UNIVERSITY OF CALGARY

UNIVERSITY OF CALGARY
Evaluating the Association between Estradiol and Quality of Life and Cardiovascular Risk and
Mortality in Healthy Women and Women with Chronic Kidney Disease
by
Sharanya Ramesh
A THESIS
SUBMITTED TO THE FACULTY OF GRADUATE STUDIES
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
GRADUATE PROGRAM IN MEDICAL SCIENCE
CALGARY, ALBERTA
AUGUST, 2016
© Sharanya Ramesh 2016
Abstract
Chronic kidney disease (CKD) is associated with a poor quality of life and high risk of
cardiovascular (CV) mortality, specifically sudden cardiac death (SCD), and an upregulated
renin angiotensin system. Women with end stage kidney disease (ESKD) experience premature
menopause, and in healthy women menopause is correlated with a poor quality of life and higher
CV mortality. A series of studies was conducted in healthy women and women with CKD to
determine the associations between menopause status, serum estradiol and 1) cardiac autonomic
tone (CAT), a surrogate marker for SCD, in a high Angiotensin II (AngII) state 2) mortality in
women with ESKD and 3) quality of life(QoL) in women with CKD. We also summarized the
impressions of healthcare workers and patients on the discussion of symptoms of low sex
hormones in a clinical setting. In healthy men and women, sex hormones did not correlate with
baseline CAT; however, men with lower testosterone levels were unable to maintain CAT in
response to AngII. At baseline, postmenopausal women had a lower CAT in comparison to
premenopausal women. In response to AngII postmenopausal women and premenopausal
women in the luteal phase were unable to maintain their CAT. Through a survey of nephrologists
we found that nephrologists recognize the impact of CKD on sex hormones in women but report
infrequently discussing sex hormone related issues with patients. In a systematic review of
studies examining the effect of postmenopausal hormone therapy on CV outcomes in women
with ESKD, hormone therapy was associated with a favourable lipid profile. However, we found
that peri- and premenopausal women with ESKD on hemodialysis had a higher risk of all-cause,
cardiovascular and non-cardiovascular mortality compared to postmenopausal women.
Furthermore menopause specific QoL scores did not correlate with kidney function in CKD
ii
women. We found that associations between menopause status and CV risk and QoL in the CKD
population are complex. This body of work can be used for hypothesis generation for future
studies and trials aimed to determine the mediators of cardiovascular risk and poor quality of life
in this population.
iii
Acknowledgements
I am very thankful for the opportunity to have spent the past four years of my life
studying this important area of research, which I am very passionate about. This would not have
been possible without the support and guidance of incredible people. First, I would like to thank
my supervisor and my valued mentor, Dr. Sofia Ahmed, from the bottom of my heart. Her
unwavering support, mentorship, kindness, guidance, and trust have carried me through these
four years, and this thesis would not have been possible without her. I would also like to extend a
heartfelt thank you to my thesis committee members, Dr. Jayna Holroyd-Leduc, Dr. Stephen
Wilton, and Dr. Matthew James, for their commitment to my academic and personal
development, valuable input and time, and mentorship throughout the years.
To my parents, Mallika and Ramesh, thank you so much for instilling in me the values of
hard-work, resilience and a passion for learning, without your love and support I would not be
who I am today. A very special thank you to my sister, Sanjana, who has been my rock for the
past few years and who held my hand through many doubtful moments, you continue to amaze
me with your understanding and kindness. To my friends near and far, who have always been
there to keep me grounded and supported, I am so thankful for your uplifting messages, votes of
confidence and wonderful company.
Last but not least, I would like to express my deep gratitude to the patients who
volunteered to participate in my studies, and the nurses, nephrologists and administrators of the
Southern Alberta Renal Program who took the time out of their busy schedules to help with my
project. Your kindness will not be forgotten.
iv
Table of Contents
Abstract ............................................................................................................................... ii
Acknowledgements ............................................................................................................ iv
Table of Contents .................................................................................................................v
List of Tables .................................................................................................................... vii
List of Figures and Illustrations ....................................................................................... viii
List of Symbols, Abbreviations and Nomenclature .............................................................x
CHAPTER ONE: INTRODUCTION ..................................................................................1
1.1 Chronic Kidney Disease ............................................................................................1
1.1.1 Chronic kidney disease prevalence and burden of disease ................................1
1.1.2 Physical and psychological symptoms of uremia ..............................................1
1.1.3 Reproductive dysfunction, sexual dysfunction and premature menopause in
women with CKD ..............................................................................................2
1.1.4 Cardiovascular (CV) mortality in chronic kidney disease ................................2
1.2 Menopause and the role of estradiol ..........................................................................4
1.2.1 Menopause associated poor quality of life and high cardiovascular risk ..........4
1.2.1.1 Menopause and quality of life .................................................................5
1.2.1.2 Menopause and cardiovascular risk .........................................................5
1.2.2 Estradiol .............................................................................................................6
1.2.3 Normal hypothalamus pituitary gonadal axis ....................................................6
1.2.4 Endogenous estradiol on quality of life .............................................................8
1.2.5 Cardioprotective role of endogenous estradiol ..................................................9
1.2.6 Cardiovascular risk and exogenous estradiol ..................................................10
