5.8. Inclusion of Children.....................................................................................216
5.9 Vertebrate Animals ........................................................................................216
Chapter V References ..........................................................................................217
Bibliography.............................................................................................................225
vii
LIST OF TABLES
Table II-1. Summarizing the clinical trials ................................................................64
Table II-2. Studies evaluating the relationship between endogenous sex
hormones and CVD/ atherosclerosis in postmenopausal women ..................75
Table II-3. Association of endogenous sex hormones to the risk factors of
CVD/atherosclerosis in women. ....................................................................94
Table III.1. Baseline characteristics of EPAT women (n=180)*.............................122
Table III.2. Serum levels of sex hormones and SHBG by treatment group.............123
Table III.3. Mixed models relating changes in sex hormones/SHBG and
carotid IMT progression...............................................................................124
Table III.4. Mixed models relating changes in the sex hormones/SHBG and
carotid IMT progression, stratified by treatment group...............................125
Table III.5. Relationship of serum sex hormone/SHBG levels to serum lipids
and triglycerides...........................................................................................128
Table IV-1. Demographic and baseline clinical characteristics of EPAT women
(n=216).........................................................................................................150
Table IV-2. Mean serum levels of sex hormones and SHBG..................................152
Table IV-3. Association of sex hormones/SHBG levels and inflammatory
markers with pro-inflammatory factors. ......................................................152
Table IV-4. Association between sex hormone/SHBG levels and
SICAM-1 (ng/ml).........................................................................................153
Table IV-5. Association between sex hormone/SHBG levels and
hs-CRP (mg/L).............................................................................................154
Table IV-6. sICAM models unadjusted and adjusted for homocysteine .................155
Table V.1. Characteristics of Five Randomized Athersclerosis Regression
Trials. ...........................................................................................................183
viii
Table V.2. Sex hormones and atherosclerosis progression in 180
postmenopausal women. ..............................................................................191
Table V.3. Relationship of serum sex hormone levels to serum lipids and
triglycerides..................................................................................................194
Table V.4. Relationship of serum sex hormone levels to carbohydrate related
factors...........................................................................................................195
Table V.5. Mediating effect of HDL- and LDL-cholesterol in sex hormones
and atherosclerosis progression association.................................................196
Table V.6. Baseline characteristics of 1006 postmenopausal women
participating in five randomized trials. ........................................................204
Table V.7. Correlation between sex hormones and IMT progression rate in
EPAT and power to detect in the proposed study........................................215
ix
LIST OF FIGURES
Figure I-1. Progression of Atherosclerosis ................................................................11
Figure I-2. Regulation of Sex Hormone Production and Transport...........................24
Figure I-3. Sex Steroid Hormone Synthesis...............................................................26
Figure II-1, Risk for Coronary Heart Disease in Estrogen Users Compared to
Nonusers.........................................................................................................43
Figure II-2. Risk for Coronary Heart Disease in Estrogen Plus Progestin Users
Compared to Nonusers...................................................................................46
Figure III-1. Change in sex hormones combined with ET and IMT progression....127
Figure V-1. Change in Sex Hormones Combined with ET and Carotid IMT
Progression...................................................................................................193
x
ABSTRACT
Cardiovascular disease is the leading cause of death in women in the United States
and in the entire world. Both incidence and prevalence of cardiovascular disease
increase significantly after menopause, indicating changes in the sex hormone milieu
may be an important contributor. More than 80 observational epidemiologic studies
have showed a significant reduction in cardiovascular disease risk and extent of
atherosclerosis with postmenopausal hormone therapy. A number of in vitro studies
and studies using animal models have demonstrated the direct and indirect
cardiovascular effect of estrogen. However, only a handful of studies have evaluated
the association of circulating estrogens and other sex hormone levels to
cardiovascular disease and risk factors of cardiovascular disease in women.
Literatures relating sex hormones and cardiovascular disease consistently
demonstrated an inverse association between total testosterone, sex hormone binding
globulin and cardiovascular disease/atherosclerosis. However, none of the studies
showed any association with estrogens. The most plausible explanation for this could
be measurement error in estrogen. Given the significant within subject variation of
estrogen levels in postmenopausal women, one single determination of estrogen may
not be enough to characterize estrogen. We used data from Estrogen in the
Prevention of Atherosclerosis Trial (EPAT) where estrogens, androgens and sex
hormone binding globulin were measured multiple times over two years.
xi
Atherosclerosis was measured by intima-media thickness of the common carotid
artery using B-mode ultrasound at the beginning and every six months during the
follow-up of the study. Markers of inflammation such as C-reactive protein,
intercellular adhesion molecule-1 and homocysteine were also measured every six
months. We report a significant inverse association between estrogens, total
testosterone and sex hormone binding globulin with atherosclerosis progression. We
also report that estrogens were significantly beneficially associated with HDL- and
LDL-cholesterol, markers of inflammation and homocysteine. We also demonstrated
that the inverse association between estrogens and atherosclerosis progression was
partially explained by their beneficial association with serum cholesterols. These
results may help understand the cardiovascular impact of estrogens and other sex
hormones in postmenopausal. Thereby reduce the bulk of morbidity and mortality of
this high risk population.
1
CHAPTER I. INTRODUCTION AND BACKGROUND ON SEX HORMONES
AND ATHEROSCLEROSIS IN POSTMENOPAUSAL WOMEN
1.1 Introduction
Cardiovascular disease (CVD) comprises the highest proportion of total disease
morbidity and mortality in the United States and in the entire world. It is the single
largest cause of mortality among women, accounting for one third (8.5 million
annually) of all deaths in women worldwide
1
. Since 1984, the number of annual
CVD deaths for females in the U.S has exceeded those for males
2
. In 2002, 53.2
percent of the deaths from CVD in the U.S occurred in females. CVD is the leading
cause of death in women. In the United States, in 2002, CVD claimed the lives of
nearly half a million women; this is more than the number of lives claimed by the
next 5 leading causes of deaths combined (lung cancer, breast cancer, chronic lower
respiratory disease, Alzheimer’s disease, and influenza/pneumonia). Although the
overall prevalence of CVD appears to be similar in men and women, nearly 34% for
both, CVD is relatively uncommon in females before 45 years of age compared to
men. After the age of 45, prevalence of CVD rates in women gradually increase and
after 55, women suffer more from CVD than men.
Coronary heart disease (CHD) represents 53% of all CVD deaths and has been
identified as the single leading cause of death in American females
1
. CHD rates are
2
2-3 times higher in women after menopause than those of women the same age who
are premenopausal
3
.
The gradual waning of ovarian sex hormone production, culminating in menopause,
has been linked to the apparent loss of cardioprotection in women after menopause.
Several epidemiologic studies have documented a steady increase in CHD incidence
in women with age and its rare occurrence before menopause
4, 5
even in high-risk
populations
6
. Women experiencing early menopause, either natural or due to
bilateral oophorectomy, show increased risk of heart disease
7-9
. An early report from
the Nurses’ Health Study suggested that the risk of non-fatal MI significantly
increases with decreasing age at menopause among women who had surgical
menopause
10
.Two prospective epidemiologic studies have indicated that early age
at menopause was a significant predictor of mortality from ischemic heart disease as
well as all CVD death
11, 12
. These data provide support to the hypothesis that the
decline in estrogen associated with natural or surgical menopause contributes to
CVD risk in women. Even among premenopausal women with CHD, serum estradiol
concentrations were significantly lower compared to controls
13
.
Atherosclerosis or thickening of the arteries due to deposition of low density
lipoprotein (LDL) cholesterol in the arterial walls is the primary underlying cause of
heart disease or stroke
14
. A large population-based study found a significant inverse
3
association between the age at menopause and both the prevalence and extent of
atherosclerosis assessed by ultrasound in 2588 postmenopausal women
15
. This
observation is supported by adverse changes in the risk factors for atherosclerosis
after menopause. Altered lipid profiles have also been reported to be associated with
menopausal status
16, 17
. While a higher percentage of men than women have high
blood pressure before age 55, after this age a much higher percentage of women than
men have hypertension
18
.
Based on the large body of evidence suggesting that endogenous sex hormones,
particularly estrogen, may be protective against CVD, a number of epidemiologic
studies have been conducted to evaluate if postmenopausal hormone therapy (HT) or
estrogen therapy (ET) can protect postmenopausal women against heart disease.
Observational epidemiologic studies over the last three decades provided substantial
evidence that exogenous estrogens and or progesterone have beneficial impact on
CVD in postmenopausal women. The most recent meta-analysis including 25
observational studies through mid-1997 reported that postmenopausal women ever
using unopposed estrogen therapy had a 30% reduced risk of CHD compared to non-
users (summary relative risk 0.70, 95% CI: 0.65-0.75)
19
.
Since 1998, ten randomized clinical trials have been conducted testing the effect of
HT or ET on CVD risk or atherosclerosis progression. Nine of these clinical trials
4
failed to support the findings from observational studies. Eight of the ten trials were
secondary prevention trials including women with known CVD. One of the two
primary prevention trials found a significant reduction in the progression of
subclinical atherosclerosis in women randomized to 1 mg of 17 β-estradiol/day
relative to placebo (p = 0.046)
20
. The other primary prevention trial conducted by the
Women’s Health Initiative (WHI) researchers, which is the largest clinical trial to
date, reported no significant cardioprotection in women receiving estrogen plus
progestin relative to placebo
21
. However, a temporal trend analysis in that report
indicated that the risk of CHD was in fact elevated among the HT treated group
within the first year after initiation of hormone use and the hazard ratios were lower
in the following years of HT use The trend toward decreasing relative risks over time
was statistically significant. A stratified analysis by years since menopause showed
that women starting HT 20 or more years after menopause had a significant higher
CVD risk compared to women using HT earlier after menopause.
Results from the two primary prevention trials suggest that HT may protect against
CHD in early stages of the disease process, particularly in the early stages of the
atherosclerotic process, which is the precursor of most of the cardiovascular end
points such as MI, cardiac death, even stroke. Early intervention with HT right
around menopause is crucial to avail cardiovascular benefit from exogenous
hormone.
5
Despite such strong evidence from approximately 80 observational epidemiologic
studies that suggest both exogenous and endogenous hormones are related to CVD
risk, research examining the relationship between circulating levels of sex hormones
and CVD have been relatively neglected in women. Of note, the association between
endogenous sex hormones and CVD has been explored extensively in men
22
.
Understanding the role of sex hormones on atherosclerosis, and the possible
mediators of this relationship in postmenopausal women may help to reduce the
morbidity and mortality from CVD in this population.
Only a handful of studies have evaluated serum levels of estrogens and androgens in
relation to atherosclerosis, CVD, or CVD risk factors in postmenopausal women
revealing conflicting results. These contradictory results may in part be explained by
use of a single measurement of the sex hormones which might lead to measurement
error since postmenopausal sex hormone concentration show high intraindividual
variation
23
. Almost all the studies in the past have measured serum sex hormone
concentrations at a single time point. To better evaluate the role of sex hormones on
progression atherosclerosis, these associations need to be examined in a prospective
study with serial measures of both atherosclerosis and sex hormones.
To understand the mechanisms of action of sex steroid hormones on different stages
of the atherosclerotic disease process, their impact on mediators of atherosclerosis
6
should also be evaluated in postmenopausal women. To explore the possible
pathways of the effect of sex hormones on atherosclerosis, impact of sex hormones
needs to be correlated with standard risk factors of atherosclerosis (including lipids
and markers of carbohydrate metabolism) as well as novel risk factors, including
markers of oxidative stress and inflammation since vascular inflammation plays a
major role in the development of atherosclerosis. The mechanisms of action of
estrogens in atherosclerosis have been well studied in the past both in pre- and post-
menopausal women
24
. However, the role of androgens in the development of
atherosclerosis in postmenopausal women needs further exploration.
This dissertation will focus on the impact of sex hormones and atherosclerosis in
postmenopausal women and will attempt to address some of these issues. The
dissertation is divided into four different chapters:
The remainder of Chapter I, the background chapter, will include a brief summary
on:
The primary ‘outcome’ of interest, ‘atherosclerosis’: Definition, pathophysiology
and risk factors for atherosclerosis and a summary of the descriptive epidemiology of
atherosclerosis in older women. The primary ‘exposure’ of interest, ‘sex hormones in
7
females’: Regulation and biosynthesis of sex hormone production, transport and
metabolism will be summarized.
Chapter II will present a complete review on sex hormones and cardiovascular
disease in postmenopausal women: This chapter present a review of the literature
evaluating the impact of both ‘exogenous’ and ‘endogenous’ sex hormones on
CVD/atherosclerosis in postmenopausal women.
Chapters III and IV will present two data analyses using data from the Estrogen in
the Prevention of Atherosclerosis Trial (EPAT). EPAT was a randomized, double-
blind, placebo-controlled clinical trial conducted at the Atherosclerosis Research
Unit at USC. EPAT was designed to evaluate the impact of 17 β-estradiol on
subclinical atherosclerosis progression in postmenopausal women. The primary
results of the trial indicated that women receiving estradiol had significantly less
progression of carotid atherosclerosis, which was assessed by B-mode ultrasound.
Both data analyses use longitudinal data on carotid intima-media thickness (CIMT)
as well as serum levels of sex hormones.
Chapter III will present the first data analysis in the form of a publishable journal
article. This independent research work evaluated the relationship between serum
levels of sex hormones and SHBG and carotid IMT progression in healthy
8
postmenopausal women.” The abstract was presented as an oral presentation in the
American Heart Association Scientific Session 2004.
Chapter IV will present the second data analysis evaluating the association between
inflammatory markers and sex hormones and SHBG in postmenopausal women. This
work was recently presented as a poster in the 45
th
Annual Conference on
Cardiovascular Disease Epidemiology and Prevention. For the quality of
examination, chapter IV is presented in its current format of poster slides. The
chapter’s final format will be as a publishable journal article.
Chapter V will be a grant proposal written in an NIH RO1 format that will propose
to measure serum levels of estrogens, androgens, and SHBG in postmenopausal
women, who participated in one of 5 trials conducted at the Atherosclerosis Research
Unit.
1.2 Background on atherosclerosis
1.2.1 Definition and clinical features of atherosclerosis
Atherosclerosis is a progressive disease characterized by accumulation of lipids and
fibrous tissue in the large arterial walls resulting in thickening of the arterial walls
and complementary occlusion of the arterial lumen.
9
The atherosclerotic process is initiated by endothelial damage, which leads to
subendothelial accumulation of cholesterol-engorged macrophages known as ‘foam
cells’. This early ‘fatty streak’ lesion can be found in the aorta, and in coronary
arteries and cerebral arteries with advancing age and beginning as early as the first
decade of life
14
. Early stages of atherosclerosis can remain undiagnosed since there
are no clinically evident signs and symptoms of the disease process. However,
atherosclerosis is the precursors of clinically symptomatic cardiovascular disease
including myocardial infarction (MI) and stroke.
Coronary heart disease (CHD) constitutes 53% of the total deaths from CVD. CHD
includes atherosclerotic diseases of coronary arteries and its complications such as
angina pectoris, acute and chronic MI and cardiac death
1
.
Coronary Artery Disease (CAD) primarily refers to atherosclerotic changes in the
coronary arteries and constitutes the basic component of CHD. CAD is often referred
to as ‘atherosclerosis’ even though atherosclerosis is a generalized condition
affecting arteries all over the body. Coronary artery atherosclerosis is the major
component of generalized atherosclerosis that leads to life threatening events such as
heart attack or myocardial infarction (MI). Since atherosclerosis remains
asymptomatic until clinical complications as described above occur, in research
practice, it is referred to as ‘subclinical atherosclerosis’.
10
1.2.2 Pathophysiology of atherosclerosis
The primary initiating event of atherosclerosis is endothelial injury, which may be
caused by a number of factors such as disturbed blood flow in regions of arterial
branching, smoking, and high concentrations of LDL-cholesterol. In response to the
injury, LDL-cholesterol and lipoprotein(a) diffuses through the endothelial cell
junction into the subendothelial matrix. The trapped LDL-cholesterol undergoes
modifications in the arterial wall including oxidation, lypolysis, proteolysis and
aggregation, which in turn initiates an inflammatory process and subsequent foam
cell formation. A number of pro-inflammatory molecules such as adhesion molecules
and growth factors are stimulated that allow lymphocytes and monocytes to
aggregate at the site of the lesion. Once LDL-cholesterol is ‘highly oxidized’ it is
rapidly engulfed by macrophages to form foam cells. Cytokines and growth factors
released by macrophages and T cells also stimulate migration of smooth muscle cells
(SMC) to the site of lesion as well as proliferation of SMC and extracellular matrix
leading to formation of ‘fibrous plaques’. In advanced lesions, intimal calcification
covers the fibrous plaques that helps maintain stability of the atherosclerotic lesion
14
. In some cases, the atherosclerotic lesion further impinges into the arterial lumen
and thereby narrowing the lumen leading to compensatory enlargement in the artery,
which ultimately can compromise blood circulation to a particular area supplied by
that particular vessel
25
.
11
In some cases, the atherosclerotic lesion further impinges into the arterial lumen and
thereby narrowing the lumen leading to compensatory enlargement in the artery,
which ultimately can compromise blood circulation to a particular area supplied by
that particular vessel
25
.
Figure I-1. Progression of Atherosclerosis
Rupture or erosion of the fibrous plaque stimulates the coagulation factors, which
leads to the process of thrombosis. The thrombus can be spontaneously resolved by
fibrinolysin followed by repair and remodeling of the arterial wall. However, if
spontaneous resolution does not take place, the thrombus can occlude the arterial
lumen. Partial occlusion can cause unstable angina and complete occlusion leads to
Alteration of endothelial function due to risk factors
Deposition and oxidation of LDL
Inflammation in the arterial wall
Thickening of arterial wall; “remodeling” of artery
Formation of fibrous cap over lipid core
Stable plaque
Increased growth
Stenosis
Unstable plaque
Plaque rupture
Thrombus/MI
12
acute MI. When a cerebral artery is affected, occlusion of the arteries leads to
cerebral infarction or stroke
26
. The steps of atherosclerosis progression are
summarized in Figure I-1.
1.2.3 Descriptive statistics of CVD in women
Approximately 70.1 million Americans suffered from one or more types of CVD in
the year of 2002
2
. Of them 37.6 million were female. In the United States, the
overall prevalence of CVD is similar in men and women, 34% for both. However,
the age specific prevalence is much lower in women than men before the age of 45.
After 45, the prevalence of CHD strikingly increases among women and remains
higher then men thereafter.
For the past few decades, CVD has been the leading cause of death in both men and
women, across all racial and ethnic groups. Nearly half a million women’s die every
year from CVD which is more than the number of lives claimed by the next five
causes of death combined. The striking decrease in CVD mortality that has been
observed over the past 30 years in men has not occurred in women and since 1983
then number of annual CVD deaths has been higher in women than in men.
13
Based on The Framingham Heart Study (FHS) in its 44-year follow-up of original
participants and the 20-year follow-up of their offspring
18
-
-CHD comprises more than half (53%) of all cardiovascular events in men
and women under age 75.
- The lifetime risk of developing CHD after age 40 is 49% for men and 32% in
women.
- The incidence of CHD in women lags behind men by 10 years for total CHD
and by 20 years for more serious clinical events such as MI and sudden deaths.
The apparent lower lifetime risk of CHD in women compared to men is primarily
influenced by low rates of CHD in women before menopause. After menopause,
CHD rates are 2-3 times higher than the rates in premenopausal women of the same
age. Although the prevalence of MI is lower in women (3%) compared to men (4%),
survival is worse among women. Thirty eight percent of women as opposed to 25%
men die within 1 year after being diagnosed with MI. Women are also more likely to
die suddenly of CHD with no previous symptoms of the disease.
The overall incidence of stroke is 1.25 times greater in men than women. Like other
cardiovascular outcomes, the difference in incidence rates between the sexes is
somewhat larger at younger ages but nonexistent after age 65. More women than
men die of stroke each year. Women accounted for 61.5% of all stroke deaths in the
14
U.S in 2002. The higher mortality rate in women from stroke and MI can be partially
explained by higher age adjusted prevalence of major CVD in women with diabetes
and heart disease death rates among adults with diabetes are 2 to 4 times greater than
the rates for adults without diabetes. These facts underscore the importance of more
extensive research on primary prevention of atherosclerosis especially in
postmenopausal women who are most vulnerable to CVD.
1.2.4 Effect of Menopause on CVD/atherosclerosis
Among premenopausal women, CHD is extremely rare, even in high risk group
6
.
The incidence of cardiovascular complication is much lower in premenopausal
women compared to men of similar age
7
. The reduced risk of CHD is gradually lost
in women after menopause, an event that marks the end of a gradual process of
waning of the ovarian sex steroid hormone production. The fact that endogenous
female sex hormones, particularly estrogen is most likely to have a protective effect
on CHD among women during reproductive life has been supported by the initial
Framingham risk study, which showed that CVD rates were lower among
premenopausal women than postmenopausal women
27
. They also reported that
women experiencing natural menopause between 45 and 49 years or who were
surgically menopaused between 40 and 44 years were more likely to develop CHD
than premenopausal women of similar age. Nurses’ Health Study reported that
women who had bilateral oophorectomy resulting in surgical menopause and did not
15
take hormone replacement therapy (HRT), had 2 times higher risk of CHD compared
to premenopausal women of the same age
8
. A recently updated report from Nurses’
Health Study including women only who had experienced natural menopause and
had not used HRT found an overall significant association between younger age at
menopause and increased risk of CHD
9
. Age at natural menopause has also been
reported to have significant association with mortality from ischemic heart disease
11
.
Surgically menopaused women taking HRT had even lower risk than the
premenopausal women, which was not statistically significant. Menopause has also
been found to influence a number of modifiable risk factors of CHD like lipids and
lipoproteins, independent of any effects of ageing. These changes may in part
explain the increased incidence of coronary heart disease seen in postmenopausal
women. The Framingham Study following a cohort of 2,873 since 1948
demonstrated that total cholesterol levels increased after menopause
4
. The increase
was primarily due to an increase in LDL-cholesterol levels and to some extent a
slight decrease in HDL-cholesterol. A cross-sectional study of 542 healthy, non-
obese women reported similar results
28
. All the evidence listed above are suggestive
of a cardioprotective effect of ovarian sex hormones particularly estrogens.
1.2.5 Risk factors of CVD/atherosclerosis
Epidemiological studies have shown that the major independent risk factors
predicting the onset of CHD in women are similar to those of men
29
. Although the
16
major risk factors are similar in men and women, the magnitude of effect is different
between male and female. The major independent risk factors for CHD in women are
age, cigarette smoking, hypertension, dyslipidemia, and diabetes mellitus. Minor risk
factors are obesity, reduced physical activity, positive family history of CHD,
hypertriglyceridemia, increased lipoprotein (a) (Lp[a]), increased serum
homocysteine, and abnormalities in coagulation factors
30
. A brief summary of these
risk factors is provided below:
1.2.5.1 Traditional risk factors
Age: Advancing age increases the absolute risk of CHD in both men and women
31-
33
. Although the atherosclerotic process begins in young adulthood, the onset of
progressive CHD symptoms are delayed in women compared to men. The incidence
of CHD in women lags behind men by 10 years for overall CHD. The lag period is
20 years for MI and sudden cardiac death
34
.
Cigarette smoking: Cigarette smoking is the most important and well-established
risk factor that increases the risk of CHD by two to four fold in both men and
women
29
. There is
a clear dose–response relationship between smoking and risk of
CHD. Compared with nonsmokers, women who smoke one to four cigarettes per
day
(defined as light smokers) have more than twice the risk of coronary disease
35
.
Cessation of smoking reduces the risk of CHD in both women and men, as early as
months of quitting and the risk falls to the level of the risk among
nonsmokers within
17
three to five years after cessation, regardless
of the amount smoked, the duration of
the habit, or the age
at cessation
35, 36
. Smoking is one of the leading preventable
causes of CHD in women as well as men
37
.
Hypertension: As in men, the strong association between elevated systolic and
diastolic blood pressure
and CHD risk in women has been documented by
a number
of prospective epidemiological studies
37
. Isolated hypertension is a common risk
factor in older women, affecting 30% of women older than 65
38
. Blood pressure
control by antihypertensive medication reduces the risk of major CVD events and
stroke in women as well as in men
39
.
Serum cholesterol: A number of prospective observational studies have reported a
positive
association between total cholesterol levels and coronary heart
disease in
women. An increased
level of high-density lipoprotein (HDL) cholesterol is a
particularly
strong predictor of a decreased risk of coronary heart disease
in women
40, 41
. HDL cholesterol was second only to age as
a predictor of CVD death among
women
in the Lipid Research Clinics Follow-up Study
42
. Among postmenopausal,
HDL-cholesterol was inversely and LDL-cholesterol was positively associated with
progression of carotid atherosclerosis
43
. Secondary prevention trials of lipid-
lowering therapy in women with CHD show substantial benefit. However, limited
trial data are available on primary prevention of CVD with lipid-lowering therapy
18
among women. A substantial proportion of postmenopausal women have elevated
cholesterol levels and a higher percentage of women than men have total blood
cholesterol of 200 mg/dl or higher after age 45
44
.
Diabetes Mellitus: Diabetes is an even stronger risk factor for coronary heart
disease
in women than in men. Mortality rates for coronary heart
disease are three to seven
times higher among diabetic women
than among nondiabetic women, as compared
with rates that are
two to four times higher among diabetic men than among those
without diabetes
37
. Diabetes exacerbates the effects
of known coronary risk factors
and may impair estrogen binding,
negating the possible protection against coronary
heart disease that
endogenous estrogens may confer on premenopausal women
45
.
Diabetes is also one of the most important risk factors for stroke in women.
According to Framingham data and several European studies, the impact of diabetes
on stroke is greater in women than in men
44
.
1.2.5.2 Predisposing risk factors
Risk factors other than the six major independent ones are primarily associated with
the risk of CHD by mediating one or more of the major risk factors described above.
Obesity & physical exercise: Direct positive associations between CHD risk and
obesity and physical inactivity have been documented by multiple
prospective cohort
19
studies of women. However, according to Framingham data, obesity and physical
inactivity exert much of their adverse influence by mediating major risk factors for
CHD risk. In the Nurses'
Health Study, even the women who were mild to moderate
overweight had nearly twice as high a risk of CHD as the lean women
46
. Although a
large portion
of the excess risk is attributable to the influence of adiposity
on blood
pressure, glucose tolerance, and lipid levels, after
adjustment for these variables a
moderate residual effect persists
that may be due to other mechanisms. Based on this
report and the fact that 20% of postmenopausal women are obese, it has been
suggested that the NCEP guidelines should include BMI>30kg/m
2
as a risk factor.
Positive family history of premature CHD: A Family history of heart disease is an
important predictor and therefore used by NCEP in defining cardiovascular risk
status
47, 48
. However, the effect of familial predisposition on CVD risk acts primarily
by influencing the major CVD risk factors like blood pressure or cholesterol levels
30
.
Although Framingham data reported that family history is correlated with CVD risk
factors, others have indicated that the effect of family history is independent of major
cardiovascular risk factors
49
.
1.2.5.3 Some novel risk factors
A new set of risk factors, called ‘conditional’ or sometimes ‘emerging’ risk factors,
are not yet fully established. The extent to which these variables are independent of
20
established risk factors is not clear
50
. Some of these conditional risk factors have
been found to be closely related to the six major independent risk factors of CHD
51
.
Further research examining the relationship between these novel risk factors and
atherosclerosis and factors mediating these relationships may provide new insights
towards understanding the process of atherogenesis. A brief summary of the novel
factors that will be evaluated in this dissertation is provided next:
1.2.5.4 Biomarkers of oxidative stress and inflammation
These factors are considered to be in the mechanistic pathway of the atherosclerotic
process and are related to most of the major CVD risk factors.
Homocysteine: Although a number of cross-sectional, case-control and cohort
studies have linked elevated levels of homocysteine with atherosclerosis/CHD, the
exact mechanism of this association is largely unknown
52
. Several hypotheses have
been postulated such as a direct oxidative stress on vascular endothelium, or some
indirect mechanism may be involved in the process. The Atherosclerosis Risk in
Communities (ARIC) reported that CHD incidence was associated
positively with
serum homocysteine levels only in women
53
.
C-reactive protein (CRP): The development of atherosclerotic lesion has been
described as a series of highly specific cellular and molecular responses
21
characteristic of inflammation. Recently, biomarkers of inflammation have been
considered to be important in prediction of atherosclerosis. CRP, a biomarker of
chronic inflammation, is emerging as a promising novel risk factor. Data are
accumulating on the positive association between c-reactive protein and CHD risk in
both men and women
52
. A graded association between hs-CRP and carotid
atherosclerosis has been demonstrated in women but not in men
54
. In
postmenopausal women, hs-CRP has been reported to improve the predictability of
coronary events significantly when included in the model in addition to lipids.
C-reactive protein was an independent predictor of cardiovascular events such as
death from coronary heart
disease, nonfatal myocardial infarction or stroke, or the
need
for coronary-revascularization procedures
55
.
Adhesion molecules: Adhesion molecules including E-selectin, P-selectin, vascular
cell adhesion molecule-1 (VICAM-1) and soluble intercellular adhesion molecule
type-1 (sICAM-1), play an important role in the early inflammatory process of
atherosclerosis
56
. Epidemiologic data evaluating the relationship between
atherosclerosis and adhesion molecules are relatively sparse. A nested case-control
study among participants of the Women’s Health Study, who were postmenopausal
with an average age of 59 years, reported significantly higher levels of SICAM-1 in
women with cardiovascular events relative to women free of cardiovascular events
55
.
22
Coagulation factors: Hemostatic factors including tissue plasminogen activator
(TPA), plasminogen activator inhibitor-1 (PAI-1), factor VII, D-dimer and
fibrinogen are positively related to the risk CVD
57-59
. The direct association of higher
concentration of TPA and PAI-1 with CHD (MI and sudden cardiac death) was not
independent of insulin resistance, inflammation, or cell damage in a prospective
study of 3043 patients with angina pectoris
58
. However, a cross-sectional analysis of
the Atherosclerosis Risk in Communities (ARIC) reported that TPA, PAI-1 and
D-dimer were positively associated with atherosclerosis, measured by carotid artery
intima-media thickness (IMT), independent of common CVD risk factors
60
.
Fibrinolytic system plays an important role in coronary thrombosis as well as in early
stages of atherosclerosis.