1.2.7 WISE classification for menopausal status .....................................................12
1.3 Abnormalities in the hypothalamic pituitary gonadal axis in CKD.........................13
1.4 Assessment of Cardiovascular Risk, Quality of Life and Menopausal Symptoms .15
1.4.1 Heart rate variability (HRV) as a predictor of CVD risk ................................15
1.4.2 Using the Menopausal Specific Quality of Life (MENQOL) survey to determine
menopausal symptoms .....................................................................................17
1.5 Current state of the science ......................................................................................17
1.6 Objective ..................................................................................................................18
1.7 Thesis Outline ..........................................................................................................19
CHAPTER TWO: TESTOSTERONE IS ASSOCIATED WITH THE CARDIOVASCULAR
AUTONOMIC RESPONSE TO A STRESSOR IN HEALTHY MEN ...................20
CHAPTER THREE: CARDIAC AUTONOMIC RESPONSE TO ANGIOTENSIN II IN
HEALTHY PREMENOPAUSAL AND POSTMENOPAUSAL WOMEN............39
CHAPTER FOUR: SEX HORMONE STATUS IN WOMEN WITH CHRONIC KIDNEY
DISEASE: SURVEY OF NEPHROLOGISTS’ AND RENAL ALLIED HEALTH
CARE PROVIDERS’ PERCEPTIONS. ...................................................................59
v
CHAPTER FIVE: HORMONE THERAPY AND CLINICAL AND SURROGATE
CARDIOVASCULAR ENDPOINTS IN WOMEN WITH CHRONIC KIDNEY
DISEASE: A SYSTEMATIC REVIEW AND META-ANALYSIS .......................80
CHAPTER SIX: MENOPAUSE STATUS IS ASSOCIATED WITH MORTALITY IN
WOMEN ON HEMODIALYSIS IN CANADA ....................................................101
CHAPTER SEVEN: CHARACTERIZATION OF SEX HORMONE LEVELS,
MENOPAUSAL STATUS AND MENOPAUSE-SPECIFIC QUALITY OF LIFE IN
WOMEN WITH CHRONIC KIDNEY DISEASE .................................................122
CHAPTER EIGHT: CONCLUSIONS ............................................................................142
8.1 Summary of Findings.............................................................................................142
8.2 Implications ...........................................................................................................144
8.3 Recommendations for future research ...................................................................147
REFERENCES ................................................................................................................149
APPENDICES .................................................................................................................171
vi
List of Tables
Table 1-1 Heart rate variability measurements and their representations ..................................... 16
Table 2-1 Baseline Characteristics................................................................................................ 35
Table 3-1 Baseline characteristics of premeopausal and postmenopausal women....................... 51
Table 3-2 Sex hormone levels in premenopausal women (follicular vs. luteal phase)................. 52
Table 3-3 Baseline cardiac autonomic tone and autonomic response to AngII infusion in
premenopausal versus postmenopausal women .................................................................... 53
Table 3-4 Baseline cardiac autonomic tone and autonomic response to AngII infusion in
premenopausal women in the follicular vs luteal phase ....................................................... 54
Table 4-1 Baseline Characteristics of Nephrologists and medical trainees in nephrology........... 75
Table 5-1 Study Characteristics .................................................................................................... 94
Table 5-2 Randomized controlled trial and observational studies risk of bias assessment .......... 95
Table 6-1 Baseline Characteristics.............................................................................................. 115
Table 6-2 Unadjusted and adjusted risk of all-cause mortality, cardiovascular mortality and
non-cardiovascular mortality by menopause status ............................................................ 116
Table 6-3 Unadjusted and adjusted risk of all-cause mortality, cardiovascular mortality and
non-cardiovascular mortality by median estradiol levels ................................................... 117
Table 7-1 Baseline Characteristics.............................................................................................. 136
Table 7-2 Serum sex hormone levels across CKD specturm ...................................................... 137
Table 7-3 Impressions of patients on discussion of symptoms associated with low sex
hormone with nephrologist ................................................................................................. 138
vii
List of Figures and Illustrations
Figure 1-1Risk of all cause mortality, cardiovascular mortality and cardiovascular events
with declining kidney function(Go, Chertow et al. 2004) ...................................................... 4
Figure 1-2 Normal Hypothalamic Pituitary Axis............................................................................ 7
Figure 1-3 Effect of loss of pulsatile GnRH secretion(Belchetz, Plant et al. 1978) ....................... 8
Figure 1-4 Abnormal sex hormone profile in CKD(Ahmed and Ramesh 2016) .......................... 14
Figure 2-1Cardiac autonomic tone at baseline and in response to AngII in men and women ..... 36
Figure 2-2Association between cardiac sympathetic and vagal response to 6ng/kg/min of
Ang II infusion and serum testosterone in men .................................................................... 37
Figure 2-3: LF(nu) and HF(nu) response to 60min of Angiotensin II challenge in men,
stratified by testosterone level............................................................................................... 38
Figure 3-1 Baseline HRV in premenopausal and postmenopausal women .................................. 55
Figure 3-2 : Response to 6ng/kg/min AngII infusion in A) premenopausal and
B)Postmenopausal women .................................................................................................... 56
Figure 3-3 Baseline HRV in premenopausal follicular and luteal phase ...................................... 57
Figure 3-4Response to 6ng/kg/min AngII infusion in A) Follicular and Luteal phase ................ 58
Figure 4-1 Nephrologist impression of sex hormone status in CKD ............................................ 76
Figure 4-2 Nephrologist impression on patient discussion of sex hormone status in CKD ......... 77
Figure 4-3 : Nephrologist impression on hormone therapy in CKD ............................................ 78
Figure 4-4 Nephrologist impression on factors to consider for hormone therapy in CKD .......... 79
Figure 5-1 PRISMA flow diagram showing the identification process for eligible studies ......... 96
Figure 5-2Forest Plot of the effect of hormone therapy on LDL cholesterol ............................... 97
Figure 5-3Forest plot of the effect of hormone therapy on HDL cholesterol ............................... 98
Figure 5-4 Forest plot of the effect of hormone therapy on total cholesterol ............................... 99
Figure 5-5 Forest plot of the effect of hormone therapy on triglyceride levels .......................... 100
Figure 6-1 CONSORT Diagram ................................................................................................. 118
Figure 6-2 Kaplan Meier survival curve all-cause mortality stratified by menopause status .... 119
viii
Figure 6-3 Kaplan Meier survival curve cardiovascular mortality stratified by menopause
status ................................................................................................................................... 120
Figure 6-4 Kaplan Meier survival curve for non-cardiovascular mortality stratified by
menopause status ................................................................................................................ 121
Figure 7-1 MENQOL score stratified by CKD Stage ................................................................. 139
Figure 7-2 Correlations between GFR and MENQOL scores .................................................... 140
Figure 7-3 Correlations between estradiol and MENQOL scores .............................................. 141
ix
List of Symbols, Abbreviations and Nomenclature
Symbol
ANCOVA
Ang II
BMI
CAT
CEE
CHD
CKD
CKDCS
CV
CVD
DBP
ERα
ERβ
ESKD
FSH
GnRH
HDL
hERG
HERS
HF
HT
ISN
LDL
LF
LH
MENQOL
QoL
RAS
RCT
SBP
SCD
VMS
WHI
WISE
Definition
Analysis of Co-variance
Angiotensin II
Body Mass Index
Cardiac Autonomic Tone
Conjugated Equine Estrogen
Coronary Heart Disease
Chronic Kidney Disease
Canadian Kidney Disease Cohort Study
Cardiovascular
Cardiovascular disease
Diastolic Blood Pressure
Estrogen Receptor Alpha
Estrogen Receptor Beta
End Stage Kidney Disease
Follicle Stimulating Hormone
Gonadotropin Releasing Hormone
High Density Lipoprotein
Human Ether A-Go-Go
The Heart and Estrogen/Progestin Replacement
Study
High Frequency
Hormone Therapy
International Society of Nephrology
Low Density Lipoprotein
Low Frequency
Luteinizing Hormone
Menopause Specific Quality of Life
Quality of Life
Renin Angiotensin System
Randomized Controlled Trial
Systolic Blood Pressure
Sudden Cardiac Death
Vasomotor Symptom
Women’s Health Initiative
Women’s Ischemia Syndrome Evaluation
x
Chapter One: Introduction
1.1 Chronic Kidney Disease
1.1.1 Chronic kidney disease prevalence and burden of disease
Chronic kidney disease (CKD) is a growing public health concern affecting approximately
3 million Canadian adults 1. The quality of life is low in CKD patients regardless of the
stage of CKD, specifically with physical functioning, pain, general health, emotional health
and mental health2. Additionally, CKD is associated with high risk of mortality and poor
cardiovascular outcomes 3, 4. Approximately 25% of deaths in individuals with renal failure
can be attributed to cardiovascular causes 5, 6. Additionally, low glomerular filtration rate
(GFR) and high albuminuria are independently associated with worse cardiovascular
outcomes7.
1.1.2 Physical and psychological symptoms of uremia
Uremia is the term used to describe signs and symptoms that accompany kidney failure
that cannot be attributed to an alternative cause8. Studies have found that in patients with
kidney disease physical symptoms such as fatigue, lack of stamina, cramps, restless legs
and sleep disturbances are common8. A study of 1,284 patients with CKD (40% women)
found a higher prevalence of fatigue and cramps with worsening kidney disease9, 10;
moreover, women with kidney disease are more likely to have worse quality of life
associated with physical symptoms of uremia compared to men9. In addition to fatigue,
women on dialysis have also been found to have a high prevalence of sleep disorders
including insomnia, sleep apnea, and restless leg syndrome11. Similarly, mood disorders
1
such as anxiety and depression are prevalent in the chronic kidney disease population10, 12,
13
.
1.1.3 Reproductive dysfunction, sexual dysfunction and premature menopause in women with
CKD
Menstrual abnormalities, poor fertility, and sexual dysfunction are common in uremic
women with CKD14-18. A study of 100 women with CKD reported that 88% had menstrual
problems or were menopausal, with 20% of menopausal women being < 40 years of age 14.
A previous cross-sectional study examining gynecological issues in 76 women on dialysis
showed that 59% of women reported irregular menses15. Furthermore, fertility rates among
women of child-bearing age with CKD are low 16-18 and complications to both mother and
fetus are high when pregnancy occurs19. Additionally, a meta-analysis of women with
CKD found that the prevalence of sexual dysfunction, as defined by decreased sexual
desire and satisfaction, ranged from 30-80%20. There is, therefore, evidence to suggest that
women with CKD demonstrate have a significant burden of disease that is attributed to
uremia.
1.1.4 Cardiovascular (CV) mortality in chronic kidney disease
Patients with CKD have a high burden of mortality with a 20% annual mortality rate
specifically for patients on dialysis21. This is particularly characterized by a higher risk of
CV mortality in patients with CKD. As kidney function declines CV disease and CV
mortality increases proportionally6 (Figure 1-1), with approximately 25% of deaths in the
end stage kidney disease (ESKD) population being attributed to CV causes. The leading
cause of CV death in the dialysis population is sudden cardiac death (SCD) which accounts
for more than 25% of cardiovascular deaths in this population22. Conventional CV disease
treatments and therapy are not effective in the CKD population. There is evidence that the
2
pathology of CV disease, and in particular SCD, is different in the CKD population and
options for prevention of SCD are not effective in these patients 23, 24. Moreover, traditional
therapies and treatments used to prevent CV mortality in the general population such as
coronary artery revascularization25, agents that affect the renin angiotensin system26, lipidlowering treatments27-29, digoxin30 and antiarrhythmic drugs31 are not effective in this high
risk population. A better understanding of the cardiovascular risk factors and novel
treatments is required in the CKD population.