1.3 Background on sex steroid hormones in women
1.3.1 Regulation of sex hormone production, transport and metabolism
In women, important circulating sex hormones are estrogens, progesterone and
androgens. During the reproductive life, the primary source of sex hormones in
women is the ovaries. Ovarian production of steroid hormones includes estrogen,
progesterone and androgens like testosterone and androstenedione. The adrenal
cortex also contributes to the circulating androgens. The production and release of
sex steroid hormones are mediated through hypothalamo-pitutary-ovarian axis. Any
stimulation (visual, olfactory, pineal, stress, signals related to hormonal feedback
23
mechanism) in the hypothalamus causes release of gonadotropin releasing hormone
(GnRH), which in turn signals the anterior pituitary to produce specific
gonadotropins (follicle stimulating hormone (FHS) or leutinizing hormone (LH)).
Gonadotropins then act on particular hormone-producing cells of the ovaries and
adrenal cortex to stimulate the production and release of sex hormones. After release,
sex hormones circulate either free or bound to specific serum proteins such as sex
hormone binding globulin (SHBG), corticosteroid-binding protein (CBG), or
albumin. SHBG has a high affinity but low capacity to bind with
dyhydrotestosterone (DHT), testosterone and estradiol whereas all steroids bind to
albumin with low affinity but high capacity. Androgens, particularly DHT have
greater affinity to bind with SHBG than estradiol. Hormones bound to SHBG are
biologically inactive. Free and albumin-bound sex hormones are bioactive and can
bind to specific receptors to exert their action [Figure I-2].
Most of the sex steroid hormones are metabolized primarily in the liver. Sex
hormones are degraded by the process of sulfonation, or glucoronidation, or
hydroxylation by a group of enzymes known as hydroxylsteroid dehydrogenase
(HSD). Elimination of the metabolites take place by conjugation and metabolites are
excreted in the urine.
24
Figure I-2. Regulation of Sex Hormone Production and Transport
1.3.2 Biosynthesis, metabolism and cardiovascular functions of sex hormones
1.3.2.1 Estrogens
Biosynthesis: The major naturally occurring steroids with estrogenic activity are
17β-estradiol and estrone
61
. Estrogens are produced by the granulosa and theca cells
of ovarian follicles as a result of a complex coordinated activities of specific
enzymes
62
. Figure I-3 displays a schematic diagram of biosynthesis of estrogens as
well as progesterone and androgens. Androgens, specifically androstenedione and
Hypothalamus
Pituitary
Ovaries Adrenals
Target Tissues
Precursor Metabolism
Hormone Action
Oral
Transport Hepatic & Intestinal
Metabolism
Elimination
Metabolism
Receptors
Androgen
Progesterone
Estrogen
SHBG
CBG
25
testosterone, produced by theca cells are aromatized into 17 β –estradiol and estrone
within the granulosa cells. More than 95% of circulating estradiol is directly
secreted by the ovaries, with a smaller contribution from peripheral conversion of
estrone to estradiol. Peripheral aromatization of adrenal androgens also contributes to
the levels of circulating estrogen. This source of estrogen is particularly important in
postmenopausal women since the ovarian source is no longer functional after
menopause.
In premenopausal women, 17 β-estradiol produced by the ovaries is the chief
circulating estrogen. During reproductive age, estradiol concentrations vary greatly
by the phases of the ovarian cycle. An average estradiol concentration is
approximately 100pg/ml in the follicular phase and 600 pg/ml at the time of
ovulation. Serum levels of estradiol may be as high as 20,000 pg/ml during
pregnancy. Since ovarian function wanes gradually after the third decade of life,
there is a gradual reduction in the estradiol levels circulating in the blood. After
menopause, serum estradiol concentrations may be as low as 5-20 pg/ml.
26
Transport: After release into the circulation, most of the estradiol binds with SHBG
and albumin. Only 2-3% circulates as free estradiol. Total concentration of estradiol
is proportional to free estradiol because the concentration of SHBG is stable and
estradiol binds with SHBG with lesser affinity compared to testosterone. Estradiol
can be converted to estrone in the peripheral circulation.
Figure I-3. Sex Steroid Hormone Synthesis
Cholesterol
Pregnenolone
17a-Hydroxy
Pregnenolone
DHEA
Progesterone
17a-Hydroxy
Progesterone
Androstenedione
Testosterone
CYP17
CYP17
CYP17
CYP17
CYP11A
HSD3B
HSD3B
HSD3B
HSD17B
Adrenal
Ovary
Estrone (E1)
Estradiol (E2)
HSD17B
CYP19
CYP19
27
Mechanism of action: Estrogen exerts its biological activity through estrogen
receptor (ER α and ER β)
63
. The ER has a DNA-binding domain N-terminal and a
ligand-binding domain for estrogens. Estrogen binding causes conformational
changes in the ER, which then translocate into the cell nucleus to bind as
homodimers or heterodimers to a specific sequence of DNA called estrogen response
elements
64
. The hormone-bound ER also interacts with transcriptional coactivators
65,
66
. The result is transcription of specific genes, leading to a change in the messenger
ribonucleic acid (mRNA) and, finally, changes in the levels and types of cellular
protein. In addition to the female reproductive system and other sites, estrogen
receptors are also found in myocardial, vascular smooth muscle cells (VSMC), and
endothelial cells
24
.
Metabolism and elimination: Peripheral estradiol and androstenedione are converted
into estrone, which is converted to estriol in the liver. Estrogens are metabolized by
desulfonation, oxidation/reduction and or hydroxylation. Metabolites are then
conjugated by process of sulfation, glucoronidation and O-methylation and,
conjugates are excreted in the urine.
Effects of estrogen on the cardiovascular system: There are indirect and direct
(genomic & non-genomic) cardiovascular effects of estrogens
24
. Genomic effects are
mediated through ERs and transcriptional mechanisms. Non-genomic effects are
28
much more rapid than genomic effects and are the result of direct estrogenic actions
on cell membranes. Non-genomic effects can also be exerted by direct interactions of
classic ER with intracellular enzymes.
1.3.2.2 Progesterone
Biosynthesis: The principal progestetional steroid hormone in human being is
progesterone. It is produced by the corpus luteum of the ovaries as a response to LH
during the luteal phase, and by the placenta during pregnancy
61
. Progesterone is an
intermediate in the synthetic pathways of androgens in the ovaries and adrenal
glands [Figure I-3]. It is also an intermediate in the pathways of production of
glucocorticoid and mineralocorticoid hormones in the adrenal cortex.
Transport: The majority of progesterone (about 80%) remains bound with albumin
in the circulation.
Mechanism of action: Progesterone acts by binding to a specific receptor protein, the
progesterone receptor (PR). Like ER, PRs are also a member of the nuclear receptor
superfamily
63
. There are two specific PR proteins, A and B which are derived from a
single gene
62
. Hormone bound PR bind to specific progesterone responsive elements
on the DNA resulting in transcription of genes.
29
Physiologic effects of progesterone: The biologic effect of progesterone is mostly
restricted to the female reproductive tract and mammary tissue. However, animal
studies show of some evidence of cardiovascular effects of progesterone opposing
the effects of estrogen. For example, progesterone inhibits estrogen-induced
endothelium-dependent vascular relaxation
67
, reduction in intimal plaque size and
cell proliferation
68, 69
.
Metabolism and elimination: Progesterone is metabolized in the liver in a process
similar to estrogen and metabolites are excreted in the urine.
1.3.2.3 Androgens
Biosynthesis: The major naturally-occurring steroid hormones with androgenic
activity are 5 α-dyhydrotestosterone (DHT), testosterone, androstenedione and
dehydroepiandrosterone (DHEA). In female, androgens are derived from the adrenal
cortex (DHEA and androstenedione) and from the ovaries (androstenedione and
testosterone). The majority of testosterone is produced by peripheral conversion of
androstenedione and DHEA which contribute to approximately 50% of the
testosterone concentrations in women. The remaining testosterone is concentrated
and secreted by the ovaries (25%) and adrenal cortex (25%)
70
. In females, peripheral
conversion of testosterone to DHT is limited because of the higher sex hormone
binding globulin (SHBG) and increased aromatization to estrogen
61
. In contrast to
30
ovarian androgens, adrenal androgen production does not vary with the menstrual
cycle. Adrenal androgens remain as the only source of estrogen in postmenopausal
women after being aromatized in the peripheral circulation. The biochemical
pathway of androgen production is similar to that of estrogen [Figure I-3].
Transportation: After release, androgens mostly bind to the carrier proteins. Only
2% of testosterone, 8% of androstenedione and 4% of DHEA remains free or
unbound in the circulation
71
. Most of the testosterone (66%) binds with SHBG
whereas androstenedione (85%) and DHEA mostly bind with albumin. Free
testosterone concentration is dependent on SHBG levels as testosterone has greater
affinity for binding with SHBG than estrogen.
Mechanism of action: The classic pathway of testosterone action involves steroid
binding to androgen receptor (AR) and ligand activated transcription. AR is
expressed in all cells of the vasculature, including endothelial cells, smooth muscle
cells, myocardial fibers, macrophages and platelets
72
. Testosterone also has non-
genomic action on macrophages, vascular smooth muscle cells and endothelial cells.
Animal studies showed that testosterone is pro-atherogenic in female.
Evidence show that all the sex hormones have biological effects on the
cardiovascular system. However, it is still not clear whether the cardiovascular
31
effects of sex hormones are different between men and women. The current
controversy on the impact of hormone replacement therapy on cardiovascular disease
has underscored the importance of more research exploring the relationship between
sex steroid hormones and CVD in postmenopausal women.
The following chapter II provides detailed review of the literature regarding the
association between sex hormones and CVD/ atherosclerosis in postmenopausal
women.
32
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39
CHAPTER II. LITERATURE REVIEW OF CVD/ATHEROSCLEROSIS AND
SEX HORMONES IN POSTMENOPAUSAL WOMEN
2.1 Exogenous hormone therapy and CVD/ atherosclerosis in postmenopausal
women
In this section, observational epidemiologic studies evaluating the association
between postmenopausal HT and CVD/atherosclerosis will be reviewed. Different
cardiovascular outcome will include CVD, CHD, atherosclerosis and cardiovascular
risk factors. Review of observational studies will be followed by a review of the
clinical trials testing the impact of HT, both estrogen alone and estrogen plus
progestin regimens, on CVD/atherosclerosis.
2.1.1 Observational epidemiologic studies
2.1.1.1 CVD or CHD in relation to HT
Estrogen therapy has been available to women since 1938 when estrogen was first
synthesized, followed by introduction of Premarin (conjugated equine estrogens or
CEE) in 1942. However, hormone therapy was not considered for the ‘menopausal
syndrome’ until the 1950s. Scientific research on the possible impact of HT on CVD
was inspired by the results of the Lipid Research Clinics (LRC) Program Follow-Up
Study in 1987. This landmark study reported a 66% (95% CI: 0.12-0.81) reduced
40
risk of mortality in women using estrogen for non-contraceptive purposes, compared
to nonusers
1
. Since then, more than 40 epidemiologic studies have reported on the
association between postmenopausal HT and CHD. Several review articles
summarizing the findings from those observational studies have also been published.
The most recent meta-analysis included epidemiologic studies published through
mid-1997, comprising 25 studies where CEE was the predominant regimen and 7
studies where women used combined estrogen and progestin
2
. This meta-analysis
reported a 30% reduction in CHD among women who had ever used CEE compared
to never users (summary statistic of relative risk = 0.70, 95% CI: 0.65-0.75). A
similar relative risk was observed among women using combination therapy
(estrogen and progestin RR = 0.66, 95% CI: 0.53-0.84).
The Nurses’ Health Study (NHS) is one of the largest and most comprehensive
prospective studies to examine HT and primary prevention of CVD in
postmenopausal women. This cohort was established in 1976 when 121,700 female
nurses replied to a mailed questionnaire with their postmenopausal and medical
history including CVD disease and risk factors. Results from the first wave of
follow-up were published in 1985
3
, followed by updates in 1991
4
, 1996
5
and most
recently in 2000
6
. The key finding was consistent in all four reports: a 40% reduction
in CHD risk among postmenopausal women who had ever used HT compared to
41
non-users. The most recent publication from NHS, including 20 years of follow-up
data on 70,533 postmenopausal women, reported that the risk for major coronary
events was lower among current users of HT compared with never-users (RR = 0.61,
95% CI: 0.52-0.71). HT also had a significant protective effect against CHD among
past users (RR = 0.82, 95% CI: 0.72-0.94) and short term current users (RR = 0.40,
95% CI: 0.21-0.77, for <1 year of use). A lower dose of oral estrogen, 0.3mg/d was
equally effective in reducing the risk of coronary events to that of women taking
estrogen 0.625mg/d. In this cohort, the risk of stroke was statistically significantly
higher among women taking CEE at a dose of 0.625mg/d or more compared to never
users. A lower dose of CEE was not associated with increased risk of stroke. CEE
alone (RR = 0.55, 95% CI: 0.45-0.68) or combination therapy with estrogen plus
progestin (RR = 0.64, 95% 0.49-0.85) were protective against the risk of coronary
heart disease. However, risk of stroke increased only with combination therapy (RR
= 1.45, 95% CI: 1.10-1.92). The authors concluded that postmenopausal estrogen
therapy reduces the risk of CHD, even at a dose lower than the standard dose but
increased the risk of stroke.
The Women’s Health Initiative observational study is the most recent prospective
cohort study that followed 16,608 postmenopausal women, on average 65.3 years,
taking estrogen plus progestin (E+P) for an average 5.5 years (maximum 8.4 years);
38,313 women on average 63 years old taking estrogen (E) alone for 7.1 years
7
.
42
Study end points were cardiovascular events such as coronary heart disease, stroke,
and venous thrombosis. The overall hazard ratio among E+P users for developing
CHD was 0.87 (95% CI: 0.72-1.05). The HR (95% CI) for less than 2 years of use
was 1.12 (0.46-2.74), 2 to 5 years 1.05 (0.70-1.58), and more than 5 years was 0.83
(0.67-1.01). It is important to note there were very few women in the study who used
HT for less than 2 years (number not mention in the manuscript), and there were
only 5 CHD cases among women who used HT for less than 2 years. In the E alone
observational study, the HR (95% CI) for women who used E alone therapy for less
than 2 years was 1.20 (0.49-2.94), 2 to 5 years 1.09 (0.75-1.60), and more than 5
years was 0.73 (0.61-0.84)
8
.
A population-based case-control study nested in a cohort of 164,769 postmenopausal
women in the U.K, included women who were 50 to 74 years of age without
clinically evident CVD
9
. A total of 1,013 acute MI cases and 5,000 age-matched
controls participated in this study. Compared to never-users, current-recent users
(used HT any time in the past 6 months) of HT had a reduced risk of acute MI (OR =
0.72, 95% CI: 0.59-0.89). The protective effect was observed among women using
unopposed estrogen (OR = 0.52, 95% CI: 0.35-0.78) as well as combination therapy
(OR = 0.79, 95% CI: 0.59-1.08). Transdermal HT was similarly effective in reducing
the risk of acute MI as oral HT (OR = 0.62, 95% CI: 0.59-1.08).
43
Figure II-1, Risk for Coronary Heart Disease in Estrogen Users Compared to Nonusers
Rosenberg, 1976
Talbott, 1977
Rosenberg, 1980
Bain, 1981
Ross, 1981
Szklo, 1984
Beard, 1989
Psaty, 1994
Rosenberg, 1993
Mann, 1994
VarasLorenzo, 2000
Gruchow, 1988
Sullivan, 1988
McFarland, 1989
Wilson, 1985
Bush, 1987
Petitti, 1987
Criqui, 1988
Avila, 1990
Sullivan, 1990
Henderson, 1991
Wolf, 1991
Falkenborn, 1992
Grodstein, 1996
Grodstein, 2000
0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 2.5 2.75 3 3.25 3.5 3.75 4 4.25 4.5 4.75 5
Cohort Studies
Angiographic Studies
Case-Control Studies
While the majority of epidemiologic studies have supported a protective association
of HT on CHD risk, a number of epidemiologic studies failed to show any significant
association between estrogen use and risk of heart disease (Figures II-1 and II-2).
Most of the studies reporting null results were case-control studies. One potential
reason for case-control studies to report null finding is the measurement bias of the
exposure which is based on memory recall. If both case and control women could not
44
recall their history of HT use properly, this could essentially lead to a non-
differential misclassification leading to a bias towards null. However, in most of
these null studies the summary relative risk estimates were less than 1(although not
statistically significant). Case-control studies nested in a cohort study is free from the
recall bias as seen in the study by Varas-Lorenzo C et al.
9, 10
[Figure II-2]. The only
epidemiologic study that reported a significant increase in the risk of cardiovascular
morbidity was the Framingham Study, a cohort study including 8 years of follow-up
data on 1234 postmenopausal women who participated in the 12
th
biennial
examination
10
. The risk of total cardiovascular morbidity was increased by over 76%
(RR = 1.76, p < 0.01) among women reporting estrogen use at one or more
examinations. Even in this study however, postmenopausal estrogen use increased
the risk of MI or total cardiovascular disease risk only among the female smokers
(RR for total CVD = 3.16, p < 0.01). No significant increased risk of CVD was
observed among nonsmoking postmenopausal estrogen users in this study (RR =
1.26, p=NS).
Data from studies using angiographically-determined coronary artery stenosis
suggest that postmenopausal estrogen treatment reduces the risk for angiographically
significant coronary artery disease
11-13
. One of the 3 studies was conducted with a
considerably large sample of postmenopausal women, 2188 women. Of them, 1444
women had >70% coronary artery stenosis and 744 women had no stenosis. The
45
odds ratio for the risk of coronary artery disease for estrogen users relative to the risk
of coronary artery disease for nonusers was 0.44 (95% CI: 0.29-0.67) after
adjustment for age, cigarette smoking, diabetes, cholesterol, and hypertension.
Postmenopausal estrogen replacement was a significant independent protective
factor for coronary artery disease in a multivariate logistic regression model (P =
0.037). Consistency in results among the 3 observational studies using coronary
angiography, as the assessment tool of CAD confirms no measurement biases related
to the outcome as opposed to subjective data used in many other observational
studies. However, these studies have an inherent selection bias issue as all of these
women had some clinical symptoms and suspected CVD that led to an angiography
in the first place. Therefore, these results may not be generalizable to women who do
not have a clinically evident cardiovascular disease.
46
Figure II-2. Risk for Coronary Heart Disease in Estrogen Plus Progestin Users Compared to Nonusers
Falkeborn,
1992
Varas
Lorenzo, 2000
Psaty, 1994
Mann, 1994
Grods tein,
1996
0 0.25 0.5 0.75 1 1.25 1.5 1.75 2
2.1.1.2 Carotid atherosclerosis in relation to HT
Relatively few observational studies have examined the association between
postmenopausal HT use and carotid atherosclerosis measured by carotid artery
intima-media thickness (IMT). In contrast to the Framingham cohort study findings,
postmenopausal women who smoke have been reported to benefit from HT in a
cross-sectional study of 140 age-matched postmenopausal smokers and nonsmokers.
This study demonstrated that smokers using HT had lower cholesterol levels, lower
carotid artery IMT and systemic arterial compliance, compared with smokers not
using HT
14
. A cross-sectional study among 623 postmenopausal women showed that
HT use was significantly associated with thinner carotid IMT equally in diabetic (n =
212) and non-diabetic women (n = 411)
15
.
Cohort
Studies
Case-
Control
Studies
47
2.1.1.3 Critique of observational studies
Observational epidemiologic studies are subject to selection as well as measurement
biases. Most of the observational studies measured hormone use only at one time
point or asked women if they have ever used HT. Thus, ever and current HT use
does not differentiate short term or long term use. Prospective studies with frequent
follow-up measurements such as the Nurses’ Health Study were able to measure
duration of HT use more accurately during the follow-up. However, the NHS
population may not necessarily represent the general population particularly in terms
of socioeconomic status. Observational studies are also subject to confounding bias
as physicians may prescribe HT to women who asked for it and are relatively more
health conscious than those who did not. Thereby, a biased protective effect of HT
could have been apparent. However, it is also possible that women who used HT had
more adverse menopausal symptoms; and these potentially are the women benefit
most from HT.
Another important issue is the time since menopause. Although age at menopause
has been taken into account in some of the observational studies, time from
menopause to start of HT use has not been considered in any of the observational
studies. If women included in the observational studies sought HT for menopausal
symptoms, they would have initiated HT at or shortly after menopause. So it is
possible that the cardiovascular benefit from HT is apparent only after a long term
48
use. Again since HT in the observational studies were self-selected, it is likely that
the great majority of women initiated HT around menopause. Cardiovascular
alteration due to lack of estrogen could be at an earlier stage around menopause.
Thereby, the detrimental changes in the cardiovascular system could be more
responsive to HT if initiated around menopause. Since observational studies are
never free of biases, clinical trials were warranted to evaluate the impact of HT on
CVD and atherosclerosis.
2.1.2 Clinical Trials
A series of clinical trials to test the effect of HT on CVD/atherosclerosis began in the
early 1990s. Most of the clinical trials, 8 out of 11 to date, were designed as
secondary prevention trials. However, the largest trial, the Women’s Health Initiative
(WHI) trial, was a primary prevention trial. Descriptions of these clinical trials are
summarized briefly in Table II-1.
2.1.2.1 Secondary prevention trials
Trials with CVD/Stroke/CHD as primary end point
The Heart and Estrogen/Progestin Replacement Study (HERS) was the first
randomized controlled clinical trial to test the effect of postmenopausal hormone
therapy on CHD
16
. HERS was a randomized double-blind, placebo-controlled trial
49
that included 2763 postmenopausal women, 80 years of age or younger with
established CHD. The mean age of the study participants was 66.7 years.
Participating women were randomized to either 0.625 mg CEE along with 2.5mg
MPA or placebo daily. At the end of the average 4.1 year follow-up period, there
was no significant difference in the primary outcome (nonfatal MI and CHD death)
between the HT and placebo groups, (Hazard ratio (HR) = 0.99, 95% CI: 0.80-1.22).
The risk for primary CHD events was in fact elevated after 1 year of HT use (HR =
1.52, 95% CI: 1.01-2.29). After the first year of randomization, the RH estimates for
CHD events decreased with increasing years of HT use. The decreasing trend in the
risk of CHD with duration of randomized HT use was statistically significant (p =
0.009) despite the overall null effect. An additional 2.7 years of follow-up (without
randomized treatment) also failed to show any overall significant difference between
the treatment group (HR = 0.97, 95% CI: 0.82-1.14)
17
. However, the time trend
analysis showed significant increased risk for CHD after 1 year (HR = 1.52, 95%CI:
1.01-2.29), and a decreasing trend in CHD risk after 1 year (trend p value = 0.18).
The incidence of stroke and peripheral arterial disease (PAD), secondary outcomes
of the trial, were also not different between randomized HT and placebo-treated
women
18, 19
.
Several smaller secondary prevention trials followed HERS. The Estrogen in the
Prevention of Reinfarction Trial (ESPRIT) included 1,017 postmenopausal women,
50
age 50-69 years who were survivors of a first heart attack and randomly assigned
subjects to either 2mg of unopposed estradiol valeriate daily or placebo
20
. After 2
years, there were no significant differences in the frequency of reinfarction or cardiac
death between the treatment groups (RR = 0.99, 95% CI: 0.74-1.41). All cause
mortality also did not differ by treatment assignment. Adherence to the estradiol
treatment at the end of 1
st
year dropped to 49% and at the end of the trial 43%
whereas in the placebo group the compliance was 69% and 63% at the two
corresponding time points. So, non-compliance was a concern in this trial, which
was particularly higher in the treatment group.
The Papworth HT Atherosclerosis Study (PHASE) was an unblinded trial in 255
women with angiographically proven heart disease
21
. The mean age of the study
participants was 66 years. Women were randomized to either a 17 β-estradiol patch (n
= 134) or no treatment (n = 121). In an intent-to-treat analysis approach, after an
average 31 months of follow-up, the rate of CHD (cardiac death, MI or unstable
angina) was slightly higher in HT group compared to non-treated women, which was
not statistically significant (event rate ratio = 1.29, 95% CI: 0.82-1.86, p = 0.24). In
the as-treated analysis, the event rate ratio was 1.49 (95% CI: 0.93-2.36, p = 0.11).
Women in the HT group had a higher, but not statistically significant, event rate than
the control group, only in the first 2 years after initiation of hormone use. Events per
patient year (%) for year 1 through 4 in the controls were 17.7, 12.0, 4.8, 3.5
51
respectively and among the HT group 23.7, 14.8, 4.3, 8.5 respectively. At the end of
the trial, there was a 60% withdrawal in the treatment group, and only 7% in the
placebo. In addition to the high drop out concerns, the final sample size of PHASE
ended up very small, less than what was required to statistically evaluate the effect of
HT on coronary events.
The Women’s Estrogen for Stroke Trial (WEST) was conducted among 664
postmenopausal women (mean age, 71 years) who recently had an ischemic stroke or
transient ischemic attack
22
. Women were randomized to placebo or 1 mg 17 β-
estradiol per day and followed for occurrence of stroke or death. After an average 2.8
years of follow-up, ET did not reduce the risk of stroke (RR = 1.1, 95% CI: 0.8-1.4)
or death (RR = 1.2, 95% CI: 0.8-1.8). A significant increase in the risk of stroke
(fatal or nonfatal) was observed at the first six months of the follow-up (RR = 2.3,
95% CI: 1.1-5.0) but was not observed in the remainder of follow-up.
The Estrogen Replacement and Atherosclerosis (ERA) trial enrolled 309
postmenopausal women diagnosed with at least one coronary artery stenosis of at
least 30%
23
. The trial end point was progression of coronary atherosclerosis assessed
by changes in the mean minimal diameter (MMD) of coronary artery from baseline
to follow-up. Follow-up angiographic data were available on 248 (80%) women. The
average age of the participants was 65.8 years at randomization. Women were
52
randomized either to 0.625mg of CEE alone (if hysterectomized), CEE with 2.5mg
of MPA daily (women with an intact uterus), or placebo. After an average follow-up
of 3.2 years, the changes in the MMD in the two HT groups were not significantly
different from that of the placebo group (p for difference=0.86). Although not
powered to detect differences in event rates, the rates of clinical cardiovascular
events such as MI, CHD death, revascularization, hospitalization for unstable angina
were also similar among treatment groups. Women randomized to estrogen alone
were significantly less adherent (74%) to their assigned medication than those
randomized to estrogen plus MPA (84%) or placebo (86%) (p for difference = 0.03).
The Women’s Angiographic Vitamin and Estrogen (WAVE) trial was a randomized
double-blind trial of 423 postmenopausal women with a mean age at entry of 65
years
24
. Eligible women had at least one 15% to 75% coronary artery stenosis
determined by coronary angiography at baseline. This trial had a 2 x 2 factorial
design where women were randomized to receive either 0.625 mg CEE daily
(women with an intact uterus received 2.5 mg MPA along with CEE) or placebo, and
400 IU of vitamin E plus 500 mg of vitamin C twice daily or placebo. Repeat
coronary angiograms were obtained in 306 (72%) women after 2.8 years. The
primary trial end point was coronary atherosclerosis progression measured as
annualized mean change in minimum lumen diameter from baseline to final
angiogram. The mean (SD) coronary progression was 0.047 (0.15) mm/year in
53
women treated with HT and 0.024 (0.15) mm/year with placebo (p for difference
0.17). Overall compliance to assigned treatment assessed by tablet/capsule counts at
each visit was relatively poor in the WAVE trial participants, an average of 67% in
the HT group and 70% in the placebo group.
The Women’s Estrogen-Progestin Lipid-Lowering Hormone Atherosclerosis
Regression Trial (WELLHART) evaluated the impact of 17 β-estradiol alone or
sequentially with MPA on the change of coronary artery stenosis measured by
quantitative coronary angiography
25
. A total of 226 postmenopausal women (mean
age 63.5 years), who had at least one coronary artery lesion occluding 30% or more
of the luminal diameter were randomized either to placebo, 1mg/day 17 β-estradiol
alone or 1 mg 17 β-estradiol daily plus 5mg MPA taken for 12 consecutive days
every month. A repeat angiogram was evaluated in 169 (75%) women at the end of
the intervention. After a median follow-up of 3.3 years, the mean change in the
percent stenosis of lesions in the coronary arteries was not significantly different
between the three treatment groups (p = 0.66). Compliance was assessed by pill
count as well as serum levels of estradiol. Over all compliance at the end of the trial
was above 92% in all the treatment groups. The authors concluded that 17 β-estradiol
either alone or along with MPA had no significant effect on progression of
atherosclerosis in older postmenopausal women with established coronary artery
atherosclerosis.
54
Trials with carotid atherosclerosis as the primary endpoint
Two secondary prevention trials used measures of carotid atherosclerosis as primary
outcomes. A secondary prevention trial in 321 postmenopausal women, 40 to 70
years of age, with increased IMT (>1mm) in 1 or more segments of the carotid
arteries, randomized women to placebo or one of two treatments arms both receiving
1mg/d 17 β-estradiol plus either continuous 0.025mg gestodene 12 days every month
or 0.025 gestodene for 12 days every 3 months
26
. Carotid artery intima-media
thickness (IMT) was measured at baseline and at the end of the trial in 3 segments:
the distal 10 mm of the common carotid artery, the carotid bifurcation from the
widening of the artery to the flow divider, and the proximal 10 mm of the internal
carotid artery. Follow-up IMT was available in 264 (82%) women. After 48 weeks of
follow-up, the mean maximum IMT in the carotid arteries increased by 0.02 mm in
the placebo, and 0.03 mm in both treatment groups; the difference was not
statistically significant (p > 0.20). The authors concluded that 48 weeks of HT was
not sufficient to slow atherosclerosis progression. This trial had a very high
compliance rate, over 95% in the two HT groups and 89% in the placebo. Analysis
restricted to compliant subjects yielded results very similar to the intent-to-treat
analysis. The trial was limited by a short intervention period of 48 weeks.
A substudy from HERS used progression of carotid atherosclerosis as a secondary
trial end point
27
. A total of 454 (16%) women of the 2763 original HERS participants
55
who agreed to have a carotid ultrasound at baseline were included in this sub-study.
Of those, 362 (80%) women had the follow-up scan at the end of the trial (average
follow-up 3.8 years). The average maximum IMT measured across 8 carotid artery
wall segments was the primary outcome of the trial. The IMT progression rate was
26 µm/y in women receiving estrogen plus progestin, and 31 µm/y in the placebo
women; the difference was not statistically significant (p = 0.44). However, IMT
progression at the bifurcation of the common carotid artery was slightly lower in the
HT group (p = 0.06).
2.1.2.2 Primary prevention trials
Trials with cardiac events as endpoint:
The Women’s Health Initiative (WHI) estrogen plus progestin (E+P) trial included
16,608 postmenopausal women aged 50-79 years with an intact uterus and 93%
without cardiovascular disease
28
. Women were randomized either to 0.625mg/d
CEE, plus 2.5 mg MPA per day or placebo. The trial was terminated after 5.2 years,
3.3 years ahead of the planned end of the trial, as the Data Safety Monitoring Board
judged that the overall risks exceeded benefits and there was no reason to expect a
future favorable effect on CVD outcomes if the trial was continued. Combined HT
was associated with a HR for CHD of 1.24 (95% CI: 0.97-1.60). A temporal trend
analysis showed that there was a substantial increase in the CHD risk within 1 year
56
of HT initiation (HR = 1.81, 95% CI: 1.09 to 3.01). A smaller and non-significant
excess risk was observed between 2 to 5 years of hormone use. In year 6 and beyond,
CHD risk was higher among the placebo group (HR = 0.70, 95% CI: 0.42-1.14). The
decreasing trend in the risk of CHD over time was statistically significant (trend p-
value = 0.02). In addition, a stratified analysis by time since menopause showed a
decreasing trend in CHD risk with shorter time since menopause although the
interaction was not statistically significant (p for interaction = 0.33). For women in
whom menopause had begun within 10 years, 10-19 years and 20 or more years of
randomization, the associated CHD hazard ratios due to HT use were 0.89, 1.22 and
1.71, respectively, the last being statistically significant.
WHI trial participants who had a hysterectomy (n = 10,739) received 0.625mg/d
CEE or placebo and followed for an average of 6.8 years in the WHI-E (estrogen
alone) arm of the trial
29
. The results of this unopposed estrogen trial showed no
higher risk of breast cancer (HR = 0.77, 95% CI: 0.59-1.01), or CHD (HR = 0.91,
0.75-1.12) in women receiving CEE compared to placebo. The CHD risk was
slightly elevated in the early follow-up periods and diminished over time with longer
HT use (HR = 1.16 in year 1, 1.20 in year 2, 0.89 in year 3, 0.79 in year 4, 1.28 in
year 5, 1.24 in year 6, and 0.74 in 7 or more years of HT use). The decreasing trend
in HRs associated with longer duration of HT was statistically significant (p = 0.02).
Stratified analysis by age at randomization indicated that women initiating HT at
57
older ages were at somewhat increased risk of CHD (trend p-value = 0.14).
Compared to women <50 years old at randomization, the HR (95% CI) associated
with CHD in women 50-59 years, 60-69 years, and >70 years were 0.56 (0.30-1.03),
0.92 (0.69 -1.23), and 1.04 (0.75-1.44) respectively. The risk of stroke was increased
in the entire CEE group (HR = 1.39, 95% CI: 1.10-1.77).
A later updated report of the WHI-E trial was published recently documented the
final, centrally-adjudicated
results for the primary efficacy outcome (myocardial
infarction
or coronary death), secondary coronary outcomes, and subgroup
analyses
30
. In this final analysis, the CHD risk did not differ between treatment
groups (HR = 0.95; 95% CI: 0.79-1.16). Among
women aged 50 to 59 years at
baseline, the HR for
cardiac events was 0.63 (95% CI,
0.36-1.08). In that age group,
coronary revascularization was
less frequent among women assigned to CEE (HR =
0.55; 95% CI: 0.35-0.86), as were several
composite outcomes, including the primary
outcome and coronary
revascularization (HR = 0.66; 95% CI: 0.44-0.97).
The
updated analysis did not find any significant trend in CHD risk over follow-up time
(p for trend = 0.14).
Trial with carotid atherosclerosis as endpoint:
The Estrogen in the Prevention of Atherosclerosis Trial (EPAT) was a randomized,
double blind, placebo-controlled trial designed to evaluate the impact of 17 β-
estradiol 1mg/d on carotid atherosclerosis progression in postmenopausal women
58
without clinically evident CVD
31
. A total of 222 women, on average 62 years old
(range, 46-80 years) were randomized to either placebo or 17 β-estradiol for 2 years.
The primary results of the trial showed a significant reduction in the progression of
carotid IMT in women receiving 17 β-estradiol compared to placebo. The rate of
carotid IMT progression was -.0017 mm/year and 0.0036 mm/year in women
randomized to the estradiol and placebo groups, respectively (p = 0.045). The
estrogen-related reduction of carotid IMT progression was observed in the subgroup
women not taking lipid-lowering medication. The treatment group difference in
carotid IMT progression was not significantly different among women taking lipid-
lowering medication (p-value for interaction = 0.007).
2.1.2.3 Summary and comments on clinical trials
Primary limitations of the clinical trials are: inability to acquire data on longer
duration of HT use, exclusion of women with menopausal symptoms because of
ethical reasons, and inclusion of much older women who had been on menopause for
a while with advanced CHD. None of the 9 secondary prevention trials, irrespective
of study end points and type of HT, demonstrated any significant impact of HT on
coronary heart disease. It is therefore possible that estrogen, either unopposed or
combined with progestin, may not help to reduce CHD risk in postmenopausal when
the underlying disease process is already advanced. Considering the impact of HT in
the primary prevention of CHD, HT may help reduce atherosclerosis progression in
59
postmenopausal women with no cardiovascular disease, i.e. when the underlying
atherosclerosis is at an earlier stage. Although the WHI E+P trial demonstrated an
overall increase in CHD risk, time trend analysis showed that the increased risk was
only apparent within the first years of HT initiation and CHD risk decreased with
longer use of combined estrogen and progestin (p = 0.02). The unopposed estrogen
intervention in the WHI trial did not show any significant impact of ET on CHD risk.
However, women between 50 to 60 years of age were more likely to benefit from ET
as this younger group of women receiving ET had a significantly lower risk of
several composite CHD outcomes.
Comparison of the EPAT and WHI-E trials generate the alternate hypothesis on the
effect of HT on CHD risk in postmenopausal women. A greater proportion of EPAT
women were less than 60 years old (47% in EPAT vs. 32% in WHI-E). In addition to
age at initiation, the type of preparation of estrogen can be a crucial factor. 17 β-
estradiol is the most potent form of estrogen that can readily bind with the estrogen
receptor and function immediately. In contrast, CEE is primarily composed of
sulfated ring B unsaturated estrogen that needs to be transformed to 17 β-estradiol to
be active
32
. It is also possible that postmenopausal estrogen therapy is protective for
atherosclerosis progression, but may not be as effective in protecting from CHD
events such as MI or cardiac death.
60
2.1.3 Discussion on the discrepancy between observational studies and randomized
trials
The bulk of observational studies established postmenopausal HT beneficial for
CHD while most of the clinical trial failed to confirm that. Several hypotheses have
been postulated to explain this outstanding divergence:
1) Healthy-user effect: The choice of healthy women to use HT was proposed as the
primary explanation by several researchers. Women who chose to use HT are more
educated, more health-conscious, have better CVD risk profile. Thereby, women
participating in the observational studies who used HT had lower risk of CHD. Due
to randomization of treatment assignment, clinical trials are not expected to have
such confounding bias. Few observational studies adjusted for socioeconomic status
in order to minimize this bias reporting a higher relative risk of CHD among HT
users
33
. Yet others continued to find that current HT use reduced CHD mortality and
incidence and had no effect on CHD risk suggesting that differences in
socioeconomic status could not completely explain the divergent findings from
observational studies. Moreover, it is interesting to note that results related to other
clinical outcomes such as breast cancer, colon cancer, even stroke were similar
between NHS and WHI, again suggesting that there is something more to HT use
and CHD risk that can explain the discrepancy
34
.
61
2) Underestimation of CHD events: Some proposed that in the observational studies
early CHD might have been ignored or not reported since women taking HT believed
that they were protected from CHD. This may not explain the difference because, if
women taking HT are more health conscious, they are more likely to report their
illness.
3) Effect of excluding silent MI: Given that approximately one third of MI are silent
with mild symptoms that are often unrecognized, excluding these events might lead
to reporting bias in the observational studies like NHS whereas WHI included silent
MI in their analysis. It is important to note that a subset analysis of WHI trial
excluding ‘silent MI cases’ revealed no changes in the results
35
.
4) Length of HT use: A number of observational studies including the most recent
studies from WHI reported that CHD risk decreased with increasing time from
initiation of E+P or E-alone over a period of 8.5 years
7, 8
. Follow-up time in most of
the clinical trials was less than 3 years, which may not be sufficient to see a
beneficial impact of HT on CHD. HERS, WHI-E+P, and WHI-E followed women
for more than 6 years. In all 3 of those trials there was significant decreasing trend in
CHD risk with longer duration of HT use. Of note, there was an increased risk within
the 1
st
year of HT use in most of the clinical trials.
62
5) Residual bias: To account for the residual bias, WHI researchers published a
combined analysis of WHI clinical trials and observational studies evaluating the
corresponding regimen of HT, either E+P or E-alone. In that report, CHD risk
patterns were not evident from E-alone or E+P observational study analyses but were
consistent with some modest increase in risk early after initiation of HT. It is
important to note that CHD risk was significantly lower in women initiating, E-alone
for >5 year (HR = 0.73, 95% CI: 0.61-0.84), and E+P for >5 years with a borderline
significance (HR = 0.83; 95% CI: 0.67-1.01).
6) Time of HT initiation: Accumulating evidence suggests that initiating HT at a
younger age around menopause could be the crucial factor in HT-CVD relationship.
Clinical trial participants are much older (mean age ranging from 62-71), whereas
observational study participants are younger and more likely to initiate HT around
menopause to alleviate menopausal symptoms. Again in younger women the
atherosclerotic process might be at an early stage. Thereby, vascular tissues could
respond better to HT compared to older age when the atherosclerosis is much more
advanced. This hypothesis is supported by a meta-analysis of 23 clinical trials
including 30,049 women contributing to 191,340 patient-years. In that report, CHD
risk was significantly lower (OR = 0.68; 95% CI: 0.48-0.96) in younger women <10
years from menopause using HT compared with HT non-users
36
. In older women no
significant association was observed. A prospective analysis of NHS data from 19-76
63
to 2000 showed that women initiating HT near menopause (within 10 years) had a
significantly reduced risk of CHD (RR = 0.66; 95% CI: 0.54-0.80 for E only, and
0.72; 0.56-0.92 in E+P)
37
. HT initiation near menopause hypothesis was also
supported in the combined WHI data analysis. For women age 50-59 at baseline,
when E-alone observational data were adjusted by using E+P clinical trial and E+P
observational data, HR estimate for more than 5 year since initiation of HT was more
precise in the E-alone observational study (HR = 0.41; 95% CI: 0.17-1.01) compared
to E-alone clinical trial (HR = 0.67; 95% CI: 0.29-1.54). Similarly CHD risk with
average 10-year use of E-alone was 0.58 (95% CI: 0.34-0.98) in E-alone
observational study, and 0.69 (95% CI=0.38-1.25) in E-alone clinical trial.
64
Table II-1. Summarizing the clinical trials
Trials Population Baseline
age mean
(range)
Intervention Follow-up
in years
Outcome Results
Secondary prevention:
Heart and Estrogen/
Progesterone
Replacement study
(HERS)
17
2763 women
with established
CHD
44-79
(66.7)
0.625 mg oral CEE
plus 2.5mg of
MPA or placebo
6.8 Primary: Non-
fatal MI or CHD
death
No significant difference
between treatment groups
RR = 0.97(95% CI: 0.82-
1.14)
Significant decreasing
trend in CHD risk with
increasing time since
initiation of HT (trend
p=0.009)
Estrogen in the
Prevention of
Reinfarction Trial
(ESPIRIT)
20
1017 women:
survivors of
heart attack
62.6(50-69) 2 mg estradiol
valereate or
placebo
2 Reinfarction or
cardiac death
No significant difference
between treatment groups
RR = 0.99(95% CI: 0.74-
1.41)
Women’s Estrogen for
Stroke Trial (WEST)
22
664 women with
recent ischemic
stroke or TIA
71 (46-91) 1 mg 17 β oral
estradiol/d or
placebo
2.8 Incidence of
stroke or death
No significant difference
in stroke or death between
the treatment groups (RR
= 1.1, 95% CI: 0.8-1.4)
Papworth HRT
Atherosclerosis Trial
(PHASE)
21
255 women with
established
CHD
66.5 Transdermal 17 β
estradiol alone or
with cyclic
norethindrone or
placebo
2.6 Cardiac death,
MI, or unstable
angina
No significant difference
between treatment group
RR = 1.49(95% CI: 0.93-
2.36)
Estrogen Replacement
and Atherosclerosis
(ERA) Trial
23
309 women with
established
CHD
65.8 (42-
80)
0.625mg of oral
CEE alone, or
0.625mg of oral
CEE and 2.5 mg
MPA or placebo
3.2 Primary:
Coronary artery
stenosis;
Secondary:
clinical events
No significant difference
in rates of change in
angiographic stenosis
between treatment groups
65
Table II-1. Summarizing the clinical trials (continued)
Trials Population Baseline
age mean
(range)
Intervention Follow-up
in years
Outcome Results
Secondary prevention:
Women’s Angiographic
Vitamin and Estrogen
(WAVE) Trial
24
423 women
with established
CHD
65 0.625mg of oral
CEE and 2.5 mg
MPA or placebo
2.8 Primary:
Coronary artery
stenosis;
Secondary: CVD
events
No significant difference
in angiographic
progression of coronary
atherosclerosis between
treatment groups (p>0.56)
Women’s Estrogen-
Progestin Lipid-
Lowering Hormone
Atherosclerosis
Regression Trial
(WELLHART)
25
226 women
with established
CHD
63.5 (48-
75)
1mg 17 β oral
estradiol alone or
1mg 17 β oral
estradiol plus 5mg
cyclic MPA or
placebo
3.3 Coronary artery
stenosis
No significant difference
in the coronary artery
percent stenosis
progression among the
three treatment groups
Postmenopausal
Hormone Replacement
Against Atherosclerosis
(PHOREA)
26
321 women
with increased
IMT in ≥1
segment of the
carotid artery
(40-70) 1mg 17 β oral
estradiol plus
continuous
0.025mg gestodene
or 1mg 17 β
estradiol plus
cyclic 0.025mg
gestodene or
placebo
0.9 Changes in the
carotid artery
IMT
No significant difference
in the mean maximum
carotid IMT among the 3
treatment groups
Heart and Estrogen/
Progesterone
Replacement study
(HERS)
321 women
with established
CHD
67 0.625 mg oral CEE
plus 2.5mg of
MPA or placebo
3.8 Progression of
carotid artery
IMT
No significant treatment
effect on IMT progression
(p = 0.44)
66
Table II-1. Summarizing the clinical trials (continued)
Trials Population Baseline
age mean
(range)
Intervention Follow-up
in years
Outcome Results
Estrogen in the
Prevention of
Atherosclerosis Trial
(EPAT)
31
222 women with
no clinical CHD
62.2
(46-80)
1mg 17 β oral
estradiol or
placebo
2 Progression of
carotid IMT
Significant reduction on
the progression of
carotid IMT in women
randomized to HT p =
0.046
Primary Prevention :
Women’s Health
Initiative (WHI)
estrogen plus progestin
trial
35
16,608 women,
93% with no
clinical CHD
63.3
(50-79)
0.625 mg oral CEE
plus 2.5mg of
MPA or placebo
5.2 Non-fatal MI or
CHD death
CHD HR = 1.24 (95%
CI: 1.00-1.54)
Time trend analysis:
significant increase
within the first year;
Significant decreasing
trend in HR over time
(p = 0.02)
Singificant increased
risk in women > 20 year
since menopause (HR =
1.71; 1.5-2.5)
Women’s Health
Initiative (WHI)
estrogen alone trial
30
10,739 women
with no clinical
CHD
63.6
(50-79)
0.625 mg oral CEE
or placebo
6.8 Non-fatal MI or
CHD death
No significant
difference in CHD risk
between treatment
group: HR = 0.91 (0.75-
1.12); Trend analysis:
significant decreasing
trend in CHD risk over
time (p = 0.02)
Age group analysis:
women <60 years HR =
0.56, 60-79 HR = 0.92,
>70 yrs HR = 1.0;
p for trend = 0.14
67
2.1.4 Impact of HT on CVD risk factors
A number of observational studies and clinical trials examining the effect of HT on
CVD risk in postmenopausal women have also evaluated the impact on HT on CVD
risk factors. Although the results of observational studies and clinical trials are
conflicting on CVD risk, reports of the impact of HT on CVD risk factors are
consistent. HT has a beneficial effect on several modifiable risk factors. A pooled
analysis of 248 prospective observational studies published up to the year 2000
revealed that all estrogen regimens raised HDL-cholesterol and lowered LDL- and
total cholesterol
38
. HT also reduced lipoprotein (a) in this pooled analysis as well as
other individual studies
39
. However, oral estrogens have been reported to raise serum
triglyceride levels
38, 40
.
The Postmenopausal Estrogen/Progestin Intervention (PEPI) Trial was a randomized,
double-blind, placebo-controlled multicenter trial. PEPI was designed to evaluate
differences in heart disease risk factors in postmenopausal women treated with
placebo, unopposed estrogen (0.625 mg/d of CEE) or one of three combined
estrogen/progestin regimens, which were CEE plus 10 mg/d of cyclic MPA for 12
d/month, CEE plus consecutive MPA 2.5 mg/d, or CEE plus cyclic micronized
progesterone 200mg/d for 12 d/mon
41
. A total of 875 healthy postmenopausal
women were randomized and followed for 3 years. The primary results showed that
women receiving active treatments had a 10-12% decrease in LDL-cholesterol and a
68
significant similar increase in HDL-cholesterol. Triglyceride concentrations
increased, while fasting/2 hour post-prandial glucose, and fasting insulin levels
decreased significantly in all active treatment arms compared with placebo. Oral
estrogen alone or in combination with MPA also reduced fibrinogen levels and
demonstrated a 25% reduction in lp(a)
42
.
Numerous relatively smaller hormone trials evaluating lipids and lipoproteins as
primary end points reported a beneficial impact of HT on these metabolic factors
consistent with the PEPI trial results
43-45
. Almost all of the clinical trials with
cardiovascular disease or atherosclerosis outcomes demonstrated a significant
decrease in LDL-cholesterol, with an increase in HDL-cholesterol and triglycerides
in women receiving any HT relative to placebo, irrespective of clinically evident
CVD among women participating in these trials.
The HERS trial also evaluated the effect of HT on fasting glucose and incident
diabetes and demonstrated that in women with CHD, HT reduced the incidence of
diabetes by 35% (HR = 0.65; 95% CI: 0.48-0.89)
46
. This finding was supported by
WHI that demonstrating a 21% reduction in the incidence of diabetes among women
receiving CEE plus MPA daily compared with placebo (HR = 0.79, 95% CI: 0.67-
0.93)
47
. WHI-E trial also demonstrated a lower incidence of diabetes in estrogen-
69
treated women (cumulative incidence rate = 8.3%) than placebo-treated women
(cumulative incidence rate = 9.3%) (HR = 0.88; 95% CI: 0.77-1.01; p = 0.07)
48
.
Studies have also evaluated the effect of HT on peripheral blood flow indicative of
an impact on vascular endothelial function. Hysterectomized women receiving
estradiol, either alone or in combination with MPA for 6 months showed decreased
peripheral flow velocity in the arteries at the wrist and ankle
49
. HT has also been
reported to increase postischemic vasodilation in postmenopausal women
50
. Another
study in postmenopausal women with CAD reported that HT with and without
lovastin, increased brachial artery flow-mediated vasodilation, particularly with HT
alone
51
. Hormone therapy with estradiol valeriate 1 mg/day for 3 months has also
been shown to improved vascular function by increasing serum nitric oxide levels in
30 Chinese women
52
. However, 17β-estradiol therapy for average 2 years did not
have any significant impact on serum NO level in EPAT women
53
. Reduction in
endothelial damage and oxidative stress by reducing serum homocysteine levels and
oxidation of LDL-cholesterol have been documented by estradiol therapy
54, 55
. Some
studies have also shown that the oxidation of LDL-C is reduced by short- and long-
term HT with 17 β-estradiol
56
. This indicates that estrogen may also act as an
antioxidant
57
. However, HT has been consistently found to increase serum levels of
highly sensitive C-reactive protein (hs-CRP) despite reduction in other inflammatory
70
markers such as soluble intercellular adhesion molecules (SICAM) and vascular cell
adhesion molecule (VCAM)
58-60
.
In summary, postmenopausal HT has been documented substantially to have
beneficial impact on metabolic cardiovascular risk factors such as LDL- and HDL-
cholesterol, glucose control, diabetes. Estradiol therapy also had antioxidant effect
by reducing LDL-C oxidation and anti-coagulant effect by reducing fibrinogen.
However, proportion of CVD risk effect of HT mediated by alteration of CVD risk
factors remains to be determined. According to a recent analysis from EPAT study,
we have shown that 30% of the effect of estradiol therapy on subclinical
atherosclerosis progression was explained by the beneficial impact on LDL- and
HDL-cholesterol
61
. Whereas, the beneficial effect of estradiol therapy on glucose,
insulin and glycosylated hemoglobin could not explain the effect of estradiol therapy
on subclinical atherosclerosis. Similar analysis of clinical trials showing null or
increased risk of CVD from HT involving adverse effect of HT on triglycerides, hs-
CRP and coagulation factors might help in understanding of their results.
2.1.5 Impact of HT on circulating levels of sex hormones
Exogenous hormone therapy induces significant changes in the circulating sex
hormone levels in postmenopausal women. A significant increase in serum estrone,
total bound estradiol concentration, and SHBG and a decrease in the percentage of
71
unbound estradiol is observed among women receiving any of the 3 HT including
conjugated equine estrogens (CEE; n = 37) or micronized estradiol (ME; n = 25) or
transdermal estradiol (TE; n = 24) for as short as 2 months
62
. Increases in SHBG
concentrations were 100% with CEE, 45% with ME and 12% with TE therapy.
Women receiving ME and TE therapy had similar and greater increases in serum
estradiol concentrations compared to women receiving CEE therapy, which is
explained by the fact that half of the estrogen in CEE being originated from estrone
sulfate.
To summarize, the effect of HT on serum concentration of sex hormones has been
evaluated by a limited number of studies, with very small sample size and short
duration of HT use. Although estrone, estradiol (total and free), and SHBG have
been consistently found to increase with HT, the effect of HT on serum androgen
concentrations are conflicting. Androgen concentrations do not decline to the same
degree as estrogens after menopause since the ovaries continue to produce androgens
and the adrenal source of androgens is also active. It is particularly important to
understand how HT, mostly containing estrogens with or without progestin, mediates
the concentrations of androgen. Sex hormone concentrations with different regimens
of HT should be studied in larger samples of women taking HT for longer periods of
time.
72
2.2 Endogenous sex hormones, CVD and atherosclerosis in postmenopausal
women
Despite the enormous body of work on exogenous sex hormones and CVD, research
involving serum levels of sex hormones and CVD is relatively sparse in women and
the results are conflicting. These relationships have been explored much more
extensively in men
63
.
2.2.1 Cardiovascular disease & mortality outcomes
Only a few prospective studies have been published evaluating the relationship
between endogenous sex hormones and death from cardiovascular and ischemic
heart disease in women. The Rancho Bernardo study was a population-based
prospective study in which 651 postmenopausal women not taking oral estrogen
were followed for 19 years
64
. Serum levels of estrone, total estradiol, bio-available
estradiol, androstenedione, and total and bio-available testosterone were measured at
the approximate middle of the study period and death from fatal cardiovascular and
ischemic heart disease was ascertained by death certificate. Age-adjusted mean
concentrations of the endogenous sex hormones were not significantly different
between women who died from CVD and those alive or who died from diseases
other than CVD. The authors concluded that neither estrogens nor androgens were
predictive of cardiovascular mortality in postmenopausal women. Another report
from the same study demonstrated that DHEAS also did not predict cardiovascular
73
death in postmenopausal women despite a direct association between higher DHEAS
and several major CVD risk factors including total cholesterol and blood pressure
64
.
Of note, low levels of DHEAS were significantly associated with an increased risk of
cardiovascular death in men
65-67
. One possible explanation for the null findings of
this study in women could be a lack of variability of estrogen levels in women after
menopause. Since estrogen levels are reduced dramatically after menopause, it is
possible that most of the women in this study were at the lower end of the
physiologic range. Furthermore, the greater levels and variability of the sex hormone
concentrations in pre- and perimenopausal women (which were not measured in this
study) have been shown to be stronger correlates of future CVD risk
68
.
Women with polycystic ovarian syndrome (PCOS) have hyperandrogenemia along
with insulin resistance and dyslipidemia
69
. Studies of CVD or CVD risk factors
among women with PCOS may reflect an association of CVD with excess androgen
levels. A prospective study of 786 PCOS women, 45 years or older, diagnosed with
PCOS between 1930 to 1979 and followed for 30 years on average, failed to
demonstrate significantly different mortality among PCOS patients compared to
national rates, (standardized mortality ratio (SMR) for CAD = 1.4, 95% CI: 0.8-
2.4))
70
.
74
In contrast to these null findings, lower levels of DHEAS (p < 0.01) and total
testosterone (p = 0.07) were significantly associated with increased risk of ischemic
heart disease mortality (IHD) among 120 postmenopausal women with diabetes
participating in the Wisconsin Epidemiologic Study of Diabetic Retinopathy
71
. The
results remained similar after controlling for other major predictors of IHD mortality
such as duration of diabetes, glycosylated hemoglobin, diuretic use and serum
creatinine. No such associations were observed among diabetic men (n = 123).
75
Table II-2. Studies evaluating the relationship between endogenous sex hormones and CVD/ atherosclerosis in postmenopausal women
First author, year N Age
Mean
(range)
Study design Hormones
studied
Endpoint Relationship
Barrett-
Conor,1995
72
651 healthy women ≥50 Prospective
cohort
8 years
E1, E2, bio
E2, A, T, bio
T
CVD mortality Null
Barrett-
Conor,1995
64
942 healthy women 65.2 Prospective
cohort
8 years
DHEA-S CVD mortality Null
Haffner SM,
1996
71
120 diabetic women;
40 cases and 80
controls
68
(60-76)
Case-control E1, E2, T,
free T,
DHEA-S,
SHBG
CVD mortality Lower levels of DHEA-S
and testosterone were
significant predictors of
CVD mortality
Rexrode KM,
2003
73
200 cases and 200
controls
63.1 Case-control,
nested in WHS
1
T, E2, FAI,
FEI, SHBG;
measured in
baseline
serum
samples
CVD events (non-
fatal MI, stroke,
CABG, CHD, stroke
death)
Lower levels of SHBG and
higher FAI were
significantly associated with
cardiovascular events
Phillips GB,
1997
74
60 women
undergoing coronary
angiography
69 Cross-sectional E2, T, free T,
DHEA-S,
SHBG
Maximum percentage
reduction in luminal
diameter of 4
coronary arteries
Free T was significantly
positively associated with
the degree of CAD
1
Women’s Health Study: a randomized, double-blind, placebo-controlled trial of low-dose aspirin and vitamine-E on prevention of CVD.
2
E1=estrone; E2=total estradiol; bio E2=bioavailable estradiol; A=androstenedione; DHEA-S=dehydroepiantrosterone sulphate; T=total testosterone;
bioT=bioavailable testosterone; FAI=free androgen index; FEI=free estrogen index; SHBG=sex hormone binding globulin; CIMT=carotid intima-media
thickness.
76
Table II-2. Studies evaluating the relationship between endogenous sex hormones and CVD/ atherosclerosis in postmenopausal women
(continued)
First author, year N Age
Mean
(range)
Study design Hormones
studied
Endpoint Relationship
Reinecke H,
2002
75
87 women
undergoing coronary
angiography
< 70
years
Cross-sectional T, DHEA-S,
SHBG
Any diameter
reduction or diffuse
lesion in any of the 8
coronary arteries
Low SHBG was
significantly associated with
CHD
Cauley JA, 1994
76
87 women
undergoing cardiac
catheterization
(51-81) Cross-sectional E1 ≥1 coronary artery
with 50% or more
occlusion
Low estrone level was not
associated with CAD.
Golden SH, 2001
77
182 cases and 182
controls
62
(45-64)
Cross-sectional E1, T, A,
DHEA-S,
SHBG
Cases:CIMT ≥ 95
th
percentile; Controls:
CIMT < 75
th
percentile
Higher T and SHBG were
inversely associated with
carotid atherosclerosis
Bernini GP,
1999
78
101 healthy women 46.7
(21-73)
Cross-sectional T, free T, A,
DHEA-S
Average CIMT A and free T were inversely
related to CIMT
1
Women’s Health Study: a randomized, double-blind, placebo-controlled trial of low-dose aspirin and vitamine-E on prevention of CVD.
2
E1=estrone; E2=total estradiol; bio E2=bioavailable estradiol; A=androstenedione; DHEA-S=dehydroepiantrosterone sulphate; T=total testosterone;
bioT=bioavailable testosterone; FAI=free androgen index; FEI=free estrogen index; SHBG=sex hormone binding globulin; CIMT=carotid intima-media
thickness.
77
A nested case-control study among the participants of the Women’s Health Study
included 200 postmenopausal women who developed CVD and 200 controls
matched on age, smoking and HT use
73
. A composite end point of CVD event
included first occurrence of nonfatal MI, coronary revascularization, nonfatal stroke,
coronary artery disease or stroke death. Sex hormones were measured in the serum
samples collected at the baseline visit of WHS. Among non HT users, (including
former and never users), cases had significantly higher median free androgen index
or FAI (calculated as the molar ratio of total testosterone/SHBG) and lower SHBG
levels than controls. After controlling for potential confounders, non HT users in the
lowest quartile of SHBG (32.4 mmol/L) had a 2.25 times (95% CI: 1.03-4.91)
greater risk for CVD relative to the highest quartile (>69.5mmol/L). There was also a
significant trend in CVD risk across FAI quartiles (p for trend = 0.03). However, the
associations of FAI and SHBG with CVD were not independent of BMI,
hypertension, diabetes and elevated cholesterol. Among current users of HT, no
association was observed between sex hormone levels or SHBG and CVD risk.
Estradiol levels were not associated with risk of CVD in either HT users or nonusers.
One potential limitation of this study was that the free androgen and free estrogen
levels were not calculated independently. Thus low SHBG is results in a higher free
androgen index and the positive association of FAI with CVD risk could be driven
by the inverse association of SHBG with CVD risk.
78
In summary, three epidemiologic studies to date have evaluated the relationship of
CVD mortality to serum concentrations of sex steroid hormones in postmenopausal
women. Endogenous levels of total estradiol or estrone were not associated with
CVD mortality. The impact of circulating levels of DHEA-S and total testosterone
on CVD mortality is conflicting. In postmenopausal women free from known CVD,
none of the endogenous sex hormones were associated with CVD mortality.