3
Figure 1-1Risk of all cause mortality, cardiovascular mortality and cardiovascular events
with declining kidney function6
1.2 Menopause and the role of estradiol
1.2.1 Menopause associated poor quality of life and high cardiovascular risk
Menopause is defined as a cessation of menstrual periods, the cause of which can be
natural (attrition of ovarian follicles), surgical (ex. oophorectomy) or pathological (ex.
chronic kidney disease). Additionally, menopause is associated with ovarian atrophy and a
decrease in estradiol levels32. The increased cardiovascular risk and decreased quality of
4
life associated with menopause33-36 make this change of sex hormone status an important
time point in the care of the female patient.
1.2.1.1 Menopause and quality of life
Natural or surgical menopause in women is accompanied by vasomotor symptoms (VMS).
This occurs in approximately 80% of women, and frequent and severe VMS have been
found to significantly affect health related quality of life35 37 35, 38
Postmenopausal women also experience sleep disturbances in the presence and absence of
hot flashes. Studies have found that the prevalence of sleep disturbance ranges from 32 to
46 percent in postmenopausal women, compared to 15 percent in the general population 37
39
. Additionally, postmenopausal women are 3.4 times more likely to report sleep disorders
compared to premenopausal women 40.
Multiple studies have also reported a significant increase in depression during menopausal
transition compared to premenopausal years, indicating an important role of estradiol in
mood-related disorders 41-45. A recent study reported a 2 to 4 fold higher risk of depression
in perimenopausal and early postmenopausal women 36.
1.2.1.2 Menopause and cardiovascular risk
Menopause affects multiple CV disease risk factors which includes changes in body fat
distribution to an android pattern 46, decreased tolerance to glucose 47, unfavourable lipid
profile48, increased blood pressure49, increased sympathetic tone50, and endothelial
dysfunction51. The prevalence of cardiovascular disease (CVD) significantly increases with
age in men; however, women have a blunted increase in CVD until menopause52 . After
menopause the CVD risk in women increases significantly to match or exceed the risk in
men52. Menopause at a younger age is associated with increased CV risk 53 in the general
population. Furthermore, there is evidence to suggest the early loss of estradiol, through
5
early menopause, in the general population is associated with higher overall mortality. In a
cohort of 12,134 women, later menopause was associated with a 2% increase in risk per
year delay in menopause onset 54. Additionally, surgical menopause in women under the
age of 45 years was associated with a 67% increase in mortality compared to controls 55.
1.2.2 Estradiol
Estradiol is a lipophilic, steroid hormone which is primarily involved in the development
of primary and secondary sexual characteristics, regulation of ovulation, and the menstrual
cycle in women56. Estradiol has also been found to influence various other functions in the
body in both men and women. First, estradiol is required for the proper functioning of the
thermoregulatory system, and a depletion of estradiol results in moderate to severe
vasomotor symptoms in women 57. Second, estradiol is required for the synthesis of
serotonin and noradrenaline, both of which are key neurotransmitters in the maintenance of
normal sleep-wake cycles and mood regulation 58. Finally, estradiol is considered to be
cardioprotective due to its vital role in the control of inflammation59, 60 , NO production60,
61
, vasodilation62, and lipid metabolism63-69.
1.2.3 Normal hypothalamus pituitary gonadal axis
The pulsatile secretion of gonadotropin releasing hormone (GnRH) from the hypothalamus
stimulates the secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH)
from the pituitary. These hormones in turn cause the ovarian secretion of estradiol (Figure
1.2). Furthermore, estradiol negatively or positively affects GnRH secretion in the follicular
or luteal phase respectively. Along with estradiol and progesterone, prolactin, a pituitary
hormone, inhibits secretion of GnRH through negative feedback. The pulsatile secretion of
GnRH is required for the secretion of LH and FSH and the subsequent release of estradiol
(Figure 1.3).
6
Figure 1-2 Normal Hypothalamic Pituitary Axis70
7
Figure 1-3 Effect of loss of pulsatile GnRH secretion71
1.2.4 Endogenous estradiol on quality of life
Estradiol plays a vital role in multiple processes that have an effect on the quality of life of
women. Initial findings of an increase in vasomotor symptoms after estradiol withdrawal
led to studying the role of estradiol in thermoregulation. The core body temperature is
maintained through an intricate system that involves heat afferents in the core body and
skin, the hypothalamus and other regulatory areas in the central nervous system, and the
peripheral efferents.
In premenopausal women the core body temperature is maintained in a thermoneutral zone,
if body temperature deviates higher or lower from the thermoneutral zone, processes such
as sweating and shivering are activated accordingly 72. A normal concentration of estradiol
is required for all three components of the thermoregulatory system to function
appropriately. First, postmenopausal women have a smaller thermoneutral zone compared
to premenopausal women, providing evidence of estradiol deficiency affecting the afferent
input 73. Second, the thermoregulatory parts of the hypothalamus are very sensitive to
estradiol, which is evidenced by the high density of estradiol receptors in these areas 57 74 .
8
Third, estradiol plays a role in the control of skin blood flow and peripheral vasculature,
thereby affecting the ability of the peripheral thermoregulatory efferents to respond quickly
and appropriately to stimuli 75.
There is also considerable evidence that suggests a role for estradiol on sleep regulation.
Estradiol is involved in the metabolism of norepinephrine, serotonin and acetylcholine, all
of which are modulatory neurotransmitters of the circadian sleep-wake cycle 58 76.
Treatment with estradiol increases rapid eye movement (REM) cycles 77 78 and total sleep
time 79 77, and decreases sleep latency78 and number of awakenings during the night 79.