However, in diabetic postmenopausal women, DHEA-S and total testosterone were
significant inverse correlates of CVD mortality. Diabetic postmenopausal women
might have a different androgenic milieu than non-diabetic postmenopausal women,
which might have impacted CVD mortality differently than the general population.
Only one epidemiologic study examined the association between endogenous sex
hormones and cardiovascular disease events reporting lower levels of SHBG and
higher FAI significantly associated with cardiovascular events. In the population of
postmenopausal women not on hormone therapy, higher free testosterone and lower
SHBG levels were associated with CVD events but none of the endogenous sex
hormones were associated with cardiovascular mortality. More epidemiologic studies
are required to be conclusive on this issue.
2.2.2 Studies with coronary atherosclerosis outcomes
A cross-sectional study among 60 postmenopausal women undergoing coronary
angiography examined the relationship between serum sex hormones and the degree
79
of coronary artery disease measured by maximum percent reduction in luminal
diameter of coronary arteries
74
. Sex hormones were measured from the blood sample
drawn at the time of angiography. Adjusted for common CAD risk factors, free
testosterone (p < 0.008) was significantly positively associated with the degree of
CAD. The results remained similar when restricted to women without a previous
history of MI. No associations were observed between estradiol or SHBG and CAD.
Estradiol and SHBG are known to have favorable effects on total cholesterol,
systolic blood pressure, and insulin levels, factors that were included in the multiple
regression models as covariates along with estradiol and SHBG. Thus it is possible
that any existing association between estradiol and SHBG was adjusted out by
including the possible mediators of the association in the model. The authors did not
test a model not including the mediators in the model. Testosterone may have an
adverse effect on CAD through mechanisms other than its action on cholesterol and
insulin. The relatively small sample size is another limitation of the study. It is also
important to notice that the sample selected in this study had known or suspected
CAD.
In contrast to the study above, low plasma levels of SHBG were associated with an
increased risk of prevalent CAD in 87 consecutive postmenopausal women admitted
for coronary angiography or percutaneous transluminal coronary angioplasty
75
. Fifty-
five women were identified with CAD from the coronary angiogram, with CAD
80
defines as diameter reduction or diffuse lesion in any of the 8 coronary segments
evaluated. Total testosterone, DHEAS and SHBG were measured from blood
samples collected immediately prior to coronary angiography. A multivariate
stepwise logistic regression model showed that SHBG was independently and
inversely associated with the presence of CAD (OR = 0.98, 95% CI: 0.96 to 0.99, per
unit of SHBG) adjusted for age, smoking and apolipoprotein B, which were
independent risk factors for CAD. No association was found between total
testosterone and DHEAS and the risk of CAD. Estrogens and testosterone levels
were not measured in this study. A stepwise regression method was used to identify
the final model for association between sex hormones and CAD. The appropriateness
of using such an approach in epidemiologic studies is not universally accepted. The
authors did not identify the criteria for variables to enter and stay within the model.
The small sample of women is another limitation.
Serum estrone concentrations did not significantly differ between 62 CAD cases and
25 control women admitted for diagnostic cardiac catheterization
76
with CAD
defined as having at least one coronary artery with a 50% or more occlusion. All
control women had 0% to 24% occlusions of all coronary arteries. A difference of 6
pg/ml in the estrone levels was associated with 1.85 times increased risk of coronary
artery disease which was not statistically significant (95% CI: 0.60 to 5.2). The null
81
finding of this study could be an issue of inadequate sample size. Nonetheless, the
mean estrone levels also did not differ by the number of occluded vessels.
In summary, studies with coronary angiographic outcomes document no association
of estrogens with the presence or degree of coronary atherosclerosis. Free
testosterone may be directly and SHBG inversely associated with CAD. The cross-
sectional nature of these studies prevents inference regarding the temporality of the
associations of sex hormones with CAD. Another inherent limitation of studies with
angiographic end point is that the controls are not representative of the general
population. In these studies, control women had some degree of CVD or suspected
for which a coronary angiography was indicated. All of these studies suffered from
inadequate sample size. Since these limitations are inevitable in angiographic
studies, alternate methods of measuring coronary atherosclerosis such as coronary
calcification, should be considered for epidemiologic studies. Calcium in the
coronary arteries can be measured non-invasively using electron beam computed
tomography or helical computed tomography scan. Both of these methods have been
validated in epidemiologic studies. Coronary calcification correlates well with
angiographically-determined coronary arterial lumen reduction or stenosis
79, 80
. In
fact, there are evidence suggesting coronary calcium outperforms carotid artery
intima-media thickness as a noninvasive index of prevalent coronary artery
stenosis
81
. Coronary calcification is a significant predictor of CVD events
82, 83
. CAC
82
has also been shown to improve CHD prediction when added to the conventional
Framingham risk score
84
. Epidemiologic studies should be designed to evaluate the
impact of sex hormones and coronary calcification to be conclusive about these
associations.
2.2.3 Studies with carotid atherosclerosis outcome
Only a few studies have examined the associations between serum levels of sex
hormones and atherosclerosis in the carotid artery in postmenopausal women and
have conflicting results. A cross-sectional study from the Atherosclerosis Risk in
Communities (ARIC) cohort reported that higher levels of total testosterone and
SHBG were inversely related to the presence of elevated carotid atherosclerosis
suggesting their potential protective role in reducing atherosclerosis risk in
postmenopausal healthy women not taking HT
77
. Cases (n = 182) were defined as
CIMT ≥ the 95
th
percentile. Controls were frequency matched to cases on age and
ARIC center and had CIMT< the 75
th
percentile. Adjusted for cardiovascular risk
factors, postmenopausal women in the highest quartile of total testosterone (OR =
0.38, 95% CI: 0.20 to 0.74) and SHBG (OR = 0.48, 95% CI: 0.24 to 0.97) had
significantly lower risk of carotid atherosclerosis compared with women in the
highest quartile of the respective hormones. The risk of atherosclerosis was not
associated with increasing levels of estrone, androstenedione, DHEA-S, or the ratio
83
of total testosterone to SHBG (a marker for free testosterone). Estradiol was not
measured in this study.
Findings of the ARIC case-control study are somewhat supportive of another cross-
sectional study that reported that endogenous androgens favorably influenced carotid
atherosclerosis
78
. This study involved a small sample of both pre- (n = 53) and
postmenopausal (n = 48) women and evaluated the relationship between androgen
levels and carotid atherosclerosis. Significant inverse relationships were observed
between CIMT and free testosterone as well as androstenedione, independent of
cardiovascular risk factors. DHEA-S was also inversely associated with carotid
atherosclerosis, but this relationship was not independent of cardiovascular risk
factors and other androgens. SHBG was not measured in this study.
In contrast to the protective effect of androgens in relation to carotid atherosclerosis
in older women, women with PCOS are at higher risk of carotid atherosclerosis,
which suggests that hyperandrogenism increases the risk of atherosclerosis. A case-
control study including 125 white PCOS cases and 142 controls demonstrated that
PCOS cases had significantly higher mean CIMT than controls (0.78 ± 0.03 vs. 0.70
± 0.01 mm, p = 0.005)
85
. The difference remained significant after controlling for
age and BMI (p = 0.05). Other relatively smaller case-control studies also showed
that women with PCOS have an increased risk of subclinical atherosclerosis
86, 87
. A
84
case-control study of 75 PCOS cases and 55 age- and BMI- matched controls
demonstrated that PCOS was a strong predictor of carotid IMT adjusted for age,
BMI, and parental history of CVD
87
.
In summary, estrogens did not have any association with carotid atherosclerosis.
Although women with PCOS are at greater risk of having carotid atherosclerosis
suggesting atherogenic effect of androgens, association of serum levels of androgens
with carotid atherosclerosis is far from conclusive. Only 2 epidemiologic studies
conducted so far have given conflicting results. More studies are required to better
understand the association of serum sex hormone levels and atherosclerosis.
2.2.4 Critique and discussion of the studies on sex hormones and
CVD/atherosclerosis in postmenopausal women
All the epidemiologic studies, irrespective of cardiovascular outcome, failed to show
any association of serum estrogens with CVD or atherosclerosis. One possible
explanation for the null associations could be that, there is no true association
between serum estrogen concentrations and CVD. Given the large body of evidence
that premenopausal women are protected from CVD and postmenopausal estrogen
therapy reducing the risk of CVD by 30%-40%, it is conceivable that estrogen
concentrations in postmenopausal women may be below those required to prevent
CVD, and that premenopausal concentrations of estrogens are required for
85
cardioprotection. In premenopausal women, hypoestrogenemia (defined as estradiol
<50 pg/ml, follicle stimulating hormone < 10 IU/l and leutinizing hormone < 10
IU/l) has been linked to CAD
68
. The Women’s Ischemia Syndrome Evaluation
(WISE) study assessed serum levels of reproductive hormones in 95 premenopausal
women undergoing coronary angiography for suspected ischemic heart disease.
Women with 70% or more luminal diameter stenosis of at least one coronary artery
were defined as cases (n = 13). Compared to women who did not have CAD, total
estradiol, bioavailable estradiol and follicle stimulating hormone were significantly
lower among CAD cases (all p < 0.05). Adjusted for the common risk factors of
CAD, hypoestrogenemia was the most powerful predictor of the presence of
angiographic CAD (OR = 7.4, 95% CI: 1.7 to 33.3). Test if postmenopausal levels of
estrogens are inadequate to protect women from CVD/atherosclerosis, epidemiologic
studies should be designed to include women on as well as not on HT to increase the
variability of estrogen levels.
Another possible explanation for the null association of estrogens with CVD could
be inability to account for the within-subject variability of estrogens in
postmenopausal women. Of note, all the epidemiologic studies done to date to test
the impact of sex hormones on CVD/atherosclerosis have used only one
measurement of estrogens and other sex hormones. Single determinations of total
estradiol may not be sufficient to measure a woman’s true serum estradiol
86
concentration. In 77 postmenopausal women, the intraclass correlation coefficient
(ICC) of 2-3 measurement of total estradiol measured over 2-3 years was only 0.51
88
.
Another study evaluating the short term (4-week) and long-term (2-year) reliability
of serum sex hormones in 174 healthy postmenopausal women reported poor
reproducibility of total estradiol (ICC for short-term = 0.45 and long-term = 0.36)
89
.
Another epidemiologic study in participants from the NHS showed somewhat higher
reproducibility for serum estradiol (ICC = 0.68) based on 3 measurements taken over
3 years. However, this study included only 79 postmenopausal women
90
. Estrone, the
primary estrogen in postmenopausal women was measured more reliably over 4
weeks (ICC = 0.74) than over 2 years (ICC = 0.56) suggesting greater variability
over longer follow-up time
89
.
These data provide evidence of a great deal of intra-individual variability of estradiol
in postmenopausal women suggesting multiple measurements of estradiol are
required to better characterize a woman’s true estradiol level after menopause. In
terms of androgens, the reproducibility was remarkably high in all of these studies
(ICC ranging from 0.88 to 0.92 for total testosterone, 0.71 for free testosterone
DHEA-S 0.85 to 0.90) suggesting one single measurement of androgens may be
sufficient to measure the true androgenic profile of a postmenopausal women. The
reproducibility of SHBG was also high in NHS participants (ICC = 0.92).
87
Methods of sex hormone measurements should also be considered when interpreting
epidemiological studies. Conventional or indirect RIA following organic solvent
extraction and purification by celite column partition chromatography are considered
the ‘gold standard’ technique for sex hormone measurement because it is expensive,
time consuming and require expert technicians, direct RIA are preferred in
epidemiologic studies. In 5 out of 9 studies reviewed in this section (Table II-2),
methods other than the conventional RIA were used to measure sex hormones.
The reliability of commercially available direct radioimmunoassay (RIA) assay kits
versus the conventional RIA is an issue of debate. One report including 125
premenopausal and 134 postmenopausal women, age 18-75, showed that total
testosterone measured by direct RIA was lower (mean + SD=0.77 + 0.54 nmol/L)
than the conventional RIA (1.05 + 0.58 nmol/L). The difference in total testosterone
levels were similar between the 2 methods in pre- or postmenopausal women, with
or without HT
91
. Reproducibility of sex hormone measurements using 5 different
direct assay kits were compared with the ‘gold standard’ conventional RIA. The
reliability of direct versus conventional RIA was high for most of the kits tested for
total testosterone (r = 0.85), androstenedione, and estrone (r > 0.55), but was high
only for 2 of the 5 kits tested for total estradiol (r < 0.46)
92
. Direct RIA kits are
primarily designed for clinical use where the main interest is usually to identify
individuals with relatively extreme hormone levels. In clinical practice, blood sample
88
collection and volumes are generally sufficient for the use of relatively less sensitive
methods. However, in epidemiologic studies the aim is to identify subjects by
relative hormone levels within the non-pathologic range.
Furthermore, in epidemiologic studies blood samples collected are often of smaller
quantity and conditions under which samples are collected are often not fully
controlled. In conclusion, since postmenopausal levels of sex hormones, especially
estrogen, are low, assays must be sufficiently sensitive to measure hormone levels
with reasonable accuracy.
2.2.5 Endogenous sex hormones and risk factors for CVD/ atherosclerosis in
postmenopausal women
Studies evaluating the relationship between sex hormones and cardiovascular risk
factors are limited in women compared to men, and conflicting results have been
revealed regarding these associations. A detailed literature review on each of the sex
steroid hormones and the extensive number of CVD risk factors evaluated is beyond
the scope of this document. A summary of the associations from observational
studies focusing on endogenous sex hormones and key cardiovascular risk factors in
women is provided in Table II-3.
89
2.2.5.1 Serum estrogen concentrations and CVD risk factors
Relatively fewer studies are available on endogenous estrogen levels and risk factors
for CVD/ atherosclerosis compared to androgens and SHBG in postmenopausal
women. This is partly because estrogen levels in postmenopausal women are too low
to measure accurately. One epidemiologic study failed to demonstrate any significant
correlations of lipids and lipoproteins with either estrone or estradiol levels in
postmenopausal women
93
, whereas another relatively smaller study reported a
significant positive association of plasma estrone concentration with serum HDL-
cholesterol and an inverse association with serum triglycerides and VLDL-
cholesterol levels
94
.
Among postmenopausal women receiving ET, serum estradiol concentrations were
significantly associated with a beneficial lipid and lipoprotein profile
95
, which is
consistent with the large body of evidence from randomized hormone trials where
exogenous HT therapy had a significant favorable impact on metabolic risk factors
for CVD.
Recently, a large multiethnic study including 3,297 pre- and perimenopausal women
reported a significant inverse relationship between endogenous estradiol and serum
levels of LDL-cholesterol and triglycerides and a significant positive association
with HDL-cholesterol levels
96
. It is possible that endogenous estradiol concentrations
90
in postmenopausal women are too low to have a beneficial impact on metabolic risk
factors, whereas postmenopausal women receiving HT have much higher estradiol
concentrations, which enable them to experience similar benefits of estrogen levels
as premenopausal women. It has been suggested that an estradiol concentration of
more than 25 pg/ml is required to reduce total cholesterol, LDL-cholesterol, and
Apo-B, and greater than 15pg/ml to induce a significant rise in HDL-cholesterol
95
. It
is also possible that serum levels of estradiol do not completely reflect estradiol
concentrations at the tissue level, an issue that clearly needs further investigation.
2.2.5.2 Serum androgens and risk factors
Several cross-sectional studies have reported a positive association between higher
levels of testosterone and the presence of hypertension in postmenopausal women
74,
97-99
. Another consistent finding of serum testosterone in women is a positive
association with BMI and WHR
97, 100, 101
. Testosterone has also been linked to
increased total cholesterol and LDL-cholesterol levels in postmenopausal women
97,
102
. A recent cross-sectional study among 3,297 pre- and perimenopausal women
reported a strong and consistent association of free androgen index (calculated as the
ratio of SHBG to total testosterone) to a list of CV risk factors including higher
insulin, glucose, hemostatic and inflammatory markers, and an adverse lipid profile
96
. However, some studies report no association of testosterone with serum lipids,
triglycerides or carbohydrate metabolites
78, 103
.
91
Endogenous serum DHEA-S was associated with a less atherogenic lipid profile in
postmenopausal women in a single cross-sectional study
103
. However, most of the
epidemiologic studies report no association between DHEA-S and metabolic risk
factors of CVD such as cholesterol, triglycerides, glucose, insulin, lipoproteins, and
BMI
64, 78, 97
. Other than a possible positive association with triglyceride levels and
BMI
101, 104
, androstenedione in general is not associated with CVD risk factors in
postmenopausal women
78, 103
.
2.2.5.3 Serum SHBG concentrations and risk factors
Higher serum levels of SHBG have been consistently found to have beneficial
effects on several CVD risk factors in women. The most reported associations are
favorable effects on lipid profiles, glucose, insulin and BMI
96, 97, 101, 103, 105-107
.
Decreased SHBG is also associated with hyperinsulinemia and insulin resistance in
postmenopausal
105, 107
as well as pre- and perimenopausal women
96
. Very low serum
levels of SHBG are considered to be an indirect marker of female androgenicity
because it is associated with higher free androgen levels, as observed in women with
PCOS
75
.
Serum SHBG was inversely associated with prevalent non-insulin dependent
diabetes mellitus in older women participating in the San Antonio Heart Study
108
and
the Rancho Bernardo Heart and Chronic Disease Study
109
. Low SHBG has also been
92
linked to carotid atherosclerosis, coronary atherosclerosis as well as cardiovascular
events like MI and CV death in postmenopausal women
73, 75, 77
. However, the
mechanism of action of SHBG is still not well understood. Further research is
required as to whether low SHBG is merely a marker of hyperandrogenism or has
direct influences at the cellular level through receptors as suggested by some
researchers
110
.
2.2.5.4 Serum sex hormone concentrations and obesity
The relationship between sex hormone levels and BMI is very intriguing. High BMI
has been associated with cancer of several organ sites and diabetes mellitus,
presumably by modulating the synthesis and metabolism of endogenous hormones,
such as steroid hormones and insulin
101
. In postmenopausal women, androgens and
some precursors of estrogen are converted into estradiol and estrone in the adipose
tissue, which is the major source of estrogens in women after menopausause
111, 112
.
This peripheral conversion is not regulated by a feedback mechanism. Several cross-
sectional studies have shown that serum estrogen concentrations are directly
associated with BMI in a dose-dependent manner in postmenopausal women
92, 101
. A
positive association between estrogens and BMI was also observed in pre-
menopausal women but to a lesser extent, possibly because of the active feedback
mechanism
113
. In contrast to estrogens, SHBG has been consistently reported to be
inversely associated with BMI, in particular with waist to hip circumference, which
93
is an indicator of central or abdominal obesity
101, 114, 115
. Central obesity has been
linked to insulin resistance, and elevated insulin concentration in turn reduces SHBG
production in the liver
116, 117
. Low SHBG concentrations result in higher
concentrations of free estradiol and testosterone concentration in overweight and
obese women. However, estrogen concentration has not been looked at in relation to
central obesity per se in postmenopausal women, which may be inversely associated
with total estradiol concentration. In the midst of the current controversy regarding
HT and CVD, it is important to understand the modifying effect of obesity on serum
sex hormone concentration in women taking postmenopausal hormone therapy.
Gradual decreases in sex hormone levels with age may also interact with obesity to
impact serum sex hormone levels, an issue demanding attention in future research.
94
Table II-3. Association of endogenous sex hormones to the risk factors of CVD/atherosclerosis in
women.
Sex Hormones Cardiovascular risk factors Direction of
association
References
↑ Estradiol Blood pressure, systolic =
96, 102
Blood pressure, diastolic =
96, 102
Cholesterol, total =/ ↓
93-96, 118
LDL-cholesterol ↓/=
93-96, 102
HDL-cholesterol ↑/=
95, 96, 102
Lipoprotein, apo-A ↑
95
Lipoprotein, apo-B ↓
95
Lipoprotein, apo-E ↓
95
Triglycerides =/ ↓
94-96
BMI, WHR ↓/ ↑
96, 101
Insulin =
96
Glucose =
96
↑ Testosterone Blood pressure, systolic =/ ↑
74, 78, 98, 99, 119
Blood pressure, diastolic =/ ↑
78, 99, 119
Cholesterol, total =/ ↑
103, 119
LDL-cholesterol =/ ↑
78, 103
HDL-cholesterol =
78, 103
Lipoprotein, apo-A =
103
Lipoprotein, apo-B
Lp(a) =
103
Triglycerides =/ ↓/ ↑
96, 103, 104
BMI, WHR ↑
100, 101, 119
Insulin =/ ↓/ ↑
78, 96, 120
Glucose =/ ↑
78, 96
↑ DHEA-S Blood pressure, systolic =
78
Blood pressure, diastolic =
78
Cholesterol, total =
78, 103, 119
LDL-cholesterol =
78, 103, 119
HDL-cholesterol =/ ↑/ ↓
64, 78, 119
Lipoprotein, apo-A =
103
Lipoprotein, apo-B =
103
Lp(a) ↓
103
Triglycerides =
103, 119
BMI, WHR =/ ↓
64, 78
Insulin =
119
Glucose =
64, 78
Note: ’=’ indicates null results; ‘ ↑’ indicates direct association; ‘ ↓’ indicates inverse association
95
Table II-3. Association of endogenous sex hormones to the risk factors of CVD/ atherosclerosis in
women (continued).
Sex Hormones Cardiovascular risk factors Direction of
association
References
↑ Androstenedione Blood pressure, systolic =
78
Blood pressure, diastolic =
78
Cholesterol, total =
78, 103
LDL-cholesterol =
78, 103
HDL-cholesterol =
78, 103
Lipoprotein, apo-A =
103
Lipoprotein, apo-B =
103
Lp (a) =
103
Triglycerides =/ ↑
103, 104
BMI, WHR ↓/ ↑
78, 101
Insulin =
78
Glucose =
78
↑ SHBG Blood pressure, systolic =
105
Blood pressure, diastolic ↓/=
105, 119
Cholesterol, total =/ ↓
96, 103
LDL-cholesterol =/ ↓
96, 103
HDL-cholesterol ↑
96, 103, 105, 107, 119
Lipoprotein, apo-A =
103
Lipoprotein, apo-B =
103
Lp (a) =/ ↑
96, 103
Triglycerides =/ ↓
103, 105
BMI, WHR ↓
96, 101, 103, 106, 107,
119
Insulin ↓
96, 105, 107
Glucose ↓
96, 105
Note: ’=’ indicates null results; ‘ ↑’ indicates direct association; ‘ ↓’ indicates inverse association
2.2.5.5 Conclusion of chapter II: Issues to be discussed in future research
While higher levels of SHBG are consistently found to have a beneficial impact on
CVD risk factors, the association of estrogens and androgens are not conclusive. It is
important to understand these associations, which may eventually shed some light on
the relationship of sex hormones to CVD/atherosclerosis. In future research, several
issues need to be considered: 1) Measurement error in characterization of sex
96
hormones: Measurement error is a big concern in epidemiologic studies with sex
steroid hormones in postmenopausal women. In postmenopausal women, the sex
hormone levels are very low and are very difficult to quantify. This is particularly
true for estrogens. Another source of measurement error is the variability of a
women’s sex hormone status. One single determination of the sex hormones may not
be adequate to characterize a woman’s true sex hormone status. Again, it is estrogen
that has been documented to have the greatest variability. The existing null findings
of studies evaluating estrogens impact on CVD could be the result of such
measurement error.
2) Outcome of interest: Most of the CVD outcomes including hypertension and MI,
are the clinical complications of atherosclerosis. Research involving atherosclerosis
would have more preventive impact than studying other clinical CVD outcomes.
Thus relating sex hormones to atherosclerosis in postmenopausal women is crucially
important. It would be particularly interesting to relate sex hormones with
atherosclerosis progression over time, something not done to date.
3) Study design and analytic strategy: A large scale longitudinal study in
postmenopausal women with multiple measurements of sex hormones and CVD risk
factors over time can measure these associations reasonably accurately. Analysis
should be performed strategically to determine if sex hormones have an effect on
97
CVD by modifying the CVD risk factors. However, in initially evaluating the
general associations of sex hormones with CVD, the CVD risk factors that are
associated with sex hormones should not be included as covariates in the models
testing the relationship between sex hormones and CVD/atherosclerosis. Inclusion of
these mediators will attenuate the true association. Unfortunately, this has occurred
in some of the studies reviewed in this chapter.
5) Serum concentrations vs. tissue concentrations of sex hormones: Correlation of
serum sex hormone concentrations with tissue concentrations of sex hormones is not
really known. Serum concentrations may or may not reflect the tissue concentration
of sex hormones. Factors modifying the tissue availability of sex hormones from
circulation are an issue of interest, which has not been investigated thoroughly.
Future research on animal models can elucidate this issue.
In the midst of the current controversy regarding HT and CVD and other chronic
disease risk, the associations of serum sex hormones and CVD/ atherosclerosis and
related risk factors in postmenopausal women needs further attention. Given that the
EPAT trial showed that ET was effective in reducing subclinical atherosclerosis
progression in healthy women, it is important to evaluate the impact of serum sex
hormones and SHBG levels on subclinical atherosclerosis progression as well as
factors related to the early stages of atherosclerotic disease process
98
(e.g. inflammatory mediators, markers of oxidative stress at the level of the vascular
endothelium including serum glucose and other markers of diabetes, homocysteine,
antioxidation of LDL-cholesterol).
99
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postmenopausal estrogen therapy and coronary heart disease. N Engl J Med.
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6. Grodstein F, Manson JE, Colditz GA, et al. A prospective, observational
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discrepancy between observational studies and the Women's Health Initiative
clinical trial. Am J Epidemiol. Sep 1 2005;162(5):404-414.
8. Prentice RL, Langer RD, Stefanick ML, et al. Combined analysis of
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112
CHAPTER III. Relationship Between Serum Levels of Sex Hormones and
Progression of Subclinical Atherosclerosis in Postmenopausal Women
Roksana Karim, MBBS, MS
1
, Howard N. Hodis, MD
1,4
,Frank Z. Stanczyk, Ph.D
1,2
,
Roger A. Lobo, MD
3
, Wendy J. Mack, Ph.D
1,4
1
Department of Preventive Medicine,
2
Department of Obstetrics and Gynecology,
3
College of Physicians and Surgeons, Columbia University, New York
4
Atherosclerosis Research Unit, Department of Medicine, University of Southern
California
Correspondence: Wendy J. Mack, Department of Preventive Medicine, University
of Southern California, 1540 Alcazar St., CHP Suite # 222, Los Angeles, CA 90033
Funded by NIH RO1 AG-18798
First author’s surname: Karim
Running Title: Sex hormones, SHBG and subclinical atherosclerosis
113
Chapter III Abstract
Background: Postmenopausal hormone therapy (HT) has been examined
extensively in relation to coronary heart disease (CHD)/atherosclerosis. An
outstanding conflict exists between the results of observational studies and clinical
trials in this regard. Despite this fact, research relating serum levels of sex hormones
to CHD/atherosclerosis is sparse, and the results are far from conclusive.
Methods: We measured sex hormones in longitudinally-collected samples of 180
postmenopausal women, 91 randomized to 17 β-estradiol and 89 to placebo, in the
Estrogen in the Prevention of Atherosclerosis Trial (EPAT). Repeated measures of
sex hormones including total and free estradiol, estrone, total and free testosterone,
androstenedione, dehydroepiandrosterone (DHEA), and sex hormone binding
globulin (SHBG) were tested for an association with serially measured carotid artery
intima-media thickness (IMT). Carotid IMT was measured using B-mode ultrasound
every six months during the 2-year trial.
Results: Mixed effects models in the combined sample showed that changes in
serum estrone (p = 0.03), total estradiol (p = 0.01), free estradiol (p = 0.02), and
SHBG (p = 0.007) were significantly inversely associated with carotid IMT
progression. These associations remained significant while adjusted for age and
BMI. IMT progression was not associated with estrogens and SHBG after
controlling for LDL- and HDL-cholesterol. A significant association between total
testosterone and IMT progression remained after controlling for cholesterols. Among
114
estradiol-treated women, only SHBG (p = 0.046) was significantly inversely
associated with IMT progression. No sex hormones were associated with IMT
progression in the placebo-treated women.
Conclusion: Estrogen and SHBG may reduce the progression of subclinical
atherosclerosis in postmenopausal women. This association is partially mediated by
their beneficial effect on serum cholesterols. The inverse association between total
testosterone and subclinical atherosclerosis was not mediated by serum cholesterols.
Key Words: hormones, atherosclerosis, lipids
Introduction
The transition to menopause initiates a remarkable change in the endogenous sex
hormone milieu in women. Atrophy of the ovaries results in markedly decreased
production of 17 β-estradiol (estradiol) after menopause, while the production of
androgens from the adrenal cortex remains fairly steady
1
. Postmenopausal women
have greater morbidity and mortality from cardiovascular disease (CVD) compared
to premenopausal women
2, 3
. In addition to age, increased CVD may be partially
attributable to the endogenous sex hormone level changes initiated by menopause.
115
Several studies have examined the relationship between endogenous sex hormones
and atherosclerosis
4-6
, CVD
7, 8
, and mortality
9-11
in postmenopausal women,
revealing conflicting results. While some studies found that higher levels of
androgens and SHBG were associated with reduced level of atherosclerosis
5, 12
,
others found a positive association between testosterone and CVD risk
7, 8
.
To our knowledge, no epidemiologic study has demonstrated an association between
serum estrogen levels and atherosclerosis/CVD in postmenopausal women. It is
possible that the low levels of postmenopausal estrogen may not truly have any
influence on atherosclerosis or CVD, relative to premenopausal levels. Another
possible reason for null findings could be use of a single serum measurement of
hormones, which may not be adequate to account for the relatively high intra-
individual variation in postmenopausal estradiol levels
13
. The Estrogen in the
Prevention of Atherosclerosis Trial (EPAT) was a randomized, placebo-controlled
trial that showed estradiol treatment significantly reduced progression of carotid
atherosclerosis in postmenopausal women
14
. We used longitudinal data from EPAT
to evaluate changes in sex hormones in relation to progression of carotid artery IMT
in postmenopausal women. We also examined how estradiol therapy (ET) altered
levels of sex hormones and SHBG over 2 years, and whether estrogen, testosterone
and SHBG interacted with each other to exert an impact on subclinical
116
atherosclerosis progression. This is the first study to relate sex hormone dynamics to
subclinical atherosclerosis progression longitudinally.