Finally, estradiol plays a role in the maintenance serotonin and noradrenaline levels in the
brain, which are important neurotransmitters of mood regulation. Estradiol increases
serotonin synthesis by increasing the production of tryptophan hydroxylase and decreases
serotonin degradation by decreasing the activity of monoamine oxidase A and B 80-82.
Moreover, estradiol also increases the serotonin receptor density in the hypothalamus,
prefrontal cortex and hippocampus 83-85. Similarly, estradiol increases norepinephrine
synthesis by upregulating the gene transcription of an important norepinephrine synthesis
enzyme, dopamine β hydroxylase86.
1.2.5 Cardioprotective role of endogenous estradiol
Estradiol is known to have multiple effects on the cardiovascular system. These include,
but are not limited to, the regulation of ion channels in the heart, contractile proteins,
reactive oxygen species production, endothelial NO synthase (eNOS) production,
cyclooxygenase production, stem cell and cardiomyocyte survival87. These discoveries of
the function of estradiol in the regulation of important cardiovascular processes have led to
the hypothesis that estradiol may have a protective function in the cardiovascular system
and in various cardiovascular pathologies. Some possible mechanisms are listed below.
9
First, estradiol via its actions on estrogen receptor-α (ER-α) has been shown to promote reendothelialization in mice 88. Endothelial damage is a key step in the development of an
atherosclerotic plaque, and endothelial integrity is important in the prevention of plaque
formation 88. Secondly, estradiol is found to prevent smooth muscle proliferation and
matrix deposition, which in turn impedes media/blood vessel thickening89. Lastly, estradiol
treatment has been found to directly inhibit and slow down the growth of an established
atherosclerotic plaque in mice 90.
Second, estradiol plays in integral role in regulating the level and activity of various ion
channels. It regulates the expression of K+ channels 91, human ether a-go-go (hERG)
channels (which are implicated in the pathophysiology of long QT syndrome)92, Na+-Ca2+
exchanger93, and L-type Ca2+ channels94. In addition, sex differences in the storage of
Ca2+ in the sarcoplasmic reticulum have been observed95. All the above indicate that
estradiol levels can control the number of ion channels in cardiac myocytes, and therefore
directly and indirectly control the contractility and the activity level of cardiomyocytes.
Third, research has shown that estradiol alters contractility by controlling the activation of
cardiac myocytes and by regulating cardiac hypertrophy91-95. Animal studies have shown
that mice that are treated with estradiol have decreased cardiac hypertrophy which is
mediated by ER-β70, 96. Fourth, estradiol administration has been found to significantly
reduce the size of a myocardial infarction in several animal studies97, 98. Possible
mechanisms for this cardioprotective effect are related to the role of estradiol in increased
stem cell99 and cardiomyocyte survival100.
1.2.6 Cardiovascular risk and exogenous estradiol
Despite overwhelming molecular evidence indicating the benefits of estradiol in the
cardiovascular system, use of exogenous estradiol in the form of postmenopausal hormone
10
therapy (HT) as a cardioprotective measure in humans is widely debated. In 1992, the
American College of Physicians recommended the use of HT for women at risk for
coronary heart disease101; however, the results of the 2002 Women’s Health Initiative
studies (WHI) were contrary to the results from the observational studies. It was observed
in the WHI study that the occurrence of coronary heart disease, stroke, and pulmonary
embolism102 was higher in women who were taking a combination of estradiol and
progestin and the occurrence of stroke was higher in women who were taking estradiol103
as compared to controls. The Heart and Estradiol/ Progestin Replacement Study (HERS)
was another randomized controlled trial that determined the effect of estradiol and
progesterone treatment on the incidence of myocardial infarction and coronary artery
disease. After an average 4.1 year follow up, no difference was found between the
treatment and control104.
The difference between the results of observational studies, suggesting the beneficial roles
of estradiol, and the randomized controlled trials (RCTs), suggesting harm, could be
attributed to various different factors. It is important to note that in observational studies
lifestyle differences, type of HT used, different diets, and various other factors could not be
controlled for. There were also some significant differences found in the hormones used
between the women enrolled in the observational study and those in the RCTs. In the
observational studies, most women used estradiol alone105-108 where as in the WHI and
HERS trials estradiol and progesterone combination treatment was used102-104.
Progesterone has been found to attenuate both lipid lowering65 and anti-atherosclerotic109
effect of estradiol. Additionally, the time of HT initiation has also been found to be an
important factor in the cardioprotective effect of HT. Studies have found that women who
11
start HT closer to menopause have a lower risk for coronary heart disease (CHD) as
compared to women who start HT later102, 110. The type of estradiol used was also found to
contribute to the difference in results observed between the observational studies and
RCTs. Conjugated equine estradiol (CEE) were used for both WHI trials and HERS102-104.
CEE is extracted from the urine of horses and contain a mixture of equine estradiol, weak
estradiolic molecules, and estrone sulfates with only 3% estradiol111. The effects of CEE,
therefore, may be very different from that of estradiol.
The abovementioned factors and various others such as route of administration of estradiol
and type of progestin used as co-treatment highlight the disadvantages of using exogenous
estradiol to determine its cardioprotective role112. There are multiple different forms of
estradiol and progesterone, regimens of HT, and routes of administration which introduce
multiple confounders into the study design113-115. The proposed study will aim to eliminate
these potential confounders by examining the role of endogenous estradiol in healthy men
and women and women with chronic kidney disease.
1.2.7 WISE classification for menopausal status
The women’s Ischemia Syndrome Evaluation (WISE) study derived an algorithm for nonmenstruating women to determine hypothalamic hypoestradiolemia.116. Based on the WISE
algorithm, women are classified as follows: 1) premenopausal if (a) FSH<10 IU/L or (b)
FSH between 10-20IU/L and estradiol>184pmol/L, 2) perimenopausal if (a) FSH 10-20
IU/L and estradiol <184 pmol/L (b) FSH 20-30 IU/L (c) FSH>30 IU/L and estradiol ≥ 184
pmol/L or (d) age≥45 years and estradiol >743 pmol/L and 3) postmenopausal if FSH >30
IU/L and estradiol <184 pmol/L.
The WISE classification has not been validated in the CKD population; however, in
comparison the commonly used Stages of Reproductive Aging Workshop (STRAW)
12
guidelines uses menstrual cycle regularity as the only principal criteria to determine
menopausal status117. Based on previous studies, irregular menses is common among
women with CKD14, 15, therefore categorization of menstrual status based on sex hormone
levels may be more applicable in this population. Furthermore, self-reported regularity of
menstruation is unreliable, subjective and is variable based on cultures, using reproductive
hormone levels (through the WISE algorithm) to determine menopausal status in women
with CKD may be more reliable118-121.
1.3 Abnormalities in the hypothalamic pituitary gonadal axis in CKD
In patients with CKD, disruptions in the GnRH production results in an abnormal sex
hormone profile (Figure 1.4). Abnormal production of GnRH, LH and FSH cause low
levels of estradiol 122 123, 124. The lack of pulsatile GnRH secretion due to kidney disease
has been demonstrated in both rats 125 126 and humans 127 128. While the pathophysiology
leading to abnormal GnRH secretion in patients with CKD is unclear, Rathi et al suggest
the role of impaired calcium and phosphate metabolism 129. This hypothesis is supported
by the calcium dependence of GnRH production and secretion 130 131. Furthermore,
treatment with clomiphene citrate has been found to restore HPG axis function in both
women128and men 132 on dialysis.
The pulsatile secretion of GnRH is further inhibited by hyperprolactinemia in patients with
CKD 133. In healthy individuals, pituitary prolactin secretion is chronically supressed by
the hypothalamus through dopamine. With worsening CKD the dopamine mediated
inhibition is compromised resulting in high levels of prolactin 133 134. Moreover, the
excretion of prolactin is lower in patients with CKD 135. This high concentration of
prolactin has a negative feedback on hypothalamic GnRH secretion, thereby decreasing
13
estradiol secretion in women with CKD 136. Treatment with bromocriptine in three women
with ESKD, however, did not result in a consistent effect on the HPG axis 128.
Menstrual abnormalities, poor fertility and sexual dysfunction are common in uremic
women with CKD14-18. However, unlike menopause in the general population, these
abnormalities in women with CKD is often reversed by intensive hemodialysis137 and
kidney transplantation138.
Figure 1-4 Abnormal sex hormone profile in CKD139
14
1.4 Assessment of Cardiovascular Risk, Quality of Life and Menopausal Symptoms
1.4.1 Heart rate variability (HRV) as a predictor of CVD risk
Heart rate is controlled by the autonomic nervous system (ANS), and in a healthy
individual there is a predominance of cardioprotective parasympathetic or vagal tone in the
neural control of heart rate (HR)140. HRV analysis considers the fluctuations in HR during
a certain period of time and can be quantified using a time domain or a frequency domain
analysis141. In time domain analysis the length of intervals between adjacent R waves is
measured and the standard deviation of successive RR intervals (SDNN) is calculated. This
helps determine HRV such that a higher SDNN is indicative of a higher HRV141. The data
collected from the ECG can also be transformed to a power-frequency plot where the
power of each component of heart rate modulation (sympathetic, vagal, baroreceptor etc.)
can be measured141. High frequency (HF) is characterized as a frequency of 0.15-0.40 Hz
and represents the vagal limb of the ANS. On the other hand, low frequency (LF) is
characterized as a frequency of 0.04-0.15Hz and represents both the sympathetic and vagal
limb of the ANS141. The LF:HF ratio therefore is thought to represent cardiosympathovagal
balance141. A summary of the different measures of HRV and their significance is
presented in Table 1-1.
LF:HF ratio has been found to be a good predictor of CVD mortality in both high risk
population (diabetes, hypertension etc.) and low risk, healthy populations142. In particular,
HRV has been found to be a good predictor of sudden cardiac death, the leading cause of
cardiovascular death in patients with end stage kidney disease143-147. In addition to
predicting CVD mortality, low LF:HF is also associated with CVD risk factors such as
hypertension148, diabetes149, and abnormal cholesterol150. Therefore, evidence suggests that
HRV (cardiosympathovagal tone as measured by the LF:HF ratio, in particular) is not only
15
a valid predictor of CVD mortality and risk but also a safe, easy and non-invasive method
to calculate cardiac autonomic activity.
Studies have found that individuals with chronic kidney disease have significantly lower
HRV in comparison to the healthy population151-153. As previous studies have suggested an
association between low estradiol and abnormal HRV in the general population154-159 160164
, we aimed to determine the association between estradiol and HRV in a high
angiotensin II (AngII) state that mimics the chronic high AngII state observed in CKD.
In the proposed studies, short term HRV will be analyzed, which has been shown to predict
CVD mortality and risk as accurately as 24 hour HRV measurements143, 145, 165.