Methods
The EPAT study design has been described elsewhere
14
. In brief, EPAT was a
randomized, double-blind, placebo-controlled clinical trial designed to evaluate the
impact of unopposed estradiol on the progression of atherosclerosis in
postmenopausal women who had no clinical evidence of cardiovascular disease. A
total of 222 women were randomized to either placebo or active treatment receiving
unopposed oral micronized estradiol (1mg/day). Women were eligible if their serum
estradiol level was <20 pg/ml, LDL-cholesterol level ≥130mg/dl, fasting blood
glucose level <200 mg/dl, and were not current smokers. Randomized participants
were followed with clinic visits every month for the first six months, then every
other month for the remainder of the 2-year trial period. Blood pressure, body
weight, and body mass index (BMI) were measured at each clinic visit. Blood
samples were collected after a fast of 8 hours in the morning at each visit. All study
participants gave written informed consent and the study was approved by the
Institutional Review Board of the University of Southern California.
117
Carotid Ultrasonography and Image Analysis
High resolution B-mode ultrasound images for IMT measurements of the right
common carotid artery were obtained, as previously described, at two pre-
randomization visits and every six months during the trial
14
. Average IMT was
measured over 70-100 points covering a one-centimeter length just distal to the
carotid artery bulb.
Laboratory Measurements
Sex hormone levels were measured at the end of the study from stored samples
frozen at -70
0
C. Serum levels of androstenedione, DHEA, testosterone, estrone and
estradiol were quantified by validated, previously described RIA methods
15, 16
.
Prior to RIA, steroids were extracted from serum with hexane:ethyl acetate (3:2).
Androstenedione, DHEA, and total testosterone were then separated by Celite
column partition chromatography using increasing concentrations of toluene in
trimethylpentane. Estrone and estradiol were separated in a similar fashion by use of
ethyl acetate in trimethylpentane. SHBG was quantified by direct immunoassays
using the Immulite analyzer (Diagnostic Products Corporation, Inglewood, CA).
Free testosterone was calculated using total testosterone and SHBG concentrations
and an assumed constant for albumin in a validated algorithm.
17, 18
Free estradiol
was calculated in a similar manner.
118
All the immunoassay methods were shown to be reliable. Specificity of the RIAs
was achieved by use of highly specific antisera and use of organic solvent extraction
and chromatography prior to quantification of the analytes. Assay accuracy was
established by demonstrating parallelism between measured concentrations of the
serially diluted analyte in serum with the corresponding standard curve. Intraassay
and interassay coefficients of variation ranged from 4 to 8% and 8 to 13%,
respectively. All assay methods were found to be sensitive. The sensitivity of an
RIA method was determined by the smallest amount of analyte that reduced the
number of counts per minute of the radiolabeled analyte at zero mass by 2 standard
deviations.
Fasting plasma total cholesterol (TC) and total triglyceride (TG) levels were
measured using an enzymatic method under the standardization program of the
Centers for Disease Control and Prevention. HDL-cholesterol levels were measured
after apolipoprotein B-containing lipoproteins were precipitated in whole plasma
with heparin manganese chloride. LDL-cholesterol levels were estimated using the
Friedewald equation.
Serum levels of fasting insulin were measured by direct RIA. Fasting serum glucose
levels were measured by the glucose oxidase technique using the Beckman GlucoseII
analyzer (Beckman Instruments, Brea CA). Hemoglobin A1C levels were measured
119
by high-pressure liquid chromatography (Bio-Rad Diamat, Bio-Rad Corp, Hercules
CA).
Statistical analysis
Baseline, average on-trial and changes from baseline to average on-trial levels of sex
hormones were compared between the treatment groups using independent t-tests.
Spearman correlation coefficients were used to assess the correlations among the
changes in the hormone levels.
Associations between the carotid artery IMT progression rate and changes in sex
hormone levels were tested using a mixed-effects model, where the serial follow-up
measures of carotid IMT were the dependent variable and the primary explanatory
variables in the model were follow-up time in years, changes in the serum hormone
levels and their interaction with follow-up time.
Changes in the sex hormone measures were modeled as continuous variables. The
average annual rate of IMT progression among women with no change from baseline
in sex hormones was estimated from the regression coefficient associated with the
main effect of the follow-up time. The rate of carotid IMT progression associated
with a per unit change in the sex hormone levels was estimated from the regression
coefficient associated with the interaction terms of change in sex hormone levels x
120
follow-up time. Each sex hormone was modeled separately for its association with
carotid IMT progression. Hormones were first modeled unadjusted for any factors.
Subsequent models included age at randomization and change in BMI (baseline to
ontrial) as covariates. Former smoking and alcohol intake were not significantly
associated with carotid IMT progression and including them in the multivariate
model did not alter the impact of sex hormones on carotid IMT progression.
Therefore, these variables were not included in the multivariate model.
Changes in lipids and carbohydrate-related factors were included in the models one
at a time in addition to age and BMI, to evaluate if these factors were significant
intermediate mediators in the relationship between serum levels of sex hormones and
SHBG and carotid IMT. Only those lipids or factors related to carbohydrate
metabolism that substantially (>10% change in the β-estimates) altered the
relationship between sex-hormones and IMT progression were included as
independent variables.
Analyses were also performed stratified by treatment groups and lipid-lowering
medication intake within each treatment group. Four women in the placebo group
who used exogenous estrogens during the trial were excluded from stratified analysis
performed among the placebo group.
121
To examine the patterns of sex hormone changes in relation to IMT progression, we
categorized the change in each sex hormone from baseline for each subject into 3
groups: increase, no change, or decrease. We used the within-subject SD (over
repeated measures) in the placebo group to define the change categories (increase ≥ 1
SD change; decrease ≤ 1 SD change, no change = between ± 1 SD). Categorical
hormone change variables were then tested in different combinations for an
association with CIMT progression using the mixed-effects model. Repeat measures
of sex hormones and serum lipids (HDL-C, LDL-C, and TG) were correlated using
generalized estimating equations (GEE). All analyses used SAS (version 8.01, Cary,
NC); 2-sided p-values are reported.
Results
Baseline and follow-up data on carotid IMT and serum levels of sex hormones and
SHBG were available on 180 women, 89 in the placebo and 91 in the estradiol
group. Table 3.1 details the baseline characteristics of the women included in this
analysis. The average (SD) age of the women was 61.5 (6.8) years and the majority
(61%) were non-Hispanic white. At baseline, the average (SD) carotid IMT was
763.5 (133.3) µm. Participants were on average overweight with an average (SD)
BMI of 28.8 (5.4) kg/m
2
. Only women with LDL-cholesterol >130 mg/dl were
122
included in the trial, by design, which is evident by the elevated baseline average
LDL-cholesterol level of 164.1 (27.9) mg/dl.
Table III.1. Baseline characteristics of EPAT women (n=180)*
Characteristics
Age 61.5 (6.8%)
†
Race
Non-Hispanic white 109 (61%)
African American 19 (11%)
Hispanic 35 (19%)
Others 17 (9%)
Smoking
Never smoker 93(52%)
Ex-smoker 87(48%)
Alcohol intake (at least weekly) 132(73%)
Carotid IMT(µm) 763.5 (133.3)
BMI (kg/m
2
) 28.8 (5.4)
Blood pressure (mmHg) 127/77 (15.4/8.7)
HDL-cholesterol (mg/dl) 53.8 (12.1)
LDL-cholesterol (mg/dl) 164.1 (27.9)
Glucose (mg/dl) 89.7 (11.9)
Insulin (µIU/ml) 9.7 (6.1)
*
Sample includes 180 women with baseline and at least one ontrial measure of carotid IMT and serum levels of
sex hormones
†
Mean (SD) or n(%)
Table III-2 compares the mean (SD) levels of sex hormones at baseline, on-trial and
on-trial changes from baseline between the treatment groups. Compared to placebo,
women randomized to estradiol had significant increases in total estradiol, free
estradiol, estrone and SHBG, and significant decreases in free testosterone,
androstenedione and DHEA levels (p-values < 0.0001). On-trial changes in total
testosterone did not differ between groups (p = 0.17).
123
Table III.2. Serum levels of sex hormones and SHBG by treatment group.
Hormones Placebo(n=89) Estradiol (n=91) p-value
*
Estrone (pg/ml)
Baseline 39.8(13.3)
†
47.0(31.2) 0.05
Follow-up 48.1(42.0) 310.3(167.2) <.0001
Change 8.4(41.9) 263.4(165.6) <.0001
Estradiol (pg/ml)
Baseline 19.1(5.3) 21.4(19.7) 0.27
Follow-up 14.7(6.3) 68.2(16.2) <.0001
Change 2.0(9.4) 46.8(28.0) <.0001
Free estradiol (pg/ml)
Baseline 0.5(0.2) 0.6(0.5) 0.25
Follow-up 0.6(0.2) 1.6(0.6) <.0001
Change 0.05(0.2) 0.9(0.7) <.0001
Total testosterone (ng/dl)
Baseline 22.0(9.1) 21.5(10.8) 0.73
Follow-up 22.3(9.0) 22.8(10.8) 0.72
Change 0.3(3.6) 1.3(6.2) 0.17
Free testosterone (pg/ml)
Baseline 4.0(1.7) 4.1(2.1) 0.93
Follow-up 4.1(1.8) 3.1(1.6) 0.0001
Change 0.05(0.9) -.9(1.4) <.0001
Androstenedione (pg/ml)
Baseline 535 (228) 544 (250) 0.09
Follow-up 522(189) 492 (180) <.0001
Change -13 (167) -51 (175) <.0001
DHEA (ng/ml)
Baseline 2.37(1.57) 2.23(1.45) 0.09
Follow-up 2.14(1.34) 1.77(0.78) <.0001
Change -0.23(0.98) -0.46(1.05) <.0001
SHBG (nmol/L)
Baseline 35.2(14.5) 35.2(18.5) 0.90
Follow-up 36.5(18.2) 58.2(23.6) <.0001
Change 1.2(12.9) 23.2(17.7) <.0001
*
P-values were from t-test for independent samples.
†
Mean (SD).
124
Table III.3. Mixed models relating changes in sex hormones/SHBG and carotid IMT progression.
Unadjusted Adjusted for age and
BMI
Adjusted for age, BMI,
LDL-C and HDL-C
On-trial Changes
in Hormones
*
Estimate
(SE)
p-value
*
Estimate
(SE)
p-value
*
Estimate
(SE)
p-value
Estrone (pg/ml) -0.018(0.01) 0.03 -0.018(0.01) 0.02
-.003(0.01) 0.73
Total E2 (pg/ml) -0.118(0.05) 0.01 -0.119(0.05) 0.01
-.01(0.05) 0.82
Free E2 (pg/ml) -4.97(2.17) 0.02 -4.95(2.18) 0.02
-.32(2.2) 0.88
Total T (ng/dl) -0.526(0.28) 0.06 -0.531(0.28) 0.06
-.51(0.25) 0.04
Free T (pg/ml) 0.384(1.1) 0.73 0.403(1.12) 0.72
-.72(1.04) 0.50
A4 (pg/ml) 0.005(0.01) 0.59 -0.005(0.01) 0.55
-.01(0.01) 0.32
DHEA (ng/ml) 0.234(1.4) 0.87 0.102(1.43) 0.94
-.62(1.34) 0.64
SHBG (nmol/L) -0.205(0.08) 0.007 -0.212(0.08) 0.005
-.08(0.08) 0.29
*IMT progression rate (µm/year) per unit change in the sex hormone levels
On-trial changes in the estrogen-related hormones, SHBG and total testosterone were
significantly positively correlated with each other (correlation coefficients range
r = 0.16 to 0.99; p-value range 0.02 to <0.0001). Changes in all androgen-related
hormones were also significantly positively correlated among each other (r range
from 0.21 to 0.89; p-value range from 0.005 to <0.0001). All changes in estrogens
and SHBG were inversely correlated with androgen-related hormones except for
total testosterone (range of r = -0.06 to –0.41; p-value = 0.41 to <.0001).
In the total sample (n = 180), model 1 showed that on-trial changes in estrone (p =
0.03), total estradiol (p = 0.01), free estradiol (p = 0.02), SHBG (p = 0.007), and total
testosterone (p = 0.06) were significantly inversely related to carotid IMT
125
progression (Table III-3). Changes in free testosterone, androstenedione and DHEA
did not have any significant association with carotid IMT progression.
Table III.4. Mixed models relating changes in the sex hormones/SHBG and carotid IMT progression,
stratified by treatment group.
*
Placebo women (n=4) who took exogenous estrogens during the trial were excluded
†
IMT progression rate (µm/year) per unit change in the sex hormone levels
Each model adjusted for age and BMI
In the total sample, models adjusted for age and changes in BMI showed that
changes in all of the estrogens (p = 0.01 for total estradiol, for others p = 0.02), total
testosterone (p = 0.06) and SHBG (p = 0.005) were significant correlates of carotid
IMT progression (Table III-3). Among women randomized to estradiol, only changes
in SHBG were significantly associated with IMT progression (p = 0.05) (Table III-
4). In the placebo group, total testosterone (p = 0.05) was the single significant
inverse correlate of IMT progression adjusted for age and changes in BMI.
Placebo (n=85)
*
Estradiol (n=91) On-trial Changes in Hormones
IMT progression
rate/year (SE)
†
p-value IMT progression
rate/year (SE)
†
p-value
Estrone (pg/ml) -.03(0.08) 0.75 -.10(0.06) 0.19
Total E2 (pg/ml) -.70(0.46) 0.13 -.09(0.07) 0.18
Free E2 (pg/ml) -22.9(15.8) 0.15 -3.29(2.9) 0.26
Total T (ng/dl) -1.23(0.64) 0.05 -.29(0.28) 0.30
Free T (pg/ml) -4.58(2.7) 0.09 0.61(1.2) 0.61
Androstenedione (pg/ml) 0.003(0.01) 0.83 -.02(0.01) 0.12
DHEA (ng/ml) 1.23(2.38) 0.60 -1.1(1.65) 0.51
SHBG (nmol/L) -.12(0.26) 0.65 -.20(0.10) 0.046
126
Stratified analysis by lipid-lowering medication intake group did not reveal any
significant interaction between any of the sex hormones and treatment group and
lipid-lowering medication intake (all interaction p-values > 0.10).
The addition of LDL-cholesterol and HDL-cholesterol as independent variables in
the models substantially attenuated the relationship between IMT progression and
levels of estrogens and SHBG (Table III-3). Total testosterone remained significantly
inversely associated with carotid IMT progression after controlling for LDL- and
HDL-cholesterol (p = 0.05, Table III-3). Adjustment for fasting glucose and insulin
levels did not alter the estimates of association of sex hormones and SHBG with
IMT progression.
Despite increased free estradiol, women with no change in SHBG and free
testosterone had an average progression in carotid IMT over the trial ((Mean (SE))
IMT progression rate 8.53 (4.72) µm/year)) (Figure III-1). Women with increased
free estradiol and SHBG, but no change or increased free testosterone had some
reduction in CIMT progression ((Mean (SE) IMT progression rate -3.95 (3.03)
µm/year). However, women with increased free estradiol and SHBG and decreased
free testosterone had the largest reduction in carotid IMT progression ((Mean (SE)
IMT progression rate -5.45 (2.77) µm/year). The decreasing trend in IMT
progression rate across the 3 groups was statistically significant (p = 0.03).
127
Figure III-1. Change in sex hormones combined with ET and IMT progression.
8.53
-3.95
-5.45
-8
-6
-4
-2
0
2
4
6
8
10
AB C
Carotid IMT progression (um/year)
A = free estradiol level increased with no change in sex hormone binding globulin (SHBG) and free testosterone
B = free estradiol level and SHBG levels increased with no change in free testosterone
C = free estradiol level and SHBG levels increased with lowering of free testosterone
For each subject, the change in each sex hormone from baseline was categorized using the within-subject SD
(over repeated measures) in the placebo group (up= > 1 SD change; down= < 1 SD change, no change= between
± 1 SD).
Estrogen, SHBG, and total testosterone were significantly inversely associated with
LDL-C and positively related to HDL-C (Table III-5). In contrast, free testosterone
and DHEA were significantly positively related to LDL-C and inversely associated
with HDL-C. A moderate positive relationship was observed between estrogens and
TG.
128
Table III.5. Relationship of serum sex hormone/SHBG levels to serum lipids and triglycerides.
Sex Hormones
LDL-C (mg/dl)
HDL-C (mg/dl)
TG (mg/dl)
β-estimate
(SE) p-value
β-estimate
(SE) p-value
β-estimate
(SE) p-value
Estrone (pg/ml) -.06(0.01) <.0001 0.02(0.002) <.0001 0.02(0.01) 0.10
Total E2(pg/ml) -.34(0.03) <.0001 0.09(0.01) <.0001 0.13(0.08) 0.10
Free E2(pg/ml) -14.1(1.4) <.0001 3.82(0.57) <.0001 5.74(3.1) 0.06
Total T (ng/dl) -.12(0.13) 0.34 0.11(0.06) 0.07 -.40(0.36) 0.28
Free T (pg/ml) 2.36(0.77) 0.002 -1.36(0.34) <.0001 0.20(1.6) 0.93
A4 (pg/ml) 0.001(0.01) 0.83 -.007(0.002) 0.0005 0.004(0.01) 0.79
DHEA (ng/ml) 2.5(0.82) 0.002 -1.58(0.38) <.0001 -1.15(1.8) 0.52
SHBG (nmol/L) -.41(0.05) <.0001 0.15(0.02) <.0001 -.10(0.13) 0.44
Note: GEE models adjusted for age, BMI, and treatment group
When changes in SHBG (p = 0.16) and total estradiol (p = 0.47) were included in a
single model, neither had any significant association with carotid IMT progression.
However, SHBG changes (p = 0.01) still had a significant impact on IMT
progression after adjusting for total testosterone (p = 0.17). Total estradiol (p = 0.02)
changes were significantly inversely associated with IMT progression after
controlling for changes in total testosterone (p = 0.09). None showed any significant
association with carotid progression when total estradiol (p = 0.41), total testosterone
(p = 0.15) and SHBG (p = 0.27) were tested in a single multivariate model (data not
shown).
129
Discussion
Our results suggest that changes in estrogens, total testosterone, and SHBG over 2
years in a combined sample of postmenopausal women with and without ET are
significantly associated with reduced progression of carotid IMT progression after
controlling for age and BMI. Possible mechanisms for these associations could be
modulation of atherosclerosis risk factors including: increasing HDL-cholesterol and
insulin sensitivity and decreasing LDL-cholesterol, glucose, insulin and HbA1C. Our
data support the mediating role of lipids but not carbohydrate related factors in this
association.
Although lower circulating estrogens have been found to be significantly associated
with more severe angiographically-determined coronary artery disease in
premenopausal women
19
, no study has been able to find such a relationship in
postmenopausal women
20
. Two prospective studies investigating the impact of sex
hormones on cardiovascular mortality did not find any of the sex hormones to be a
significant predictor of cardiovascular death in postmenopausal women
9, 10
. This
null finding may be in part explained by lack of prospective data measuring serum
estrogens more than one time, since postmenopausal estradiol levels show relatively
high intraindividual variation
13
. In addition, the low serum estrogen levels (<25
pg/ml) in postmenopausal women result in lower interindividual variability in these
physiological levels than evident in premenopausal women, making it difficult to
130
determine relationships between estrogen and atherosclerosis/CVD. Our data support
this fact as we did not observe any effect of estrogens on IMT progression among the
placebo women (although the regression estimates indicate an inverse association,
Table III-4).
Most of the previous studies evaluating the relationship between estrogens and
cardiovascular disease in postmenopausal women excluded women taking HT. A
nested case-control study performed among women in the Women’s Health Study
included both HT users and non-users to examine the relationship between sex
hormone levels and risk of cardiovascular events
8
. Results were reported stratified
by HT use showing no association between estradiol and cardiovascular events in
either of the groups, which is consistent with the null finding in the placebo and
estradiol groups of our study. However, the authors did not report results in the
combined population, which could depict the impact of a wide range of estrogen
levels (physiological and pharmacological) on CVD.
In addition to estrogens, our results indicate that total testosterone and SHBG are
also inversely related to progression of carotid atherosclerosis. This finding is
consistent with the existing literature. A case-control study from the Atherosclerosis
Risk in Communities cohort reported that postmenopausal women (not on HT) in the
highest quartiles of total testosterone and SHBG had significantly lower odds of
131
atherosclerosis measured by a single carotid artery IMT>95
th
percentile adjusted for
a multitude of cardiovascular risk factors
4
. The inverse association between SHBG
and CHD/ CAD among postmenopausal women has also been reported
5, 8
. At
physiologic concentrations, total testosterone has been found to be protective against
carotid atherosclerosis and cardiovascular disease in postmenopausal women
12, 21
.
Our longitudinal results extend these indicate that a higher total testosterone level is
protective against subclinical atherosclerosis progression at physiologic
concentrations (in the placebo group) as well as in the total sample, half of whom
were on HT. This association was independent of age, BMI, HDL- and LDL-
cholesterol (p = 0.04, Table III-3).
Total testosterone also showed a beneficial association with LDL-C and HDL-
cholesterol whereas free testosterone had a detrimental association with serum
cholesterol (Table III-5). It is interesting to note that at least two epidemiologic
studies in postmenopausal women with established cardiovascular disease found a
positive association between free testosterone and angiographically-determined
coronary artery disease
7
8
. Although we did not observe any significant association
of free testosterone with IMT progression, we did observe a diverging relationship of
total vs. free testosterone on serum lipids. It is known that unbound or free
testosterone is the functionally active form that can bind with the androgen receptor
whereas the bound form is not functionally active. It is conceivable that higher levels
132
of total testosterone, 98% of which is bound with SHBG or albumin, are an indicator
of increased SHBG concentration. SHBG was significantly inversely correlated with
free testosterone concentration as well as carotid IMT progression and had a
beneficial association with serum cholesterols. In support of this hypothesis, we
found that the inverse association between total testosterone and carotid IMT
progression was not independent of SHBG, whereas SHBG remained significantly
inversely related with IMT progression when adjusted for total testosterone (data not
shown).
SHBG regulates the concentration of bioavailable estrogens and androgens by
binding with testosterone and estradiol proportionately
22
. As exogenous estrogen
induces SHBG production, evidenced in this study and by others
23
, and testosterone
binds to SHBG with a greater affinity than estrogen, there may be a relative
reduction in the concentration of free testosterone. Therefore, any detrimental effect
of free testosterone on cardiovascular disease and/or risk factors, as reported by some
investigators
7, 8
, may also be reduced. Our results support this hypothesis, as among
the women receiving estradiol, free testosterone levels were significantly decreased
(Table III-2).
Our results point toward interactive dynamics of estrogen, testosterone and SHBG.
To explore this concept, we modeled the changes in these three components of the
133
sex hormone milieu among estradiol-treated women. We found that women who had
increased free estradiol and SHBG and decreased free testosterone showed the
largest regression in carotid IMT compared to women who had increased free
estradiol only (Figure III-1). There was a significant trend (p = 0.03) in the reduction
of IMT progression among the three groups of women shown in Figure III-1.
However, the interaction term (free estradiol x SHBG x free testosterone) was not
statistically significant (p = 0.17), with small sample size being an issue in this
analysis.
Cardiovascular effects of estrogen can be classified as indirect and direct
24
. Indirect
effects include increased HDL and reduced LDL, whereas direct effects include
genomic and non-genomic mechanisms. In this study we only measured the indirect
effects. Multivariate models including LDL-cholesterol and HDL-cholesterol
attenuated the relationship between serum levels of estrogens, SHBG and carotid
IMT progression (Table III-3) indicating the effect of estrogen and SHBG levels on
carotid IMT progression is partly mediated by alteration in serum lipid levels,
particularly LDL-cholesterol and HDL-cholesterol (Table III-3). These results
support our previous finding that reduction in carotid atherosclerosis progression
among women treated with estradiol in EPAT was explained by decreases in LDL-
cholesterol and increases in HDL-cholesterol
25
. In that analysis, we also observed
that alterations in glucose and insulin did not explain the beneficial effect of estradiol
134
on subclinical atherosclerosis progression despite significant reduction in these
metabolites among women randomized to estradiol treatment. These carbohydrate-
related factors also did not play a role in the effect of serum sex hormone levels on
subclinical atherosclerosis progression in the current analysis.
The most important strength of our study is the longitudinal data collection with
serial measurements of sex hormones and carotid IMT over 2 years. The
intraindividual variability of the sex hormone levels, particularly estrogens, are
expected to be better captured by multiple measurements over time than a single
measurement. According to our data, among women not taking exogenous estradiol,
within-subject variability of total estradiol over the 2-year trial was 39% and free
estradiol was 33%, whereas that of total testosterone and free testosterone was 15%
and 18% respectively. Among women taking exogenous estradiol, the within-subject
variability of total estradiol, free estradiol, total testosterone and free testosterone
was 36%, 37%, 18% and 25% respectively. Estrogen concentrations had much
greater variability than testosterone, indicating that a single measurement of estrogen
in postmenopausal women may not be sufficient for epidemiologic studies. These
data are consistent with a previous report showing lower reliability for estradiol
compared to testosterone
13, 26
. As half of our study participants were treated with
estradiol, we were able to observe the influence of sex steroid hormones and SHBG
on subclinical atherosclerosis at pharmacologic levels (active treatment group) as
135
well as physiologic levels (placebo treated group). Combining these two groups, we
also had the capability to study the effect of a wide range of sex hormone levels on
atherosclerosis progression.
One limitation of the analysis is that 62% of the women were on lipid-lowering
medication. However, stratified analysis by lipid-lowering medication intake within
each treatment group did not show significant variation in the sex-hormones and
atherosclerosis association (all p-values for interactions > 0.10). However, the
relatively small sample size to detect such interactions is another limitation of this
analysis. The power was particularly limited in stratified analysis by treatment group
and in analysis of the interaction of changes in sex hormones and SHBG among the
estradiol-treated women. These data need to be reproduced in a larger group of
women.
The current uncertainty regarding the effect of hormone therapy on cardiovascular
disease (CVD) has underscored the importance of research evaluating the association
between serum sex hormone levels and atherosclerosis in postmenopausal women.
To our knowledge, no prior study has used multiple measurements of sex hormones,
SHBG and carotid IMT to examine their relationship over time in postmenopausal
women. We examined an extensive panel of sex steroid hormones for their
136
association with subclinical atherosclerosis. Further longitudinal studies are required
to support and extend the findings of this study.
137
Chapter III References
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year prospective population-based study. The Melbourne Women's Midlife
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140
CHAPTER IV. Circulating Levels of Sex Hormones, Markers of Inflammation,
and Proinflammatory Factors in Postmenopausal Women
Roksana Karim, MD, MS
1
, Frank Z. Stanczyk, PhD
1,2
, Howard N. Hodis, MD
1,3
,
Mary Cushman, MD
4
, Roger A. Lobo
5,
Juliana Hwang PhD
6
, Jacob Selhub PhD
7
,
Wendy J. Mack, PhD
1,3
1
Department of Preventive Medicine,
2
Department of Obstetrics and Gynecology,
3
Atherosclerosis Research Unit, Department of Medicine,
6
Department of Molecular
Pharmacology and Toxicology, University of Southern California;
4
University of Vermont, Colchester, Vermont;
5
College of Physicians and Surgeons, Columbia University, New York, NY;
7
Human Nutrition Research Center on Aging at Tufts University, Boston,
Massachusetts.
Correspondence: Wendy J. Mack, Department of Preventive Medicine, University
of Southern California, 1540 Alcazar St., CHP Suite # 222, Los Angeles, CA 90033
Funded by NIH RO1 AG-18798
First author’s surname: Karim
Running Title: Sex hormones, SHBG and inflammatory markers
Key Words: estrogens, androgens, CRP, sICAM, LDL-oxidation
141
Chapter IV Abstract
Background: The incidence of CHD in postmenopausal women is significantly
higher than that of premenopausal women of the same age suggesting a
cardioprotective effect of endogenous sex hormones, particularly estrogen.
Inflammation is a critical step of atherosclerosis and markers of inflammation rise in
women after menopause. Only a limited number of studies evaluated the relationship
of estrogens and other sex hormones with markers of inflammation and all of those
studies were cross-section.
Methods: We used longitudinal data from Estrogen in the Prevention of
Atherosclerosis Trial where sex hormones, markers of inflammation (hs-CRP and
sICAM-1), and homocysteine were measured at the beginning and every six months
during the two-year follow-up of the study. Linear regression models with
generalized estimating equations approach were used to analyze the data.
Results: Adjusted for age, BMI, and treatment group, homocysteine was significantly
inversely associated with estrone (p=0.001), total estradiol, (p=0.0006), and free
estradiol (p=0.001) and directly associated with sICAM-1 (p<.0001). A significant
positive association was observed between sICAM-1 and free testosterone (p=0.03).
Total testosterone, A4 and DHEA were not associated with sICAM-1. In the total
sample, hs-CRP was significantly positively associated with estrone (p=0.0001),
142
total E2 (p<.0001), free E2 (p=0.0002) and SHBG (p=0.02), adjusted for age, BMI
and treatment group (Table 5). A significant inverse association was observed
between hs-CRP and DHEA (p=0.006). The remaining androgens were not
associated with hs-CRP. The association of hs-CRP with SHBG was significantly
different between the treatment groups (p-for interaction=0.0003).
Conclusion: Estrogens and SHBG are beneficially associated with markers of
inflammation and homocysteine. In postmenopausal women without cardiovascular
symptoms, higher circulating levels of estrogens and SHBG may reduce the extent of
inflammation, which is a critical step in atherosclerosis progression.
Introduction
Inflammation is a critical step in the process of atherosclerosis. Early stages of
atherosclerosis involve an inflammatory process consisting of migration and
deposition of leukocytes and monocytes in the subendothelial matrix. Adhesion
molecules such as intercellular adhesion molecule-1 (ICAM-1), vascular cell
adhesion molecule-1 (VCAM-1), E-selectin, and P-selectin mediate adhesion and
migration of leukocytes into the arterial wall.
1
Circulating adhesion molecules are
positively associated with levels of atherosclerosis
2, 3
and risk for cardiovascular
events.
4, 5
143
Coronary heart disease (CHD) is rare in women before menopause compared to men
of the same age. The incidence of CHD in postmenopausal women is significantly
higher than that of premenopausal women of the same age
6, 7
suggesting a
cardioprotective effect of endogenous sex hormones, particularly estrogen. In a
cross-sectional study, circulating inflammatory markers including ICAM-1, VCAM-
1, E- and P-selectins were significantly higher in postmenopausal women not on
hormone therapy (HT) compared with premenopausal women controlling for age and
BMI.
8
The impact of postmenopausal HT on markers of vascular inflammation such as
adhesion molecules and cytokines have been studied by a number of investigators
revealing an overall decrease in vascular inflammation among HT users.
9-12
In
contrast, high sensitivity C-reactive protein (hs-CRP), a marker of generalized
inflammation increased with oral HT.