Table 1-1 Heart rate variability measurements and their representations
Name
Domain
Representation
Low Frequency (LF)
Frequency (0.04-0.15Hz)
Cardiac sympathetic tone and
baroreceptor reflex
High Frequency (HF)
Frequency (0.15-0.4Hz)
Cardiac parasympathetic or
Vagal tone
LF:HF
Frequency
Sympathetic and
parasympathetic balance in
regulation of heart rate
Standard Deviation of the
Time
Standard deviation of all
normal wave (SDNN)
normal to normal RR intervals
16
Standard Deviation of the
Time
Standard deviations of the
average normal wave
average of NN intervals in
(SDANN)
multiple segments of the
recording
1.4.2 Using the Menopausal Specific Quality of Life (MENQOL) survey to determine
menopausal symptoms
The debilitating physical and psychological symptoms of low sex hormones led to the
development of several menopause related quality of life questionnaires in order to
evaluate the effect of symptoms associated with menopause. The Menopause Specific
Quality of Life Questionnaire (MENQOL) is one such questionnaire that was developed in
1996 and has since been validated in different cultural populations 166 167 (Appendix 1). In
2005, this test was further modified in order to improve the validity and the test-retest
reliability of the questionnaire 168. This has been found to be a valid questionnaire for the
physical and psychological symptoms with a test-retest validity of 0.79 169.
1.5 Current state of the science
Chronic kidney disease is highly prevalent and is associated with a high risk of mortality,
specifically sudden cardiac death, and poor quality of life1-4, 146, 147. Additionally, women
with CKD have an abnormal sex hormone profile which is believed to be characterized by
low levels of estradiol122. Estradiol is associated with CVD, and sudden cardiac death risk
is low in premenopausal women and increases significantly after menopause52, 170.
Estradiol has been implicated in various important cardiovascular processes that directly
affect cardiovascular pathologies87. Cardiac autonomic tone (CAT), measured through
heart rate variability, is a strong predictor of sudden cardiac death risk145. Previous studies,
17
including preliminary work from our group, have shown that low estradiol is associated
with poor cardiovascular physiological profiles 171. We sought to first understand the
associations between estradiol and sudden cardiac death risk in healthy women in response
to angiotensin II (Ang II) infusion. Since the renin angiotensin system is found to be
activated in high risk CVD population172, it is important to understand the potentially
protective role of estradiol in mediating the CAT both at baseline and in response to a
challenge to high Ang II. Studying this would help us further understand the association
between estradiol and CVD risk in CKD.. Low levels of estradiol results in physical and
psychological symptoms such as hot flashes, sleep disturbances and mood changes which
further decrease quality of life in women. Low estradiol is also associated with an increase
in cardiovascular mortality 173. Many of these symptoms in women with CKD are
classified as symptoms of uremia; however, the associations between estradiol levels and
menopause-associated symptoms and quality of life are currently unknown8. Furthermore,
it is currently unknown if menopausal status is associated with morality (cardiovascular
and non-cardiovascular) in women with end stage kidney disease.
1.6 Objective
The overarching objective of the full course of the work presented herein was to (1)assess
the associations between estradiol and cardiac autonomic tone in healthy women in
response to angiotensin II (Ang II) infusion, as high Ang II levels are found in CKD
patients and (2) understand the associations between CKD-mediated low estradiol levels
and menopausal status and cardiovascular disease, quality of life, and gaps in the literature
and clinical care with respect to the management of symptoms of low estradiol in the CKD
population.
18
1.7 Thesis Outline
This manuscript based thesis is composed of 6 manuscripts. The manuscripts presented in
chapters 2 and 3 assess the relationship between baseline endogenous sex hormone levels
and cardiac autonomic tone in healthy men and women. In particular, the difference in
baseline cardiac autonomic tone and the cardiac autonomic response in men and women,
postmenopausal and premenopausal women, and premenopausal women in the follicular
(low estradiol) and luteal (high estradiol) phase is determined.
Chapters 4-7 aim to summarize the role of estradiol in women with CKD. Chapter 4
presents the results from an international survey of nephrologists and allied healthcare
workers which aimed to summarize the impressions and perspectives of renal healthcare
workers on the impact of low estradiol and the management of sex hormone status in
women with chronic kidney disease. Chapter 5 presents a systematic review and metaanalysis which sought to summarize current knowledge regarding use of postmenopausal
hormone therapy and (1) cardiovascular outcomes, and (2) established surrogate measures
of cardiovascular risk in women with CKD. Chapter 6 presents the results from the
Canadian Kidney Disease Cohort Study (CKDCS), a prospective cohort study, which
aimed to determine the association between menopausal status and all cause and
cardiovascular mortality in women with end stage renal disease (ESRD). Finally, Chapter 7
presents the results from an exploratory cross-sectional study that aimed to determine the
prevalence of menopausal symptoms and menopause related quality of life in women with
chronic kidney disease and its association with menopausal status.
19
Chapter Two: Testosterone is associated with the cardiovascular autonomic response to a
stressor in healthy men
Ramesh S, Wilton SB, Holroyd-Leduc JM, Turin TC, Sola DY, Ahmed SB. Testosterone is
associated with the cardiovascular autonomic response to a stressor in healthy men. Clinical
and Experimental Hypertension. 2015;37:184-191.
20
Abstract
Objective: Men have high cardiovascular risk and unfavourable cardiac autonomic tone
compared to premenopausal women. The role of sex hormones in control of autonomic tone is
unclear. We sought to determine the association between sex hormones and
cardiosympathovagal tone at baseline and in response to a physiological stressor.
Methods: Forty-eight healthy subjects (21 men, 27 premenopausal women) were studied in
high–salt balance. Cardiac autonomic tone was assessed by heart rate variability, calculated by
spectral power analysis (Low Frequency (LF, a measure of sympathetic modulation), high
frequency (HF, a measure of vagal modulation) and LF:HF (a measure of cardiosympathovagal
balance)) at baseline and in response to graded Angiotensin II (AngII) infusion
(3ng/kg/min×30min, 6ng/kg/min×30min) were measured. The primary outcome was association
between endogenous sex hormone levels and measures of cardiac autonomic tone.