11, 13, 14
It is unknown how serum
concentrations of estrogens, androgens, and SHBG correlate with markers of
inflammation in postmenopausal women.
Homocysteine, a metabolite of methionine, is considered a potent proinflammatory
substance. Levels of serum homocysteine are positively associated with increased
circulating levels of adhesion molecules, coagulation markers, and atherosclerosis.
15-
18
In the atherosclerosis process, endothelial dysfunction is followed by migration
144
and oxidation of low density lipoprotein cholesterol (LDL-C) in the subendothelial
matrix.
19
Oxidized LDL-C has been shown to stimulate inflammation in the vascular
wall.
20
The antioxidant properties of estrogen have been demonstrated with respect
to hormone therapy in clinical as well as in vivo studies.
21, 22
The association of
serum homocysteine levels or LDL-oxidation with sex hormone concentrations has
not been examined.
We used data from the Estrogen in the Prevention of Atherosclerosis Trial (EPAT) to
evaluate in a post hoc analysis the association of serum sex hormone levels,
including estrogens, androgens, and sex hormone binding globulin (SHBG), with
markers of inflammation and pro-inflammatory factors (homocysteine and LDL-
oxidation) in postmenopausal women. We hypothesized that an effect of sex
hormones on markers of inflammation is partly mediated by their effect on the
proinflammatory agents. In EPAT, serum levels of sex hormones, hs-CRP and
soluble intercellular adhesion molecule (sICAM), LDL-oxidation, and homocysteine
were measured longitudinally over the 2-year trial. We evaluated these associations
in postmenopausal women with sex hormone at physiologic and pharmacologic
levels. To our knowledge, this is the first study to relate sex hormone concentrations
to inflammatory markers and pro-inflammatory factors longitudinally.
145
Methods
The EPAT study design has been described elsewhere.
23
In brief, EPAT was a
randomized, double-blind, placebo-controlled clinical trial designed to evaluate the
impact of unopposed 17 β-estradiol on subclinical atherosclerosis in postmenopausal
women who had no clinical evidence of cardiovascular disease. A total of 222
women were randomized to either placebo or active treatment receiving unopposed
micronized 17 β-estradiol (1mg/day). Women were eligible if their serum estradiol
level was <20pg/ml, LDL-cholesterol level ≥130mg/dl, fasting blood glucose level
was <200mg/dl and were not current smokers. Randomized participants were
followed with clinic visits every month for the first six months, then every other
month for the remainder of the 2-year trial period. Participants fasted for eight hours
before sample collection. All study participants gave written informed consent and
the study was approved by the Institutional Review Board of the University of
Southern California.
Laboratory Measurements
Hormone Assays
Serum sex hormone concentrations were measured from fasting serum samples
drawn at baseline and every 6 months during the trial period and stored at -70
0
C.
Serum levels of androstenedione, dehydroepiandrosterone (DHEA), testosterone,
estrone (E
1
) and estradiol (E
2
) were quantified by validated, previously described
146
RIAs.
24, 25
Prior to RIA, steroids were extracted from serum with hexane:ethyl
acetate (3:2). Androstenedione (A4), DHEA, and testosterone were then separated
by Celite column partition chromatography using increasing concentrations of
toluene in trimethylpentane. E
1
and E
2
were separated in a similar fashion by use of
ethyl acetate in trimethylpentane. SHBG was quantified by direct immunoassay
using the Immulite analyzer (Diagnostic Products Corporation, Inglewood, CA).
Free testosterone was calculated using total testosterone and SHBG concentrations
and an assumed constant for albumin in a validated algorithm.
26, 27
Free E
2
was
calculated in a similar manner.
All immunoassay methods were shown to be reliable. Specificity was achieved by
use of highly specific antisera and/or use of organic solvent extraction and
chromatographic steps prior to quantification of the analytes. Assay accuracy was
established by demonstrating parallelism between measured concentrations of a
serially diluted analyte in serum with the corresponding standard curve. Intra- and
interassay coefficients of variation ranged from 4 to 8% and 8 to 13%, respectively.
All assay methods were found to be sensitive. The sensitivity of an RIA method was
determined by the smallest amount of analyte that reduced the number of counts per
minute of the radiolabeled analyte at zero mass by 2 standard deviations.
147
Inflammatory markers
Serum concentrations of inflammatory markers were measured from fasting serum
samples drawn at baseline and every 6 months during the trial period and stored at -
70
0
C. Markers of inflammation were quantified at the Laboratory for Clinical
Biochemistry Research (University of Vermont, Burlington, Vermont). hsCRP was
measured in fasting blood specimens by colorimetric competitive immunoassay (C-
reactive protein antibodies and antigens from Calbiochem, La Jolla, California). The
intra- and interassay coefficient of variation using this method was 3% and 6%
respectively (Macy EM 1997). sICAM-1 was measured using a commercially
available ELISA assay (Parameter Human sICAM-1 Immunoassay; R&D Systems,
Minneapolis, MN); intra- and interassay CVs were 4.5% and 6.2% respectively.
In vitro oxidation of LDL
The kinetics of LDL oxidation were analyzed by adding 10 µΜ CuSO
4
to 200
µg/mL LDL protein extracted from fresh serum collected in a fasting state.
Formation of conjugated dienes was monitored continuously at 234 nm for up to
sixteen hours using a Beckman DU-650 spectrophotometer equipped with a six
position automated sample changer. Three measures of oxidation kinetics were
analyzed: 1) the oxidation lag time which was defined as the interval between
initiation of oxidation and the intercept of the tangent for the slope of the absorbance
148
curve during the propagation phase, 2) the rate of oxidation during the lag time,
defined as the initial oxidation rate before the onset of the propagation phase, 3) the
rate of oxidation during the propagation phase (the log rate) defined as the maximal
rate of oxidation calculated from the slope of the absorbance curve during the
propagation phase.
Homocysteine
Total fasting homocysteine concentrations were measured from stored frozen blood
samples by reverse phase HPLC with a C18 column on a Waters HPLC instrument
equipped with a WISP automatic injector and attached to a fluorimeter. Solutions
containing 40 nmol/ml homocysteine and 200 nmol/ml cysteine were used as
standards and for column calibration. For quality control, pooled plasma spiked with
different quantities of cysteine and homocysteine were used. The coefficient of
variation for this assay was 7.8%.
Statistical analysis
Mean on-trial levels of the sex hormones were compared between estradiol- and
placebo-treated groups using t-test. Associations of sex hormone concentrations with
inflammatory markers and pro-inflammatory factors were tested using linear
regression with Generalized Estimating Equations (GEE). The serial measures of
inflammatory markers (hsCRP, and SICAM-1), proinflammatory factors
149
(homocysteine, and LDL oxidation kinetics) were the dependent variables and time-
dependent measures of sex hormones/SHBG were the primary explanatory variables.
Both the outcome and explanatory measures were modeled as continuous variables.
Inflammatory markers/proinflammatory factors at a given follow-up time were
regressed on the corresponding sex hormone measure accounting for the within-
subject effect. All models were adjusted for age, BMI and treatment group; age was
significantly inversely and BMI was directly associated with most of the sex
hormones. SHBG was significantly inversely associated with BMI and was not
associated with age. Both age and BMI were positively associated with sICAM and
CRP. Models were stratified by treatment group and tests for interactions by
treatment group were performed.
The inflammatory markers were tested for associations with proinflammatory factors
using similar linear regression models. To examine if LDL-oxidation or
homocysteine levels were intermediate factors in the association between serum sex
hormone levels and inflammatory markers, separate linear regression models
included LDL-oxidation parameters and homocysteine as covariates along with sex
hormone levels, where the dependent variable was the inflammatory marker.
Regression coefficients associated with sex hormone levels were compared between
the models that did and did not include LDL-oxidation kinetic parameters or
150
homocysteine. All analyses used SAS (version 9.1, Cary, NC); 2-sided p-values are
reported.
Results
Data on markers of inflammation and sex hormones were available on 216 women
on a total of 735 trial visits (median 4 (range 1-5) per subject). The baseline
demographic and clinical characteristics of the study subjects are listed in Table IV-
1.
Table IV-1. Demographic and baseline clinical characteristics of EPAT women (n=216).
Treatment group
Placebo 109
17 β-estradiol 107
Age (years) 61(7)*
Race
White 109 (61%)
African-American 19 (11%)
Hispanic 35 (19%)
Others 17 (9%)
BMI (kg/M
2
) 29 (6)
Smoking
Never 88 (48%)
Former smokers 92 (51%)
Blood pressure (mmHg)
Systolic 128 (14)
Diastolic 78 (7)
Total cholesterol (mg/dl) 249.5 (31.3)
HDL-cholesterol (mg/dl) 54 (12)
LDL-cholesterol (mg/dl) 164 (28)
Glucose (mg/dl) 92 (21)
sICAM-1 (ng/mL) 285(59)
hs-CRP (mg/L) 2(2.0)
Homocysteine (µmol/L) 8(3)
*Mean (SD) or n (%)
151
The average (SD) age of the EPAT participants was 61 (7) years and the majority of
the women were non-Hispanic White (61%). The women were overweight with an
average (SD) BMI of 29 (5) kg/M
2
and the mean (SD) LDL-cholesterol was 164 (28)
mg/dl. The median (range) levels of inflammatory markers were: sICAM 282 (107-
472) ng/mL, hs-CRP 1.5 (0.37-13.4) mg/L, homocysteine 7.4 (4.1-40.2) µmol/L.
Women treated with estradiol (1mg/day) had significantly higher estrone, total and
free E2, and SHBG levels (all p-values <.0001; Table IV-2). In contrast to the
estrogens and SHBG, androgens including free testosterone (p = 0.0001), A4 (p
<0.0001), and DHEA (p < 0.0001) levels were significantly lower in estradiol-treated
women compared with the placebo group. Serum levels of total testosterone were not
different between the two groups (p = 0.72).
Adjusted for age, BMI, and treatment group, homocysteine was significantly
inversely associated with estrone (p = 0.001), total E2 (p = 0.0006), and free E2 (p =
0.001) and directly associated with sICAM-1 (p < 0.0001) (Table IV-3). Only
DHEA was significantly inversely associated with LDL oxidation lag time (p = 0.03)
(Table IV-3). None of the other sex hormones or inflammatory markers was
significantly related to LDL oxidation lag time (all p values > 0.10). Lag rate and log
rate, two other kinetics of LDL oxidation, were not associated with any of the sex
hormones or inflammatory markers (data not shown). sICAM was significantly
positively associated with homocysteine ( β estimate ± SE = 1.80 ± 0.38; p < 0.0001)
152
but not with LDL oxidation lag time. hsCRP was not associated with homocysteine
or LDL oxidation lag time (data not shown).
Table IV-2. Mean serum levels of sex hormones and SHBG.
Sex hormones Placebo
(n=89)
Estradiol
(n=91)
p-value
*
Estrone (pg/ml) 48.1(42.0)
†
310.3(167.2) <.0001
Estradiol (pg/ml) 14.7(6.3) 68.2(16.2) <.0001
Free E2 (pg/ml) 0.6(0.2) 1.6(0.6) <.0001
Total T (ng/dl) 22.3(9.0) 22.8(10.8) 0.72
Free T (pg/ml) 4.1(1.8) 3.1(1.6) 0.0001
A4 (pg/ml) 522(189) 492 (180) <.0001
DHEA (ng/ml) 2.14(1.34) 1.77(0.78) <.0001
SHBG (nmol/L) 36.5(18.2) 58.2(23.6) <.0001
*
P-values were from t-test for independent samples.
†
Follow-up mean (SD).
Table IV-3. Association of sex hormones/SHBG levels and inflammatory markers with pro-
inflammatory factors.
LDL oxidation lag time Homocysteine (µmol/L ) Hormones
β estimate p-value β estimate p-value
Estrone (pg/ml) 0.01(0.01) 0.25 -.002(0.005) 0.001
Total E2 (pg/ml) 0.07(0.05) 0.18 -.01(0.003) 0.0006
Free E2 (pg/ml) 2.9(2.4) 0.23 -.46(0.14) 0.001
Total T (ng/dl) -.13(0.14) 0.34 -.01(0.008) 0.07
Free T (pg/ml) -.48(0.66) 0.47 0.03(0.05) 0.58
A4 (pg/ml) -.005(0.007) 0.19 0.0001(0.0004) 0.85
DHEA (ng/ml) -2.5(1.2) 0.03 0.03(0.06) 0.59
SHBG (nmol/L) -.03(0.07) 0.65 -.006(0.005) 0.16
Note: GEE model description: Outcomes (LDL oxidation lagtime/homocysteine) and exposures (sex
hormones/SHBG) are time dependent. Models included age, BMI, and treatment group as covariates.
153
Adjusted for age, BMI, and treatment group, sICAM-1 was significantly inversely
associated with estrone (p = 0.04), total E2 (p = 0.004), free E2 (p = 0.01) and SHBG
(p = 0.02), (Table IV-4). A significant positive association was observed between
sICAM-1 and free testosterone (p = 0.03). Total testosterone, A4 and DHEA were
not associated with sICAM-1. Although these associations of sICAM-1 with
estrogens, free testosterone, and SHBG were only significant among estradiol-treated
women, the interactions between treatment group and these sex hormones were not
statistically significant (Table IV-4).
Total testosterone and DHEA showed differential associations with sICAM between
placebo and estradiol-treated women (p-for interaction 0.05 and 0.04 respectively;
Table IV-4). In the total sample, total testosterone or DHEA were not associated with
sICAM concentrations.
Table IV-4. Association between sex hormone/SHBG levels and SICAM-1 (ng/ml)
Hormones Total sample Placebo Estradiol
β estimate p-value β estimate p-value β estimate p-value
Estrone (pg/ml) -.02(0.01) 0.04 -.06(0.11) 0.56 -.02(0.008) 0.05
Total E2 (pg/ml) -.12(0.04) 0.004 0.03(0.15) 0.84 -.12(0.04) 0.008
Free E2 (pg/ml) -4.4(1.8) 0.01 2.7(4.5) 0.55 -4.3(1.8) 0.02
Total T (ng/dl)* -.11(0.19) 0.56 0.35(0.31) 0.25 -.34(0.25) 0.16
Free T (pg/ml) 2.2(1.0) 0.03 0.90(1.9) 0.63 2.4(1.2) 0.04
A4 (pg/,l) 0.01(0.008) 0.11 0.006(0.091) 0.50 0.02(0.01) 0.19
DHEA (ng/ml)
†
1.6(1.7) 0.33 -1.2(1.5) 0.40 5.0(2.6) 0.06
SHBG (nmol/L) -.17(0.07) 0.02 -.10(0.27) 0.72 -.17(0.07) 0.03
*p-value for interaction by treatment group = 0.05
†
p-value for interaction by treatment group = 0.04
154
In the total sample, hs-CRP was significantly positively associated with estrone (p =
0.0001), total E2 (p < 0.0001), free E2 (p = 0.0002) and SHBG (p = 0.02), adjusted
for age, BMI and treatment group (Table IV-5). A significant inverse association was
observed between hs-CRP and DHEA (p = 0.006). The remaining androgens were
not associated with hs-CRP. The association of hs-CRP with SHBG was
significantly different between the treatment groups (p-for interaction = 0.0003;
Table IV-5). The relationship of hs-CRP with total testosterone was significantly
different between the treatment groups (p-for interaction = 0.05; Table IV-5) despite
a null association in the total sample.
Table IV-5. Association between sex hormone/SHBG levels and hs-CRP (mg/L).
Hormones Total sample Placebo Estradiol
β estimate p-value Β estimate p-value β estimate p-value
Estrone
(pg/ml)
0.002(0.001) 0.0001 0.01(0.01) 0.32 0.002(0.0005) 0.0003
Total E2
(pg/ml)
0.008(0.002) <.0001 0.02(0.01) 0.09 0.007(0.002) <.0001
Free E2
(pg/ml)
0.44(0.12) 0.0002 0.93(0.47) 0.05 0.41(0.12) 0.001
Total T
(ng/dl)
*
-.004(0.01) 0.71 -.02(0.01) 0.03 0.02(0.01) 0.27
Free T
(pg/ml)
-.08(0.04) 0.06 -.02(0.05) 0.67 -.09(0.06) 0.12
A4 (pg/ml) -.0002(0.0004) 0.63 -.0001(0.0004) 0.90 -.0002(0.0005) 0.67
DHEA
(ng/ml)
-.15(0.05) 0.006 -.10(0.06) 0.10 -.22(0.08) 0.006
SHBG
(nmol/L)
†
0.01(0.004) 0.02 -.02(0.006) 0.004 0.01(0.004) 0.002
*
p-value for interaction by treatment group = 0.05
†
p-value for interaction by treatment group = 0.002
155
Table IV-6. sICAM models unadjusted and adjusted for homocysteine
Hormones Adjusted for homocysteine
β estimate p-value
Estrone (pg/ml) -.01(0.01) 0.09
Total E2 (pg/ml) -.10(0.04) 0.02
Free E2 (pg/ml) -3.5(1.80) 0.05
Total T (ng/dl) -.08(0.19) 0.67
Free T (pg/ml) 1.99(0.97) 0.04
A4 (pg/ml) 0.01(0.01) 0.13
DHEA (ng/ml) 1.59(1.7) 0.36
SHBG (nmol/L) -.15(0.07) 0.03
*GEE model description: Outcome=sICAM; exposures= sex hormones levels
Models included age, BMI, treatment group, and homocysteine as covariates.
When homocysteine was included as a covariate in the model testing the association
between sICAM and sex hormones (Table IV-6), all statistically significant β
estimates showed substantial change compared with the corresponding models that
did not include homocysteine (Table IV-4). Because LDL-oxidation lag time was not
associated with the inflammatory markers, inclusion of this variable as a covariate
did not alter the associated β estimates (data not shown). Similarly, inclusion of
homocysteine or LDL-oxidation lag time did not change the β estimates for the
relationship between sex hormones and CRP (data not shown).
Discussion
Among postmenopausal women, half of whom were taking oral estradiol therapy, we
report an inverse association between sICAM-1 and circulating levels of estrone,
156
total E2, free E2, and SHBG supporting an anti-inflammatory role of estrogen.
According to a cross-sectional study, postmenopausal women (n = 74) not on HT
had 24% higher mean levels of circulating ICAM-1 compared to premenopausal
women (n = 60) (p <0.001).
8
Our results support the evidence that levels of
inflammatory markers rise after menopause due to a sharp decline of estrogen. Our
findings also support the results from multiple studies showing favorable effects of
HT on soluble markers of vascular inflammation.
9-12
Several in vivo studies have
also demonstrated an inhibitory effect of estradiol on cytokine-induced intercellular
adhesion molecule.
28
Only a handful of studies have examined the relationship of inflammatory markers
with sex hormones.
17, 29
and only one have evaluated sICAM as an outcome. This
was a cross-sectional study among a subset of participants (n = 623) from
Postmenopausal Estrogen Progestin Intervention (PEPI) Trial.
29
Endogenous serum
levels of total estradiol measured prior to HT initiation were inversely but not
significantly associated with IL-6 and sICAM.
Lower serum concentrations of SHBG have consistently been found to be associated
with increased risk of atherosclerosis and CVD.
30-32
However, the mechanisms
involved in the cardioprotective effect of SHBG are largely unknown. Several
hypotheses have been proposed including maintenance of the estrogen to androgen
157
ratio, and a direct effect of SHBG on endothelial cells mediated by cyclic AMP.
30
Our analyses showed that higher SHBG concentration was significantly associated
with lower sICAM, which may partly explain the cardioprotective effect of SHBG.
The molecular mechanism of such an effect remains to be explored.
We observed a significant positive association between estrogens/SHBG and CRP.
Of note, half of the women received oral estrogen therapy (ET). Oral ET has been
shown to raise C-reactive protein in postmenopausal women despite decreasing the
levels of other markers of inflammation such as E-selectin, VCAM, sICAM, serum
amyloid A, and IL-6.
11, 13, 14
Therefore, this apparent increase in CRP observed with
oral ET can be explained by a first-pass hepatic effect rather than a pro-inflammatory
response. However, in our analysis, the positive associations between serum estrogen
and hsCRP levels were evident in women taking or not taking oral estradiol therapy.
Among women not on ET, there was a significant inverse relationship between
SHBG and hsCRP, which is consistent with previous reports.
29, 33-35
In contrast to
this, SHBG levels were significantly positively associated with hsCRP in estradiol-
treated women. This divergent association that we observed between SHBG and
hsCRP among estradiol users vs. non-users was statistically significant (p for
interaction = 0.0003).
158
Our finding of a significant positive association of free T with sICAM is intriguing.
The cross-sectional report from PEPI demonstrated an inverse association between
endogenous levels of bioavailable testosterone and sICAM, which was of borderline
significance (p-value = 0.07).
29
These author also reported a significant positive
association between bioavailable testosterone and hsCRP, a finding supported by a
longitudinal study in women of mid-life.
35
In contrast, we found a borderline
significant inverse association between hsCRP and free testosterone. In addition to
the cross-sectional nature of the PEPI sub-study, PEPI substudy subjects were much
younger than ours (mean + SD age 56 + 4 years vs. 61 + 7 years) and half of our
women received estradiol whereas in the PEPI sub-study women were not on
hormone therapy at baseline. However, when stratified by treatment group, we did
not find any significant relationship between free testosterone and either of the
inflammatory markers.
Androgens constitute an important fraction of the sex hormone milieu in women
after menopause, since androgen concentrations do not plummet, as does estrogen
after menopause. Yet little is known about the impact of testosterone or other
androgens on CVD in postmenopausal women, and the existing data are
conflicting.
32, 36-38
It is interesting to note that in epidemiologic studies of
postmenopausal women, total and free testosterone do not relate to cardiovascular
end points in a similar direction; total testosterone is inversely
30, 37
while free
159
testosterone is positively related to CVD/atherosclerosis in postmenopausal
women.
32, 38
In vitro, testosterone may have a detrimental effect on human vascular
endothelial cells.
39
In contrast, evidence suggests that testosterone inhibits TNF- α-
induced VCAM-1 expression in human endothelial cells
40
and conversion of
testosterone to estradiol by aromatase is a possible mechanism.
41
We also noted a
significant inverse association of DHEA with CRP. Further evaluation of the role of
testosterone in vascular inflammation and risk of cardiovascular disease in
postmenopausal women is warranted.
Inflammation is a critical step in the process of atherosclerosis that initially involves
an interaction between cells of the arterial wall (endothelium and smooth muscle
cells) and blood cells that migrate into the arterial wall. Homocysteine, an
intermediate metabolite of methionine, can cause endothelial dysfunction and has
been suggested to be a potent inducer of inflammation.
42, 43
Elevated levels of
homocysteine have been associated with increased IL-6 production in monocytes,
and can enhance monocyte adhesion to the vascular endothelium by upregulating
VCAMs.
44, 45
Oxidation of LDL-C can also initiate a cascade of inflammatory
pathways. Considering homocysteine and oxidation of LDL-C as proinflammatory
factors, we hypothesized that an effect of sex hormones on inflammatory markers
could be at least partly mediated by their effect on these proinflammatory agents.
Our data showed that estrogens were significantly inversely associated with
160
homocysteine concentration (all p-values < 0.0005) but not with LDL oxidation lag
time. We also observed that the significant inverse associations of estrogens with
sICAM were appreciably attenuated (>10% change in the estimates) when the model
included the association of estrogens with homocysteine. Several HT trials have
shown that oral estrogen use, alone or combined with progesterone, lowered serum
homocysteine levels in postmenopausal women.
46-49
We extend these findings to
report the relationship between serum homocysteine and sex hormone levels with
linkage to sICAM.
Our data showed that DHEA was significantly inversely related to CRP, independent
of its prolongation effect on the lag time of LDL oxidation. The anti-inflammatory
associations of DHEA have not been studied well. A recent in vitro study reported
that DHEA inhibits the expression of adhesion and chemoattractant molecules
involved in the inflammatory process.
50
This issue demands further investigation.
In conclusion, our results support the anti-inflammatory effect of estrogens in
postmenopausal women as estrogens were inversely associated with sICAM. The
anti-inflammatory effect of estrogen was partly mediated by reduction in
homocysteine. The positive association of estrogens with CRP could not be
explained by these mechanisms. Testosterone may enhance inflammation in
postmenopausal women as evidenced by the positive association between free
161
testosterone and sICAM concentration in this study. However, the underlying
mechanism of the inverse relationship of testosterone and DHEA with CRP needs
further attention.
162
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17. Folsom AR, Nieto FJ, McGovern PG, et al. Prospective study of coronary
heart disease incidence in relation to fasting total homocysteine, related
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21. Skafar DF, Xu R, Morales J, et al. Clinical review 91: Female sex hormones
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effects in animals and man. New York. Vol 165: Academic Press; 1979.
25. Goebelsmann U HR, Mestman JH, Arce JJ, Nagata Y, Nakamura RM,
Thorneycroft IH, Mishell DR, Jr. Male pseudohermaphroditism due to
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26. Vermeulen A, Verdonck L, Kaufman JM. A critical evaluation of simple
methods for the estimation of free testosterone in serum. J Clin Endocrinol
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27. Sodergard R, Backstrom T, Shanbhag V, et al. Calculation of free and bound
fractions of testosterone and estradiol-17 beta to human plasma proteins at
body temperature. J Steroid Biochem. Jun 1982;16(6):801-810.
28. Caulin-Glaser T, Watson CA, Pardi R, et al. Effects of 17beta-estradiol on
cytokine-induced endothelial cell adhesion molecule expression. J Clin
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29. Crandall C, Palla S, Reboussin B, et al. Cross-sectional association between
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30. Golden SH, Maguire A, Ding J, et al. Endogenous postmenopausal hormones
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31. Reinecke H, Bogdanski J, Woltering A, et al. Relation of serum levels of sex
hormone binding globulin to coronary heart disease in postmenopausal
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cardiovascular events in postmenopausal women. Circulation. Oct 7
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37. Haffner SM, Moss SE, Klein BE, et al. Sex hormones and DHEA-SO4 in
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tumor necrosis factor-alpha-induced vascular cell adhesion molecule-1
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3):129-132.
41. Mukherjee TK, Dinh H, Chaudhuri G, et al. Testosterone attenuates
expression of vascular cell adhesion molecule-1 by conversion to estradiol by
aromatase in endothelial cells: implications in atherosclerosis. Proc Natl
Acad Sci U S A. Mar 19 2002;99(6):4055-4060.
42. Kanani PM, Sinkey CA, Browning RL, et al. Role of oxidant stress in
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in humans. Circulation. Sep 14 1999;100(11):1161-1168.
43. McCully KS. Homocysteinemia and arteriosclerosis: failure to isolate
homocysteine thiolactone from plasma and lipoproteins. Res Commun Chem
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44. Silverman MD, Tumuluri RJ, Davis M, et al. Homocysteine upregulates
vascular cell adhesion molecule-1 expression in cultured human aortic
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45. van Aken BE, Jansen J, van Deventer SJ, et al. Elevated levels of
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46. Mijatovic V, Kenemans P, Netelenbos C, et al. Postmenopausal oral 17beta-
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168
CHAPTER V. Sex Hormone Levels and Subclinical Atherosclerosis Progression
in Postmenopausal Women
5.1. Specific Aims
5.1.1. Overall Objectives
Coronary heart disease (CHD) is characterized by atherosclerotic lesions and
accounts for more than half of cardiovascular disease (CVD) deaths in women.
Changes in the sex hormone milieu associated with menopause coincide with the
increased risk of CHD in women. Several epidemiologic studies have documented a
steady increase in CHD incidence in women with age and its rare occurrence before
menopause
1, 2
, even in high-risk population
3
, supporting to the hypothesis that the
decline in estrogen, associated with natural or surgical menopause, contributes to
CVD risk in women. Despite the convincing evidence that menopause plays an
important role in CHD in women, research linking sex steroid hormone levels to
CHD has been very limited in women and the results are conflicting. None of the
existing studies were able to confirm the hypothesis that decreased estrogen levels
were associated with the increased risk of CHD in postmenopausal women. Possible
explanations for the null finding could be 1) lack of power due to small sample size,
2) inability to characterize estrogen levels in postmenopausal women with one single
measurement, 3) sex hormones in addition to estrogen may provide the necessary
hormonal milieu for CVD benefit. We hypothesize that androgens and sex hormone
169
binding globulin (SHBG) may be related to CHD in postmenopausal women either
independently or by modifying the role of estrogens since androgens and SHBG
levels do not decline to the same degree as estrogens after menopause and they
constitute major fractions of the postmenopausal sex hormone milieu in
postmenopausal women
4
. Our preliminary analysis revealed a fascinating
finding that increased estradiol levels alone were not sufficient to reduce
progression of carotid intima-media thickness in postmenopausal women
receiving estradiol therapy; rather women with increased SHBG and decreased
free testosterone levels in addition to increased estradiol levels had the
maximum reduction in atherosclerosis progression. Therefore, understanding
the interaction of estrogens, androgens and SHBG could provide important
insights related to cardiovascular health of postmenopausal women. We propose
to study the relationship of estrogens, androgens and SHBG with subclinical
atherosclerosis progression in 1006 postmenopausal women participating in 5
different clinical trials. Subclinical atherosclerosis was the primary end point of all 5
trials, and was assessed by common carotid artery intima-media thickness (CIMT)
measured longitudinally over the follow-up period. All trials were single centered
(conducted in the same institution) and data collection occurred under the same
standard protocol. Blood samples have been collected from these participants and
stored at each study visit. The proposed work will give us the unique opportunity to
relate repeat measures of sex hormones to progression of atherosclerosis. Currently
170
there is no data available on progression of atherosclerosis in relation to sex
hormone levels in postmenopausal women. Such data could help reduce the
burden of morbidity and mortality in postmenopausal women. We propose to address
the following specific aims:
5.1.2. Specific aims
1. To evaluate the relationship of serum estrogens, androgens and SHBG
concentrations with atherosclerosis progression in postmenopausal women.
2. To assess the association between sex hormones and CVD risk factors in
postmenopausal women (including cholesterol, triglycerides, glucose, insulin
and lipoproteins) and relate these associations to atherosclerosis progression
in postmenopausal women.
3. To evaluate the extent to which associations between sex hormones and
CVD risk factors mediate the associations between sex hormones and
atherosclerosis progression.
5.2. Background and significance
5.2.1. Sex hormones in postmenopausal women
Estrogens, including estrone (E1) and estradiol (E2) are the primary sex steroid
hormones in women, produced predominantly by the ovaries, and much less from the
171
adrenal cortex. Circulating androgenic hormones in women include testosterone,
dehydroepiandrosterone (DHEA) and androstenedione (A4), which also derive from
the ovaries and the adrenal glands. After menopause, the ovarian source of estrogen
(95% of circulating E2) is negligible and the remaining sources are the adrenal
glands and peripheral tissues (primarily subcutaneous fat). However, it is believed
that the ovarian follicles continue to produce androgens
4
. For this reason, after
menopause the estrogenic hormone levels plummet, whereas the androgen
concentrations remain fairly steady which serves as a potential source of estrogen via
aromatase conversion. Since androgens constitute a major fraction of the sex
hormone distribution after menopause, it is exceedingly important to examine the
impact of postmenopausal androgens on cardiovascular health in addition to
estrogens. We hypothesize that the complete hormonal milieu, not just estradiol,
plays a role in atherosclerosis and cardiovascular disease outcomes. Androgens and
sex hormone binding globulin (SHBG) are possibly important in the cardiovascular
health of postmenopausal women either independently or by modifying the role of
estrogens.
5.2.2. Coronary Heart Disease in Postmenopausal Women
According to Heart Disease and Stroke Statistics-2005 provided by the American
Heart Association, the overall prevalence of CVD is similar in men and women in
the United States, 34% for both. However, the age-specific prevalence is much lower
172
in women than men before the age of 45. After 45, the prevalence of CVD strikingly
increases among women and remains higher then men thereafter
5
. The gender-based
differences in the incidence of CHD also narrows after menopause. These
epidemiological data support the hormone-heart hypothesis relating estrogen
depletion to increased CVD risk in postmenopausal women. Ovarian function wanes
gradually after the third decade of life causing a gradual reduction in circulating
estradiol levels. After menopause, serum estradiol concentrations may be as low as
5-20 pg/ml as opposed to 100-600 pg/ml before menopause. Women experiencing
early menopause, either natural or due to bilateral oophorectomy, show increased
risk of heart disease
6-8
. Early age at menopause is also associated with increased risk
of MI, and cardiovascular mortality.
9-11
The risk of atherosclerosis is also increased
when estrogen production by the ovaries ceases, either naturally or after surgical
removal of the ovaries.
12, 13
The large body of observational studies evaluating the effect of hormone therapy
(HT) after menopause adds further support to the hormone-heart hypothesis.
According to a meta-analysis including studies until 1998, women who ever used
postmenopausal HT had a 30% (95% confidence limit 0.65-0.75) reduced risk of
CHD compared to never users
14
. A twenty years of follow-up data on 70,533
postmenopausal women in the Nurses’ Health Study reported a 29% reduced risk of
major coronary events among current users of estrogen therapy (RR = 0.71, 95% CI:
173
0.61-0.83), and a 32% reduced risk in current estrogen plus progestin therapy users
(RR = 0.68, 95% CI: 0.55-0.83) compared with never-users
15
. The Women’s Health
Initiative observational study is the most recent prospective cohort study that
followed 53,054 postmenopausal women, aged 50-79 years, taking estrogen alone or
estrogen plus progestin for an average 7.1 and 5.5 years, respectively
16, 17
. They also
reported a 32% and 50% reduction in CHD risk in estrogen alone and estrogen and
progestin therapy users, respectively, compared with nonusers. However, 10 out of
11 randomized clinical trials have failed to support the hypothesis that
postmenopausal HT is protective against CHD
18, 19
. Although much of this
discrepancy has been hypothesized to be related with early vs. late initiation of HT
since menopause, as well as duration of HT use
15, 20
, it is extremely important to
determine the underlying mechanism of increased CHD risk posed after menopause.
Understanding the effect of sex hormone levels, individually and in combination, on
cardiovascular health may shed some light on the current controversy regarding the
cardiovascular benefit of HT.
5.2.3. Significance of Studying Atherosclerosis Progression
Atherosclerosis, or thickening of the arteries due to deposition of low density
lipoprotein (LDL) cholesterol in the arterial walls, is the primary underlying cause of
most of cardiovascular disease or stroke
21
. The only clinical trial showing a
beneficial effect of postmenopausal estrogen therapy was the Estrogen in the
174
Prevention of Atherosclerosis Trial (EPAT) that studied progression of subclinical
atherosclerosis measured by carotid intima-media thickness (IMT) in healthy
postmenopausal women
22
. EPAT results indicate that estradiol is capable of altering
the process of atherosclerosis in women who do not have clinically evident CVD.
Two other clinical trials, HERS
23
, and ERA
24
also studied atherosclerosis
progression among women with pre-existing CVD but did not find treatment benefit.
WELL-HART
19
and WAVE
25
examined the effect of HT on angiographically
determined coronary atherosclerosis, both reporting null associations. The most
likely reason for this discrepancy could be that all of those trials (other than EPAT)
included women with established CVD. A randomized controlled trial in
ovariectomized female monkeys showed significant reduction in atherosclerosis
progression with conventional doses of HT
26, 27
, and even smaller doses (CEE
0.30mg/day)
28
. It is possible that exogenous sex hormones are most effective in
altering the process of atherosclerosis when administered in an early stage as
opposed to advanced atherosclerosis characterized by the presence of clinically
evident CVD. Animal studies provide strong evidence in favor of this hypothesis
20,
29
. Therefore, it is likely more relevant to understand the association of sex
hormone levels with progression of atherosclerosis over time in women without
clinically evident CVD compared to population without CVD. We will be able to
evaluate the relationship of sex hormones and atherosclerosis progression in
postmenopausal women with or without clinically evident CVD as 22% of our study
175
population has advanced CHD while the remaining 88% were free of CHD at the
beginning of the study. Thereby, we will be able to test if sex hormones are
associated with differential atherosclerosis progression depending on the disease
status. Studying atherosclerosis progression as an end point will enable us to
understand the effects of different sex hormones on atherosclerosis over time rather
than cross-sectionally with a single atherosclerosis measurement. A more complete
understanding of the process of atherosclerosis progression in postmenopausal
women will contribute towards primary prevention of CVD and its complications.
5.2.4. Circulating sex hormone levels and CHD
Despite the convincing evidence that cardiovascular disease risk is increased after
menopause, translational studies relating circulating sex hormone levels to
cardiovascular disease are sparse in women as opposed to men
30
. Only a limited
number of studies have tested the associations of sex hormones and CVD/
atherosclerosis in postmenopausal women and the results are far from conclusive.
Most of these studies had limited sample size, and may have suffered from
measurement error in determination of sex hormone levels as hormones were
measured only once in all of these studies.
Circulating levels of SHBG and total testosterone have been inversely associated
with CVD
31-33
; while free testosterone has been linked with increased CVD risk
33
176
and CHD
34
in postmenopausal women. None of the studies found any significant
association between serum estrogen levels and CVD risk in postmenopausal women.
It is not known if there is any interaction among estrogen, testosterone, and SHBG
levels in relation to CHD/atherosclerosis. Low estrogen levels alone may not be the
sole correlate of increased CHD risk in postmenopausal women- a hypothesis that is
yet to be tested. Another possible explanation for the null associations of estrogen
concentrations with CHD risk could be the very low estrogen levels in
postmenopausal women, which may not be sufficient to impact the risk. Thus, it may
the comparative decline in estrogens from pre- to postmenopausal state i.e. the range
of physiologic variability rather than variability in levels evident in postmenopausal
women, that are required to detect the impact of circulating estrogen on
atherosclerosis and CVD risk. In support of this hypothesis, hypoestrogenemia has
been associated with coronary atherosclerosis in pre- and peri-menopausal women
35
.
Another explanation could be potential measurement error in determining estrogen
levels. Estrogens were measured only once in all of those studies which may not be
adequate to characterize a woman’s hormone status given the large within-individual
variability of sex hormones, particularly estrogens. Evidence shows that one single
determination of sex hormones may not be adequate to characterize the hormone
status of a postmenopausal woman, particularly when estrogen is very low. A
sensitive and reliable assay is mandatory to quantify such low levels of sex hormones
in postmenopausal women, which was a limitation in most of the studies.
177
5.2.5. Measurement Error in Sex Hormone Determination:
There are two major potential sources of measurement error in sex hormone
determinations of postmenopausal women: (1) inability to capture the within-subject
variability of sex hormones; (2) inaccuracy in measurement due to use of insensitive
methods.
5.2.5.1. Within subject variability of sex hormones in postmenopausal women
Single determinations of total estradiol are insufficient to measure a women’s true
serum estradiol concentration after menopause. In 77 postmenopausal women, the
intraclass correlation coefficient (ICC) of 2-3 measurements of total estradiol over 2-
3 years was 0.51
36
. Another study evaluating the short term (4-week) and long-term
(2-year) reliability of serum sex hormones in 174 healthy postmenopausal women
reported poor reproducibility of total estradiol (ICC for short-term = 0.45 and long-
term=0.36)
37
. In participants from the Nurses’ Health Study, the reproducibility for
serum estradiol was somewhat higher (ICC = 0.68) based on 3 measurements taken
over 3 years. However, this study included only 79 postmenopausal women
38
. On the
other hand, estrone, the primary estrogen in postmenopausal women, has less
variation (ICC ranging from 0.56 to 0.74) than other biological variables which have
been shown to predict cardiovascular disease, including plasma cholesterol
37
. These
data provide evidence of a great deal of intra-individual variability of estradiol in
postmenopausal women suggesting multiple measurements of estradiol are required
178
to characterize a woman’s true estradiol level after menopause. In terms of
androgens, the reproducibility was remarkably high in all of these studies (ICC
ranging from 0.88 to 0.92 for total testosterone, 0.71 for free testosterone, 0.85 to
0.90 for DHEA-S) suggesting one single measurement of androgens may be
sufficient to measure the true androgenic profile of a postmenopausal women. The
reproducibility of SHBG was also high in Nurses’ Health Study participants (ICC =
0.92). Consistent with the literature, EPAT participants receiving placebo (n = 111)
had lower reproducibility of estradiol measurement (ICC = 0.30), and relatively
higher reproducibility of testosterone (ICC = 0.68) and SHBG measurement (ICC =
0.56).
5.2.5.2. Sensitivity of sex hormone assay methods
Sex steroid hormones are often measured using commercially available direct
radioimmunoassay (RIA) kits. The reliability of commercially available direct RIA
assay kits are questionable, particularly in epidemiologic studies where the objective
is not only to identify extreme levels but to identify subjects by relative hormone
levels within the non-pathologic range. Conventional or indirect RIA after organic
solvent extraction and purification by celite column partition chromatography are
considered the ‘gold standard’ technique for sex hormone measurement. However,
because it is expensive, time consuming and requires expert technicians, direct RIAs
are often used in epidemiologic studies. One report including 125 premenopausal
179
and 134 postmenopausal women, ages 18-75, showed that total testosterone
measured by direct RIA was lower (mean + SD=0.77 + 0.54 nmol/L) than the
conventional RIA (1.05 + 0.58 nmol/L). Reproducibility of sex hormones using 5
different direct assay kit was compared with the ‘gold standard’ conventional RIA.
The relative validity of direct versus conventional RIA was high for most of the kits
tested for total testosterone, androstenedione, and estrone, but was high only for 2 of
the 5 kits tested for total estradiol
39
. Since postmenopausal levels of estrogens are
low, assays must be sufficiently sensitive to measure hormone levels with reasonable
accuracy.
5.2.6. Circulating Sex Hormone Levels and CVD Risk Factors
Although menopause has been associated with hypertension, and increased LDL-
and decreased HDL-cholesterol, only a limited number of studies have evaluated the
relationship between sex hormones and cardiovascular risk factors in
postmenopausal women. Several cross-sectional studies showed a significant
positive association between serum estrogens and HDL-cholesterol
40-42
. However,
some studies failed to support these associations
43, 44
. A major weakness of all of
these studies was their cross-sectional design. Longitudinal assessment of serum
estrogens and lipids will provide a clearer understanding of these associations. In
contrast to estrogens, serum testosterone has been consistently reported to have an
adverse impact on blood pressure, total, LDL- and HDL-cholesterol, glucose, insulin
180
and BMI
34, 45-48
. Higher serum levels of SHBG have been consistently found to have
beneficial association with several CVD risk factors in women including BMI, blood
pressure, HDL-cholesterol, LDL-cholesterol, glucose, and insulin
40, 46, 47, 49-52
.
The role of CVD risk factors as mediators of the relationship between sex hormones
and atherosclerosis has not been evaluated. We propose to examine if the
association of sex hormones with atherosclerosis can be partially explained by
hormone-related alterations in the CVD risk factors such as lipids, and
carbohydrate metabolites.
5.2.8. Importance of the proposed work
Despite the convincing evidence relating menopause to increased risk of CHD in
postmenopausal women, the exact mechanism of this association is yet to be
understood. Postmenopausal levels of estrogens, androgens, and SHBG need to be
examined in relation to atherosclerosis, the primary underlying pathology of CHD.
Epidemiologic studies in the past have failed to link these decreased estradiol levels
to the risk of CHD in postmenopausal women. Given the potential sources of
measurement error in determining sex hormones, we are proposing to measure
estrogens, testosterone and SHBG longitudinally using the conventional celite
column partition chromatography method. This work will give us an excellent
opportunity to relate the longitudinally-measured sex hormone data to the repeated
181
measures of carotid IMT and a panel of CVD risk factors. Repeat measures of
carotid IMT will enable us to assess the progression of atherosclerosis in relation to
the sex hormone levels. The proposed work will be conducted with immense cost
efficiency, since all but the sex hormone data are currently available on the
sample of 1006 postmenopausal women proposed to be studied. We will also
examine if there are interactions among estrogen, testosterone, and SHBG in
their association with atherosclerosis progression as we hypothesized that
decline in estrogen may not be the sole mechanism in the
hormone/atherosclerosis. The diversity of our population in terms of HT use,
pre-existing cardiovascular conditions, age, and BMI will increase the scope of
this research. Results from this proposed work can lead to a new direction
toward prevention of atherosclerosis progression in postmenopausal women,
thereby reducing the burden of morbidity and mortality from CHD in this
population.
5.3. Previous Work/Preliminary Studies
5.3.1. Randomized Controlled Trials in Postmenopausal Women
We are proposing to measure serum sex hormone concentrations using stored blood
samples from postmenopausal subjects participating in 5 clinical trials that were
conducted at the Atherosclerosis Research Unit (ARU) of USC. All of the clinical
trials measured subclinical atherosclerosis by carotid intima-media thickness (CIMT)
182
as either a primary or a secondary trial end point. Primary and secondary endpoints
as well as the ancillary data (including CVD risk factors) were collected under the
same standard protocol in all 5 trials. The sample characteristics of each trial in
relation to the postmenopausal women proposed for this study are summarized in
Table V-1.
5.3.1.1. Estrogen in the Prevention of Atherosclerosis Trial (EPAT) (NIH R01
AG-18798, PI: Howard N. Hodis, M.D)
EPAT was a randomized, double-blind, placebo-controlled, carotid artery ultrasound
trial designed to test whether unopposed oral micronized 17 β-estradiol (1 mg daily)
versus placebo reduces progression of subclinical atherosclerosis (carotid IMT) in
healthy postmenopausal women without preexisting CVD with LDL-C levels of at
least 130 mg/dL. All subjects were non-smokers at randomization and remained so
on-trial. After randomization, subjects were treated for 2 years with unopposed
estradiol or placebo and with lipid-lowering medication (primarily HMG-CoA
reductase inhibitors) if needed, to maintain LDL-cholesterol levels <160 mg/dL. The
carotid artery ultrasonography was performed at baseline (2 visits) and every 6
months on-trial. The mean age of the 222 subjects randomized to EPAT was 61.1
years with a range of 47 to 80 years. 42% of the EPAT cohort was comprised of
women from a racial minority group. Women randomized to 17 β-estradiol had a
significantly lower carotid IMT progression rate relative to placebo group
22
.
183
Table V.1. Characteristics of Five Randomized Athersclerosis Regression Trials.
EPAT
WELL-HART VEAPS BVAIT WISH
Intervention unopposed
oral 17 β-
estrodiol
1mg/day
Daily 1 mg 17 β-
estrodiol alone or
daily 1mg 17 β-
estrodiol plus 5mg
cyclic medrox-
yprogesterone
acetate or placebo
DL- α-
tocopherol
400 IU/day
Folic acid 5
mg, B
12
0.4
mg and B
6
50
mg
Soy protein
25 g soy
protein,
150mg
isoflavone/
day
Key inclusion
criteria
≥ 45 years
old;
Postmenopau
sal;
No existing
CVD;
LDL-C ≥
130mg/dL
≤ 75 years old;
Postmenopausal;
LDL-C 100-250
mg/dL;
TG < 400 mg/dL;
≥ 1 coronary artery
lesion
≥ 40 years
old;
No existing
CVD;
LDL-C ≥
130 mg/dL
≥ 40 years
old;
Postmenopaus
al (for
women);
No existing
diabetes,
CVD or
cancer;
Elevated
homocysteine
>40 years
old;
Postmenop
ausal;
No existing
CVD;
Study period Apr. 1994 –
Nov. 1998
Jun. 1995 – Oct.
2000
Jul. 1996 –
Sep. 2000
Nov. 2000 –
Aug 2006
Apr.2004-
ongoing
Follow-up
(years) 2 3 3 2.5 Æ 4.5 2.5
Randomized 222 226 353 506 350
Female, n(%) 222 (100) 226 (100) 172 (52) 197 (39) 350 (100)
Post-
menopausal,
n(%of
female)
222 (100) 226 (100) 120 (66) 197 (100) 350 (100)
Evaluable
subjects
(Tx / Pl)
180(81%)
(81/80)
226(100%)
(150/76)
114(95%)
(55/59)
191(97%)
(98/93)
295(84%)
EPAT (Estrogen in the Prevention of Atherosclerosis Trial)
WELL-HART (Women's Estrogen-progestin Lipid-Lowering Hormone Atherosclerosis Regression Trial)
VEAPS (The Vitamin E Atherosclerosis Prevention Study)
BVAIT (The B Vitamin Atherosclerosis Intervention Trial)
WISH (Women’s Isoflavone Soy Health)
†
Evaluable subjects=postmenopausal women with at least one repeat visit.
Tx=active treatment group, Pl=placebo.
184
5.3.1.2. Women's Estrogen-progestin Lipid-Lowering Hormone Atherosclerosis
Regression Trial (WELL-HART)(U01-HL-49298, PI : Howard N. Hodis, M.D).
WELL-HART was a randomized, double-blind, placebo-controlled, serial coronary
angiographic trial. A total of 226 postmenopausal women 48 to 76 years old (mean
age of 63.5 years) with established coronary artery disease were randomized to 1 of 3
treatment arms: daily micronized 17 β-estradiol 1 mg per day plus
medroxyprogesterone acetate (MPA) placebo (ERT), daily micronized 17 β-estradiol
1 mg per day plus active MPA 5 mg per day (PERT), and daily 17 β-estradiol placebo
plus MPA placebo. Coronary angiograms were obtained prior to randomization and
repeated 3 years after randomized treatment under a standardized protocol. The
primary trial end point was the mean per patient change in percent diameter stenosis
of coronary artery lesions measured by quantitative coronary angiography (QCA). A
secondary trial end point was carotid artery IMT measured by-model ultrasound at
baseline and every 6 months throughout the trial. 70% of the WELL-HART cohort
was comprised of women from a racial minority group. Results describing the effects
of HT on coronary atherosclerosis were published
19
. In older postmenopausal
women with established coronary-artery atherosclerosis, 17 β-estradiol either alone or
with sequentially administered medroxyprogesterone acetate had no significant
effect on the progression of coronary atherosclerosis. Analysis of the secondary
outcome revealed that unopposed 17 β-estradiol significantly reduced the progression
185
of carotid IMT progression in non-diabetic women compared with placebo treated
women.
53
5.3.1.3. The Vitamin E Atherosclerosis Prevention Study (VEAPS) (R01- NIA
AG13860, PI: Dr. Howard N. Hodis, M.D)
VEAPS was a randomized, double-blind, placebo-controlled, carotid IMT prevention
trial of vitamin E 400 IU per day versus placebo in 353 healthy men and women >40
years of age with LDL-C >130 mg/dL and triglycerides <500 mg/dL; 353 subjects
were randomized and followed every 3 months for an average of 3 years. The
primary end point of the trial was progression of subclinical atherosclerosis
measured by carotid artery IMT using B-mode ultrasonography. The published
results demonstrated that vitamin E supplementation had no significant impact on
progression of carotid artery IMT in healthy men and women without CVD,
However, compared to placebo, α-tocopherol supplementation significantly raised
plasma vitamin E levels (P <0.0001), reduced circulating oxidized LDL (P =0.03),
and reduced LDL oxidative susceptibility (P <0.01)
54
. Of the 172 women
participating in VEAPS, 120 postmenopausal women will be included in the
proposed study.
186
5.3.1.4. The B Vitamin Atherosclerosis Intervention Trial (BVAIT) (R01-NIH
AG RO1 17160, PI: Dr. Howard N. Hodis, M.D)
BVAIT is a randomized, double-blind, placebo-controlled, clinical trial designed to
evaluate the effect of vitamin B on progression of carotid atherosclerosis. A total of
309 eligible men and 197 postmenopausal women age >40 years, fasting
LDL-cholesterol levels >130 mg/d, fasting plasma homocysteine >8.5 µmol/l and
without clinical evidence of CVD were randomized. The rate of change in common
carotid artery IMT in computer image processed B-mode ultrasonograms was the
primary end point. Participants were randomized to either placebo or a daily pill
containing oral folic acid 5 mg + vitamin B
12
0.4 mg + vitamin B
6
50 mg for 2.5
years. All 197 women participating in the trial were postmenopausal. Data collection
on this trial are completed, analysis of the primary end point data are in progress.
5.3.1.5. Women’s Isoflavone Soy Health (WISH) Trial (NIH U01-AT001653, PI:
Dr. Howard N. Hodis, M.D).
WISH is an ongoing randomized, double-blind, placebo-controlled, noninvasive
ultrasonographic trial designed to test the effect of soy isoflavones and soy protein
on progression of subclinical atherosclerosis in postmenopausal women without
clinical evidence of CVD. A total of 350 postmenopausal women, >40 years of age
with serum estradiol level <20 pg/ml without diabetes mellitus, uncontrolled
187
hypertension and thyroid disease were randomized to either placebo or 25 g soy
protein daily. The rate of change in common carotid artery IMT in computer image
processed B-mode ultrasonograms is the primary trial end point. Repeat measure of
CIMT and serum sample are currently available on 295 women. The trial is expected
to be complete by October 2008.
5.3.2 Ultrasound measures of carotid IMT
Ultrasound scanning has been included in ARU clinical trials since 1982 and has been
accompanied by development of new methods
55-59
. In 1993, this group published the
first clinical trial data from CLAS (Cholesterol Lowering Atherosclerosis Study),
demonstrating a significant benefit of lipid-lowering (with colestipol-niacin therapy) on
progression of carotid artery IMT
60
. We confirmed this original report with a second
clinical trial MARS (Monitored Atherosclerosis Regression Study) indicating the
benefit of lipid-lowering (with lovastatin therapy) on progression of carotid IMT
61
. We
have since published a number of papers including EPAT
22
and VEAPS
54
demonstrating the usefulness of our carotid IMT methodology as an end point for
quantifying subclinical atherosclerosis under funding from R01HL-49885, R01HL-
45005, R01AG-18798, R01AG13860, R01AG-18012, R01HL-51913 and R01AG-
15139. We showed a significant relationship between progression of CCA IMT and
progression of coronary artery atherosclerosis
62
. Additionally, we have demonstrated
that progression of CIMT is predictive of clinical coronary events over a 12-year period
188
in men with CAD
63
, results consistent with other such studies in asymptomatic men and
women
64-67
. The image processing program used to measure carotid IMT at ARU
utilizes an automated computerized edge detection algorithm to track the lumen-intima
and media-adventitia ultrasound echoes
58, 59
, first identified by Pignoli et.al
68
as
representing carotid IMT. Results from CLAS, MARS, EPAT and VEAPS indicate
that small therapeutic effects on carotid IMT can be measured with relatively small
sample sizes with as little as 2 years of follow-up.
5.3.3 Determination of Sex Steroid Hormones
Serum sex hormone assays will be measured in the The Reproductive Endocrine
Research Laboratory, under the direction of Frank Z. Stanczyk, Ph.D. This
laboratory has extensive experience in measuring steroids, peptides, proteins and
prostanoids in biological fluids (serum, plasma and/or urine) and/or tissues using
immunoassay methods. The laboratory is part of the Department of Obstetrics and
Gynecology, located at Women’s and Children’s Hospital, which is part of the
University of Southern California (USC) Keck School of Medicine and Los Angeles
County Medical Center, and has been in operation since it opened in 1973. It is
considered solely as a research laboratory; no clinical diagnostic testing is conducted
there. From 1973 - 1979, RIAs for a variety of natural and synthetic compounds
were developed and validated. For most of the steroid assays, organic solvent
extraction and chromatographic steps preceding the RIAs were incorporated into the
189
assay methods, in order to achieve greater assay specificity. Most of the steroid
assay methods that were developed and validated in the 1970s are essentially the
same as the ones that are used presently. However, one important modification that
was made in all the steroid assay methods was to use an iodinated tracer, instead of
tritium, in the RIAs to achieve greater assay sensitivity. Also, a much shorter
counting time is required with the iodinated tracer, resulting in a shorter turnaround
time for the assays.
From the late 1970s to the present time, many different analytes in thousands of
samples have been quantified at the Reproductive Endocrine Research Laboratory.
Samples have been obtained from investigators at the National Cancer Institute, the
National Institutes of Health, Fred Hutchinson Cancer Research Center, Cancer
Center of Hawaii, The Jean Hailes Foundation (Victoria, Australia), Cancer Research
UK (London, England), Norris Cancer Hospital (USC), and a variety of
pharmaceutical companies. Data from those analytes have been reported in
numerous peer review articles. In most instances, Dr. Stanczyk has worked closely
with the investigators to assist in data interpretation and writing of abstracts and
manuscripts. The same collaboration will be available for data generated from the
proposed study.
190
5.3.4 Preliminary Data on Sex Hormones, Subclinical Atherosclerosis, and CVD
Risk Factors
5.3.4.1 Sex hormone concentrations and CIMT progression
The research questions of this grant proposal are based on the preliminary data from
the Estrogen in the Prevention of Atherosclerosis Trial (EPAT). Using longitudinal
data from EPAT, we examined the association between sex hormones and
atherosclerosis progression in 180 postmenopausal women.
5.3.4.1.1. Intraclass correlation coefficients (ICCs) of sex hormones in the EPAT
participants not on estradiol therapy: The ICCs were: for estradiol 0.30, for
testosterone 0.68 and for SHBG 0.56. These data are consistent with the ICCs
reported in several other studies
36, 37
.
5.3.4.1.2. Association of sex hormones to IMT progression: Mixed effects
regression models were fitted with serial measures of carotid IMT as the outcome
and the difference from baseline to average ontrial measures of sex hormones as the
exposures of interest. A total of 180 women contributed data on 859 trial visits.
Results are summarized in Table V-2 below. Adjusted for age and BMI, estrogens,
total testosterone and SHBG were significantly inversely related to the progression
of carotid IMT in postmenopausal women. When stratified by treatment group, total
191
testosterone (p=0.05) in the placebo group and SHBG (p=0.05) in the estradiol group
were significantly inversely associated with CIMT progression.
Table V.2. Sex hormones and atherosclerosis progression in 180 postmenopausal women.
Total sample (n = 180) Placebo (n = 85)
*
Estradiol (n = 91) Changes in
hormones IMT
progression
rate/year (SE)
†
p-value IMT
progression
rate/year (SE)
†
p-value IMT
progression
rate/year (SE)
†
p-value
Estrone (pg/ml) -0.018(0.008) 0.02 -.03(0.08) 0.75 -.10(0.06) 0.19
Total E2 (pg/ml) -0.119(0.05) 0.01 -.70(0.46) 0.13 -.09(0.07) 0.18
Free E2 (pg/ml) -4.953(2.18) 0.02 -22.9(15.8) 0.15 -3.29(2.9) 0.26
Total T (ng/dl) -0.531(0.28) 0.06 -1.23(0.64) 0.05 -.29(0.28) 0.30
Free T (pg/ml) 0.403(1.12) 0.72 -4.58(2.7) 0.09 0.61(1.2) 0.61
Androstenedione
(pg/ml)
-0.005(0.008) 0.55 0.003(0.01) 0.83 -.02(0.01) 0.12
DHEA (ng/ml) 0.102(1.43) 0.94 1.23(2.38) 0.60 -1.1(1.65) 0.51
SHBG (nmol/L) -0.212(0.08) 0.005 -.12(0.26) 0.65 -.20(0.10) 0.046
†
IMT progression rate (µm/year) per unit change in the sex hormone levels
*
Placebo women (n=4) who took exogenous estrogens during the trial were excluded
Each model adjusted for age and BMI.
5.3.4.2. Patterns of sex hormone changes in relation to IMT progression in
estradiol-treated women: We tested the free components of estradiol and
testosterone, which are the functionally active forms and SHBG for a particular
pattern in their associations with carotid IMT progression in women taking estradiol
therapy. For each subject, we categorized their change in sex hormones from
baseline to ontrial (increase, no change, decrease). We used the within subject SD
(over repeated measures) in the placebo group to define change (increase ≥ 1 SD
increase; decrease ≤1 SD decrease; no change = within + 1 SD). Categorical
hormone change variables were then tested in different combinations for an
192
association with CIMT progression using mixed effects model (results summarized
in Figure V-1).
These data indicate that among women with increased free E2, SHBG and decreased
free testosterone had the maximum reduction in carotid IMT progression (mean (SE)
IMT progression rate= -5.45(2.77) µm/year). Women with increased free E2 and
SHBG but no change in free testosterone had moderate reduction in carotid IMT
progression (mean (SE) IMT progression rate=-3.95(3.03) µm/year). Despite
increased free estrogen, women with no change in SHBG and free testosterone had
on average the largest progression in carotid IMT (mean (SE) IMT progression
rate=8.53 (4.72) µm/year). The small sample size was clearly a limitation of this
analysis. Expansion of these analyses to the larger cohort of postmenopausal women
will greatly increase study power enabling us to examine the complete patterns in the
sex hormonal milieu.
193
Figure V-1. Change in Sex Hormones Combined with ET and Carotid IMT Progression.
8.53
-3.95
-5.45
-8
-6
-4
-2
0
2
4
6
8
10
AB C
Carotid IMT progression (um/year)
A = free estradiol level increased with no change in sex hormone binding globulin (SHBG) and free testosterone
B = free estradiol level and SHBG levels increased with no change in free testosterone
C = free estradiol level and SHBG levels increased with lowering of free testosterone
For each subject, the change in each sex hormone from baseline was categorized using the within-subject SD
(over repeated measures) in the placebo group (up= > 1 SD change; down= < 1 SD change, no change= between
± 1 SD).
5.3.4.3. Sex hormone concentrations and serum lipids: Fasting serum levels of
triglycerides (TG), LDL-C, and HDL-C were measured at baseline and every 6
months in the EPAT trial. To assess the association between serum sex hormones
and lipids over time, we used linear regression with generalized estimating equations
(GEE) that accounted for the within subject correlation. In the GEE models, serum
lipids were the dependent variable and the sex hormone levels were modeled as the
independent variables. Separate models were fitted for each sex hormone and each
194
model included age, BMI and treatment group as adjustment variables. Results are
summarized in Table V-3.
Table V.3. Relationship of serum sex hormone levels to serum lipids and triglycerides.
Sex Hormones
LDL-C (mg/dl)
HDL-C (mg/dl)
TG (mg/dl)
β-estimate
p-
value β-estimate
p-
value β-estimate
p-
value
Estrone (pg/ml) -.06(0.01) <.0001 0.02(0.002) <.0001 0.02(0.01) 0.10
Total E2(pg/ml) -.34(0.03) <.0001 0.09(0.01) <.0001 0.13(0.08) 0.10
Free E2(pg/ml) -14.1(1.4) <.0001 3.82(0.57) <.0001 5.74(3.1) 0.06
Total testosterone (ng/dl) -.12(0.13) 0.34 0.11(0.06) 0.07 -.40(0.36) 0.28
Free testosterone (pg/ml) 2.36(0.77) 0.002 -1.36(0.34) <.0001 0.20(1.6) 0.93
Androstenedione (pg/ml) 0.001(0.005) 0.83 -.007(0.002) 0.0005 0.004(0.01) 0.79
DHEA (ng/ml) 2.5(0.82) 0.002 -1.58(0.38) <.0001 -1.15(1.8) 0.52
SHBG (nmol/L) -.41(0.05) <.0001 0.15(0.02) <.0001 -.10(0.13) 0.44
Note: Models adjusted for age, BMI, and treatment group
Estrogens and SHBG were significantly inversely associated with LDL-C and
directly associated with HDL-C. Free testosterone and DHEA were significantly
positively associated with LDL-C and inversely related to HDL-C. Free E2 was
positively associated with TG with borderline significance, none of the other sex
hormones or SHBG were associated with TG. With the proposed study, we will be
able to extend the analyses relating sex hormone levels to serum lipids longitudinally
in 1006 postmenopausal women.
5.3.4.4. Sex hormones and factors related to carbohydrate metabolism: Fasting
glucose, insulin and glycosylated hemoglobin (HbA1C) were determined at baseline
195
and every 6 months. Each of the sex hormones and SHBG were tested for association
with carbohydrate-related factors measured over 2 years using linear regression with
GEE. The sex hormones were modeled one at a time and each model included age,
BMI, and treatment group as adjustment variables. Results are summarized in Table
V-4.
Table V.4. Relationship of serum sex hormone levels to carbohydrate related factors
Glucose (mg/dl)
Insulin (IU/L)
HbA1C (%)
Sex hormones
β-estimate
p-
value β-estimate
p-
value β-estimate
p-
value
Estrone (pg/ml) -.002(0.01) 0.78 -.006(0.001) <.0001 -.0003(0.0001) 0.06
Total E2(pg/ml) -.03(0.03) 0.31 -.04(0.01) <.0001 -.002(0.001) 0.02
Free E2(pg/ml) -.73(1.3) 0.57 -1.5(0.31) <.0001 -.06(0.03) 0.07
Total T (ng/dl) -.32(0.13) 0.02 0.02(0.04) 0.61 -.007(0.004) 0.05
Free T (pg/ml) -.14(0.39) 0.72 0.44(0.16) 0.008 0.03(0.01) 0.05
Androtenedione (pg/ml) 0.007(0.004) 0.09 -.0004(0.001) 0.79 -.0002(0.0001) 0.11
DHEA (ng/ml) -1.4(0.75) 0.07 -.03(0.21) 0.90 -.03(0.02) 0.08
SHBG (nmol/L) -.16(0.07) 0.03 -.05(0.01) <.0001 -.004(0.001) 0.004
Note: Models adjusted for age, BMI, and treatment group
Estrogens were not associated with fasting glucose concentrations. Total testosterone
and SHBG were significantly inversely related with glucose. Estrogens and SHBG
were significantly inversely associated with insulin levels. Free testosterone was
directly associated with serum insulin concentration. Total E2, total testosterone, and
SHBG were significantly inversely associated with HbA1C. Free testosterone was
directly associated with HbA1c. We will be able to test the relationship between sex
196
hormones and carbohydrate related factors longitudinally in a much bigger sample of
postmenopausal women (n=1006) in the proposed work.
5.3.4.5. Mediating effects of serum lipids in sex hormone-carotid IMT
progression association: In EPAT participants, longitudinal assessment of serum
lipids revealed a strong direct correlation between LDL-cholesterol and IMT
progression and a significant inverse correlation between HDL-cholesterol and IMT
progression
69
. Based on this finding, we evaluated the role of serum lipids and
carbohydrate related factors in mediating the relationship between sex hormones and
carotid IMT progression. We compared the parameter estimates from models
including and not including serum lipids and carbohydrate related factors. A 10% or
greater alteration in the estimates was considered a substantial mediation. The results
are presented in Table V-5.
Table V.5. Mediating effect of HDL- and LDL-cholesterol in sex hormones and atherosclerosis
progression association.
On-trial Changes
in Hormones
Adjusted for age and BMI Adjusted for age, BMI, LDL-C and
HDL-C
*
Estimate(SE) p-value
*
Estimate(SE) p-value
Estrone (pg/ml) -0.018(0.008) 0.02
-.003(0.008) 0.73
Total E2 (pg/ml) -0.119(0.05) 0.01
-.01(0.05) 0.82
Free E2 (pg/ml) -4.95(2.18) 0.02
-.32(2.2) 0.88
Total T (ng/dl) -0.531(0.28) 0.06
-.51(0.25) 0.04
SHBG (nmol/L) -0.212(0.08) 0.005
-.08(0.08) 0.29
*IMT progression rate (µm/year) per unit change in the sex hormone levels
Note: Free testosterone, androstenedione, and DHEA was not significantly associated with carotid IMT
progression
197
Including glucose, insulin, or HbA1C did not have any mediating effect in the
association between sex hormones and IMT progression (data not shown). However,
LDL- and HDL-cholesterol were significant mediators in the association between
IMT progression and estrogens and SHBG levels. The association between total
testosterone levels and atherosclerosis progression did not change substantially (4%
difference) after controlling for LDL- and HDL-cholesterol. These results are novel
and need to be tested in bigger sample of women as we are proposing in this study.
5.3.4.6. Summary of Preliminary data: To summarize the data from our
preliminary analysis in EPAT, estrogens and SHBG significantly reduced subclinical
atherosclerosis progression (Table V-2), partially by their beneficial impact on serum
LDL- and HDL-cholesterol (Table V-5). The beneficial effect of estrogens and
SHBG on carbohydrate related factors did not mediate this relationship (results not
shown). We are proposing to reproduce these results in 1006 postmenopausal women
who participated in 5 different clinical trials at our institution. Carotid IMT had been
measured at each scheduled visit in these women.
Although the beneficial effects of estrogens and SHBG on lipids and carbohydrate
related factors (Tables V-3 and V-4) are consistent with previous reports, these
associations have not been confirmed in longitudinal studies. Our proposed work
will be able to examine these associations longitudinally by linking the hormone data
198
to the existing longitudinally-collected data on serum lipids, carbohydrate related
factors.
Although testosterone constitutes and important fraction of the sex hormone milieu
after menopause, the role of testosterone on atherosclerosis and CVD risk factors in
postmenopausal women are far from clear. Our preliminary EPAT analyses
demonstrated a detrimental influence of free testosterone on serum LDL-cholesterol,
HDL-cholesterol, insulin and glycosylated hemoglobin (Tables V-3 and V-4).
However, in our data on 180 postmenopausal women we did not find any significant
relationship between free testosterone and CIMT progression. Total testosterone on
the other hand was inversely associated with CIMT progression with a borderline
significance. The diverging effect of total vs. free testosterone needs to be addressed
more critically, which our proposed work will be able to do.
We also found that among women receiving estradiol therapy in EPAT, estradiol,
testosterone and SHBG interact with each other in a dynamic process to have an
impact on CIMT progression (Figure V-1). Rise in the serum estradiol levels was
necessary but not sufficient to effect CIMT progression in postmenopausal women.
Women with an increased free estradiol and SHBG, and a decreased free testosterone
benefited had the maximum reduction in CIMT progression. It is interesting to note
that free testosterone alone did not reveal a significant association with IMT
199
progression (Table V-2), but consideration of patterns of hormonal change (Figure
V-1) suggests that free testosterone may have a role in carotid IMT progression, in
conjunction with E2 and SHBG. This intriguing analysis definitely suffered from
small sample size and needs to be re-examined in a larger group of women. We will
be able to examine the dynamics of sex hormones in our participants receiving HT.
5.4. Research Design and Methods
5.4.1. Design of the Proposed Study
We propose to use existing data to conduct a post-hoc longitudinal observational
analysis to evaluate the association of serum sex steroid hormones with subclinical
atherosclerosis progression and CVD risk factors. We will measure sex steroid
hormones in stored samples of 1006 postmenopausal women participating in 5
atherosclerosis progression trials from whom 8 to 12 hour fasting blood samples
were collected at baseline and every 6 months during the trial period. We restricted
our sample to those who at least had one follow-up visit in addition to the baseline
visit because we are interested to look at progression of carotid IMT as the outcome
of interest.
200
5.4.2. Study subjects
Tables V-1 and V-6 summarize the study design and baseline characteristics of 1006
participants. In brief, all 5 clinical trials were conducted at the Atherosclerosis
Research Unit at USC. Duration of follow-up ranged from 2 to 4.5 years. There was
a homogenous distribution of age across the 5 trials. The age distribution of the
proposed study subjects has a wide variation ranging from 40 to 88 years, with a
mean of 62 years.The study sample is ethnically diverse, 57% White, 12% African
American, 21% Hispanic and 8% Asian. Forty percent (n=406) of the women were
HT users, providing a large sample sample to examine the patterns of changes in
estrogen, testosterone and SHBG in relation to carotid IMT progression among HT
users. Twenty-two percent women (n=226) with clinically evident CVD in this study
population adds further strength to this study enabling us to test the sex hormone-
atherosclerosis progression association in women with and without CVD. Only 5%
women were current smokers during the trials, 38% were former smokers. The
majority of non-smoking subjects in this sample will reduce the confounding effects
of smoking on atherosclerosis progression; the very small number of current smokers
will similarly limit the confounding effects of current smoking on sex hormone
levels.
201
5.4.3. Study Protocols
Similar trial design and data collection protocols were shared across the 5 trials.
Eligible participants based on the screening visits were randomized and then
followed up for the duration of the trial. Ultrasound examinations to measure CIMT,
laboratory tests, and sample storage were performed every 6 months. In addition to
the 6-month visits, participants were seen at the clinic once a month for the first 6
months and every 2 months thereafter for the remainder of the trial. The purpose of
the frequent clinic visits was to insure compliance to randomized interventions and
close follow-up.
5.4.4. Ancillary Data Collection
The same structured questionnaires were used in the 5 clinical trials to collect
demographic data. At baseline and scheduled on trial visits, smoking, dietary intake
(3 day dietary diary), physical activity (7 day recall) complete information on
vitamins, herbs, and other dietary supplements were collected. Detailed information
on HT use including type, dose, and duration was documented at each visit in
BVAIT and VEAPS women. In EPAT, WELLHART, and WISH, women were
required to be off of any HT use for at least a month. Previous history of HT use of
the EPAT, WELLHART and WISH participants was recorded at study entry. Blood
pressure, pulse rate, body weight, adverse events, compliance with study intervention
treatment were also recorded at baseline and each clinic visit. At the 6 month visits,
202
claudication, Rose, and 7-day physical activity recall questionnaires were
administered, and hip to waist ratios were determined. We therefore have a large
array of possible confounders, collected longitudinally and with the same methods in
all trials, to consider as adjustment variables in the proposed analyses.
5.4.5. Laboratory measurements
1) Plasma lipids: At baseline and every 6 months during the trial, the routine fasting
lipid/lipoprotein panel was performed in the Atherosclerosis Research Lipid
Laboratory by enzymatic assay methodology that is standardized to the CDC using
Lipid Research Clinic protocol. Participants fasted for 8 hours before sample
collection. Total
plasma cholesterol and triglyceride levels were measured by
using
an enzymatic method of the Standardization Program of
the National Centers for
Disease Control and Prevention. High-density
lipoprotein cholesterol levels were
measured after lipoproteins
containing apolipoprotein B were precipitated in whole
plasma
by using heparin manganese chloride. Low-density lipoprotein
cholesterol
levels were estimated by using the Friedewald equation
70
.
2) Plasma glucose, insulin, and hemoglobin A1: Carbohydrate metabolism related
factors were measured at baseline and every 6 months by UniLab in Los Angeles
California. Serum fasting insulin levels were measured
by using radioimmunoassay.
Fasting serum glucose levels were
measured by using the glucose oxidase technique
203
on a Beckman
Glucose II analyzer (Beckman Instruments, Brea, California).
Hemoglobin A
1c
levels were measured by using high-performance
liquid
chromatography (Bio-Rad Diamat, Bio-Rad Corp., Hercules,
California).
3) Chemistry panel: Blood chemistries were determined by standard analytical
methods by UniLab in Los Angeles California annually.
5.4.6. Storage of blood samples
At baseline and every 6-month study visit, 8-12 hour fasting blood samples were
collected. For each participant, plasma, serum and buffy coat samples were stored.
Samples were placed in cryogenic tubes and stored at –70
o
C in the USC Core Lipid
Laboratory.
204
Table V.6. Baseline characteristics of 1006 postmenopausal women participating in five randomized
trials.
Baseline
characteristics
EPAT WELL-
HART
VEAPS BVAIT WISH Proposed
study
Mean age (range),
Years
61.5
(48 - 80)
63.5
(48 – 75)
61.1
(44 -85)
61.4
(40 – 88)
61.0
(41-92)
61.7
(40-92)
CIMT in µm,
Mean (sd)
1
763.5
(133)
845
(219)
766
(109)
738
(116)
807
(100)
784.5
(135)
Diabetes, n(%) 5 (3) 115 (51) 0 (0) 0 (0) 0 (0) 120 (12)
CVD, n(%) 0 (0) 226 (100) 0 (0) 0 (0) 0 (0 ) 226 (22)
Ethnicity, n(%)
White 109(61) 69 (31) 85 (75) 124 (65) 186 (63) 573(57)
Black 19(11) 38 (17) 14 (12) 34 (18) 18 (6) 123(12)
Hispanic 35(19) 100 (44) 8 (7) 21 (11) 47 (16) 211(21)
Asian 17(9) 19 (8) 6 (5) 10 (5) 32 (11) 84(8)
Other 0(0) 0 (0) 1 (1) 2 (1) 12 (4) 15(1)
Education, n(%)
<12 y 27 (15) 91 (40) 3 (3) 4 (2) 12 (5) 137 (14)
≥12 y 153 (85) 135 (60) 111 (97) 187 (98) 283 (95) 869 (86)
Smoking History,
n(%)
Current smoker
2
0 (0) 26 (12) 6 (5) 8 (4) 6 (2) 46 (5)
Former smoker 87 (48) 89 (39) 34 (29) 65 (34) 112 (38) 387 (38)
Never smoked 93 (52) 111 (49) 74 (65) 118 (62) 177 (60) 573 (57)
Number (%) of
women on HT
91(51) 150 (75) 92 (77) 73(37) None 406 (40)
1
Mean CIMT at baseline
2
Current smoker is defined as having smoked daily for at least 6 months upon study entry.
5.4.7. Primary End point Measurement- Carotid Artery Intima-Media Thickness
High resolution B-mode ultrasound carotid artery images for IMT measurement were
acquired with a Toshiba SSH-140A/C ultrasound imager using a linear array 7.5
MHz probe. The ECG, time code information, and ultrasound images were
simultaneously recorded on 1/2 inch tape with a Panasonic AG-7355 SVHS
videotape recorder. Subjects were placed supine and positioned in a 45 degree
molded head block to present the optimal angle for ultrasound examination. Using
B-mode, the common carotid artery (CCA) was imaged in cross section and the
205
scanhead moved laterally until the jugular vein and the CCA were stacked with the
former above the latter. In this position, the central image line passed along the
common diameter of both vessels. The scanhead was then rotated around the central
image line 90 degrees maintaining the jugular vein stacked above the CCA while
obtaining a longitudinal view of both vessels. In this longitudinal view, the CCA far
wall was horizontal. The proximal portion of the carotid bulb was included in all
images as an anatomical reference point for standardization of IMT measurements.
Stacking the jugular vein and the CCA determines a repeatable probe angle which
allows the same portion of the wall to be imaged at each examination
71
. Minimum
gain necessary for clear visualization of structures was used and once the transducer
was positioned video tape was recorded for a minimum of 10 seconds. Images were
acquired from the carotid bulb and internal carotid artery, but emphasis of ultrasound
imaging was on the distal centimeter of the CCA because least variability occurs in
this area
72
. The far wall was used for statistical purposes since measurement of near
wall thickness was less accurate
73
. To assure security of data, each ultrasound scan
was recorded on video tape and a duplicate was made. Additionally, processed
images were stored on jaz disks.
For each subject, ultrasound input power, echo detector gain, and dynamic range
values were automatically recorded on the ultrasound image and were recorded
separately to establish identical technical conditions for each subsequent follow-up
206
examination. This established standardization for instrument setup that encompasses
the full dynamic range of the ultrasound echo across all examinations within the
same subject. If it was necessary to change these factors to obtain an adequate
follow-up examination, the electronics were checked by a Toshiba technician. In
addition, a hardcopy of each individual's baseline image was used as a guide to
match the vascular and surrounding soft tissue structures of the follow-up
examinations. This was a direct visual aid method for reproducing probe angulation.
The brightness and contrast settings of the image display were checked daily and
standardized from a video tape recorded gray-scale pattern but otherwise did not
change throughout the trial. This avoids the problem of an improperly set low
monitor brightness and contrast that leaded to excessive power or gain to obtain a
visually satisfactory image resulting in high gain noisy images unsuitable for
computer processing. These techniques resulted in significant reductions in
measurement variability between scans
58, 59
.
5.4.8. Determination of Serum Sex Hormone levels
We are proposing to measure estrogens, androgens and SHBG in the stored blood
samples collected from 826 postmenopausal women. Sex hormone levels of 180
EPAT participants are already determined. Serum sex hormone assays will be
conducted at the Reproductive Endocrine Research Laboratory at USC under the
supervision of Dr. Frank Stanczyk. Serum levels of androstenedione (A),
207
dehydroepiandrosterone (DHEA), testosterone (T), estrone (E
1
) and estradiol (E
2
)
will be quantified by validated, previously described RIAs
74
. Prior to RIA, steroids
will be extracted from serum with hexane:ethyl acetate (3:2). A, DHEA, and T will
then be separated by Celite column partition chromatography using increasing
concentrations of toluene in trimethylpentane. E
1
and E
2
will be separated in a
similar fashion by use of ethyl acetate in trimethylpentane. SHBG will be quantified
by direct chemiluminescent immunoassays using the Immulite analyzer (Diagnostic
Products Corporation, Inglewood, CA). Free T will be calculated using total T and
SHBG concentrations, and an assumed constant for albumin in a validated
algorithm
75, 76
. Free E
2
will be calculated in a similar manner. All samples from same
woman (over study visits) will be processed in batch to minimize inter-batch
variability. There will be quality control samples in each batch. Serum sex hormones
of 180 EPAT women were measured in the same endocrine lab using the exact same
assay method. All immunoassay methods are shown to be reliable. Specificity was
achieved by use of highly specific antisera and/or use of organic solvent extraction
and chromatographic steps prior to quantification of the analytes. Assay accuracy
was established by demonstrating parallelism between measured concentrations of a
serially diluted analyte in serum with the corresponding standard curve. Intraassay
and interassay coefficients of variation ranged from 4 to 8% and 8 to 13%,
respectively. All assay methods were found to be sensitive. The sensitivity of an
RIA method was determined by the smallest amount of analyte that reduced the
208
number of counts per minute of the radiolabeled analyte at zero mass by 2 standard
deviations.
5.4.9. Data co-ordination and external oversight of the randomized trials
5.4.9.1. Data co-ordination of the randomized trials: The Data Coordinating
Center for each of the 5 trials was led by Dr. Wendy Mack. All trial data remain
housed within the Statistical Consultation and Research Center (co-directed by Dr.
Mack) in the Department of Preventive Medicine at USC. All electronic data reside
on the same dedicated server, and back-up copies of all data are securely stored off-
site both locally and out-of-state using the services of an electronic data storage
company. The data entry and quality control of new data originating from this
proposal, and linkage to existing data will be supervised by Dr. Mack.
5.4.9.2. External oversight of the randomized trials: All trials were reviewed,
approved and internally monitored by the USC Institutional Review Board. All
subjects provided written informed consent for participation in the trial. For all
trials, an External Data and Safety Monitoring Board were established according to
NIH guidelines. Each committee met yearly to oversee the data collection and
management procedures and conduct and reporting of the respective trials.
209
5.4.10. Statistical Analysis
5.4.10.1. Specific aim 1: To evaluate the relationship of serum estrogens,
androgens and SHBG concentration to atherosclerosis progression in
postmenopausal women.
General Regression Model: We will use general linear mixed effects models for
longitudinal data available on 1006 postmenopausal women. To estimate the
temporal trend of carotid IMT over the trial follow-up, the entire set of baseline and
on-trial carotid IMT measures will be used to fit the mixed effects model. The serial
measures of carotid IMT will be the dependent variable and the primary explanatory
variables in the model will be the follow-up time in years (i.e., time of the CIMT
measurement from randomization), absolute changes in the serum hormone levels
and their interaction with follow-up time. The regression coefficient associated with
the main effect of follow-up time will estimate the average annual rate of IMT
progression among women whose hormone levels do not change over the trial. The
rate of carotid IMT progression associated with a per unit change in the hormone
levels will be estimated from the regression coefficient associated with the
interaction terms of: change in hormone levels x follow-up time. The test that this
regression coefficient equals zero (null hypothesis) is the test that the sex hormone is
associated with IMT progression over the trial.
210
Modeling of Hormone Measures: Each hormone will be modeled separately for its
association with carotid IMT progression. Our preferred approach will model the
hormone measures as continuous variables. Regression coefficients associated with
the continuous hormone measure will thus be interpreted as the change in the annual
IMT progression rate, per unit of the hormone measure. However, we recognize that
the associations between hormones and CIMT progression may not be linear. For
this reason, we will also model each hormone as a 4-level class variable, with the
levels defined by quartiles determined over the entire study sample. Regression
coefficients of the interaction of this class variable with follow-up time will thereby
estimate the carotid IMT progression rate in each quartile of the hormone measure.
This ordered categorical model will be the preferred approach if the relationships are
not linear. Even if the hormone-atherosclerosis associations are linear, the ordered
categorical variable will provide a descriptive estimate of IMT rates by hormone
levels. In the presence of non-linearity, we will also consider transformations of the
continuous hormone variables, such as quadratic terms.
Consideration of Confounders: Potential factors to be considered as confounders in
the analysis include age, ethnicity, smoking (current, former), BMI, waist-hip ratio,
physical activity, previous history of CVD, time since menopause, type of
menopause and prior use of HT. These variables will be tested for an association
with the sex hormones (exposure) and carotid IMT progression (outcome). Factors
211
associated with both exposure and outcome will be identified as potential
confounders and will be included in the mixed effect model as covariates. These
variables will be modeled as continuous or categorical variables based on their
nature of association ((linear or non-linear) with IMT progression. To control for
possible heterogeneity of associations among studies, a study indicator variable will
be included in the model as a random effect.
Multivariate Hormone Models: Multivariate hormone models will include
estrogen, testosterone and SHBG together in the model to test the independence of
associations. Possible interactions among estrogen, testosterone and SHBG will be
evaluated (e.g., to test if the associations of estrogens with carotid IMT progression
differs by the level of testosterone) with the inclusion of product interaction terms
considering the hormones as continuous as well as ordered categorical variables as
described above.
Among women using HT (n=406), the sex hormone changes over the follow-up will
be categorized by using the distribution of within-subject SD (over repeated
measures) of the sex hormone in women not using HT. For each woman, we will
categorize each hormone as ‘increasing’ (>1 within-subject SD increase),
‘decreasing’ (<1 within-subject SD decrease), and ‘no change’ (within ± 1 within-
212
subject SD). Categorical sex hormone change variables will be tested in different
combinations for an association with CIMT progression using mixed effects models.
Subgroup Analyses: Subgroup analyses will use the same statistical approach
described above. Stratified analysis will be performed in 780 women who did not
have clinical CVD and 226 women with pre-existing CVD at the beginning of
follow-up.
5.4.10.2. Specific aim 2: To assess the association between sex hormones and
risk factors of CVD including cholesterol, triglycerides, glucose, insulin and
glycosylated hemoglobin longitudinally and relate these associations to
atherosclerosis progression.
General Regression Model: Time dependent data on serum lipids and carbohydrate
related factors will be correlated with sex hormone levels measured at the same visit
using linear regression model with Generalized Estimating Equations (GEE). Using a
GEE approach will account for the within individual correlation in the measures. An
exchangeable correlation matrix will be initially assumed between the repeated
measures of sex hormones over time points. We have generally found an
exchangeable correlation matrix to be appropriate for these data in our preliminary
analyses of EPAT data. However, we will explore the within-subject correlations in
213
each of these CVD risk factors (dependent variables) over trial visits, to determine
the most appropriate working correlation matrix. In sensitivity analyses, we will also
assess if our regression model results (estimates, standard errors) are substantially
altered using alternative working correlation structures.
Modeling of Hormone Measures: Each hormone will be modeled separately for its
association with each CVD risk factors. Our preferred approach will model the
hormone measures as continuous variables. Regression coefficients associated with
the continuous hormone measure will thus be interpreted as the change in the CVD
risk factor per unit of the hormone measure. However, we recognize that the
associations between hormones and CVD risk factors may not be linear. For this
reason, we will also model each hormone as a 4-level class variable, with the levels
defined by quartiles determined over the entire study sample.
Consideration of confounders: Age, race, smoking, BMI, physical activity,
previous history of CVD, time since menopause, and type of menopause will be
tested for potential confounding effects. Only factors associated with both serum
hormone levels and CVD risk factors will be included in the model as an adjustment
variable. To control for possible heterogeneity of associations among studies, a study
indicator variable will be included in the model as a random effect.
214
5.4.10.3. Specific aim 3: To evaluate the extent to which associations between
sex hormones and CVD risk factors mediate the associations between sex
hormones and atherosclerosis.
To test the mediating effect of lipids and carbohydrate related factors in the
association between sex hormones and carotid atherosclerosis progression, estimates
will be compared between models with and without the mediating factors in it. The
mediating factors will be included in the models one at a time as a covariate and
changes in the β-estimates associated with atherosclerosis progression will be
compared to the model without the mediating factor. Factors causing >10% change
in the β-estimates will be considered a significant intermediate factor.
5.4.11. Power Considerations
We computed the minimum detectable correlations for various study samples at a 2-
sided α of 0.05 and 80% power. For continuous analyses of study variables using the
entire sample (n=1006), we will be able to detect correlations of at least 0.09. We
will have 90% power to detect a correlation of 0.11 in the total sample of 1006.
Table V-7 shows the power to detect the correlations, observed between sex
hormones and IMT progression in the EPAT women, in the proposed study. With the
proposed sample, we will have 99% power to detect the meaningful correlations
observed between IMT progression and estrogens, total testosterone and SHBG in
EPAT.
215
Table V.7. Correlation between sex hormones and IMT progression rate in EPAT and power to detect
in the proposed study.
Hormones Correlation with IMT progression in
EPAT (N=180)
[r]
Power to detect in the proposed study
(N=1006)
Estrone 0.16 99
Total estradiol 0.17 99
Free estradiol 0.15 99
Total testosterone 0.13 98
Free testosterone 0.03 15
Androstenedione 0.04 24
DHEA 0.02 9
SHBG 0.20 99
5.4.12. Timeline
We project to complete the sex hormone assays during the 1
st
grant year. Data entry
and cleaning is also expected to be completed within the same year. During the 2
nd
year, data analysis and manuscript writing related to specific aim 1 will be worked
on. Specific aim 2 and 3 will be focused in the last grant year.
5.5. Exempt Human Subjects Research
We are claiming exemption for human subject research. Serum levels of sex
hormones will be measure in the stored blood samples collected as part of the
clinical trials under written informed consent for collection and future use of these
samples. We will be using existing data on CIMT and potential risk factors of CVD
from 4 completed and 1 ongoing clinical trial. Subject identification of these data is
by a study specific numeric identifier that is not directly linked to the individual. No
subject-specific personal identifiers such as social security number or patient
216
identifier are used in the database. The computerized databases are well secured with
protected passwords and limited access to authorized personnel only. The hard
copies of the data are also well protected from public access under the supervision of
Dr. Mack.
5.6. Inclusion of Women
This study involves women only because of specific research questions related to
postmenopausal women. All the women included in this study are postmenopausal.
5.7. Inclusion of minorities
The study population has ethnic diversity. 16% of the women are African-American,
and 19% are from Hispanic ethnicity.
5.8. Inclusion of Children
This proposal does not include individuals under the age of 21 years since it is not
relevant to children. This is a study of postmenopausal women only.
5.9 Vertebrate Animals
Vertebrate animals are not involved.
217
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Abstract (if available)
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Asset Metadata
Creator
Karim, Roksana
(author)
Core Title
Sex hormones and atherosclerosis in postmenopausal women
School
Keck School of Medicine
Degree
Doctor of Philosophy
Degree Program
Applied Biostatistics
Publication Date
03/23/2007
Defense Date
12/01/2006
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
atherosclerosis,Lipids,OAI-PMH Harvest,sex hormones
Language
English
Advisor
Mack, Wendy J. (
committee chair
), Crimmins, Eileen M. (
committee member
), Hodis, Howard Neil (
committee member
), Pearce, Celeste Leigh (
committee member
), Stanczyk, Frank Z. (
committee member
)
Creator Email
rkarim@usc.edu
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329023
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Karim, Roksana
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texts
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Repository Email
cisadmin@lib.usc.edu
Tags
atherosclerosis
sex hormones