Results: All subjects had sex hormone levels in the normal range. No associations were
observed between sex hormones and baseline cardiac autonomic tone in men or women. Men
with lower testosterone levels, however, were unable to maintain both cardiosympathetic
(p=0.045) and cardiovagal tone (p=0.035) in response to AngII even after adjustments for
covariates. No association was observed between estradiol and progesterone and cardiac
autonomic response to AngII in either sex.
Conclusion: An unfavourable shift in the cardiac autonomic tone in men with lower testosterone
levels was observed in response to a stressor. Understanding the role of sex hormones in
modulation of cardiac autonomic tone may help guide risk reduction strategies in men.
Key words: Heart Rate Variability, Sex Hormones, Cardiac Autonomic Tone
21
Introduction
Men are at a higher risk for sudden arrhythmic death than premenopausal women; however,
there is a steep increase in the risk of sudden arrhythmic death in women after menopause,
suggesting a role for sex hormones in mediating these gender differences170. Although sudden
arrhythmic death has a complex etiology, impaired resting and dynamic changes in autonomic
tone appear to modulate the development of sudden arrhythmic death in various at-risk
populations174. The contribution of gender and sex hormones in the risk of sudden arrhythmic
death remains unclear.
Previous studies have shown that women have a more cardioprotective autonomic profile with
less cardiosympathetic and more cardiovagal activity compared to men156, 174, 175. However, the
contribution of individual endogenous sex hormones in the control of autonomic tone is unclear.
Limited studies have suggested a role for estradiol176, 177 and for testosterone176, 177 in control of
cardiac autonomic tone. These studies report only baseline values of cardiosympathovagal
balance and thus the association between sex hormone levels and the response to a stressor is
unknown. As such, we sought to determine the role of endogenous serum sex hormone levels on
cardiac autonomic tone at baseline and in response to a stressor in the form of an acute
angiotensin (Ang) II challenge in healthy men and premenopausal women.
Methods
Subjects
Forty-eight (21 male, 27 premenopausal female) healthy, normotensive, nonsmoking subjects
were enrolled. The University of Calgary Conjoint Health Research Ethics Board approved the
study and each subject provided written informed consent. Participants underwent a detailed
22
history and physical examination. None were on regular medications including oral contraceptive
medications. To account for variations in sex hormone levels and renin angiotensin system
(RAS) activity through the menstrual cycle 178, premenopausal women were studied 14 days after
the 1st day of the menstrual cycle, which was verified by measuring sex-hormone levels.
All subjects were counselled to adhere to a diet that maintained their normal daily caloric intake,
while maintaining a high-salt state (150 mmol/day) for 3 days prior to the study day to ensure
maximal RAS suppression179 . Compliance with the high-salt diet was verified by urine
collection. Each study commenced at 0800h following an overnight fast in a quiet, temperaturecontrolled room with subjects in a supine position.
Study protocol
An 18-gauge peripheral venous cannula was inserted into each antecubital vein for infusion and
blood sampling. Systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean
arterial pressure (MAP) were collected via an automated sphygmomanometer (Dinamap, GE
Healthcare, Waukesha WI) every 15 minutes. Subjects were infused with graded, incremental
doses of Ang II (3 ng·kg-1·min-1 × 30 min; 6 ng·kg-1·min-1 × 30 min), followed by a 30-min
recovery period.
Heart Rate Variability Measurements
Ambulatory ECG data were collected with a commercially available Holter monitor (SEER MC
Recorder, GE Healthcare). Holter data were collected continuously for 180 min (baseline, each
dose of Ang II (time 30min, 3ng/kg/min and time 60min, 6ng/kg/min), and recovery). Frequency
domain measures of heart rate variability (HRV) were calculated using standard methods
(MARS version 7, GE Healthcare)141. Frequency domain parameters were derived using power
spectral analysis, where cardiosympathetic power was partially represented by the low-frequency
23
(LF; ms2) band within the range of 0.04–0.15 Hz, and cardiovagal power was represented by the
high-frequency (HF; ms2) band within the range of 0.15- 0.40 Hz. The LF:HF ratio component
was calculated by comparing non-normalized, absolute LF and HF parameters, representing total
cardiosympathovagal balance. Some controversy exists over the ideal method of reporting LF
and HF, with representation of LF and HF in normalized units emphasizing the controlled and
balanced behavior of the two branches of the autonomic nervous system, though the
normalization tends to minimize the effect of the changes in total power on the values of LF and
HF components. As such, we reported both absolute and normalized measures of HRV141.
Analytical methods
Plasma estradiol, progesterone and testosterone levels were determined using radioimmunoassay
(Roche E170 Modular, Roche Diagnostics, Indianapolis, In). The lower limit of detection for the
measurement of plasma progesterone is <1nmol/L. As such, we assigned a value of 0.99 nmol/L
for these data points. Plasma renin activity (PRA) was determined by the quantification of
plasma Angiotensin I, a surrogate measure of PRA, by radioimmunoassay (PRA 125I, DiaSorin,
Stillwater, MN). Ang II plasma levels were measured by standard laboratory immunoassay
techniques (Quest Diagnostics, San Juan Capistrano, CA). Serum aldosterone levels were
determined by radioimmunoassay techniques (Aldosterone Coat-A-Count kit, InterMedico
Diagnostic Products, Markham, Ontario, Canada). Serum creatinine, cholesterol, and triglyceride
(TG) assays were quantified by enzymatic colorimetric assay techniques (Roche/Hitachi
Creatinine Plus; CHOD-PAP, HDL-C Plus, and TG GPO-PAP kits, Roche Diagnostics,
Indianapolis, IN, respectively).
Statistical methods
24
Values are presented as mean ± SD, unless otherwise indicated. Using linear regression we
examined the associations between serum sex hormone levels and cardiac autonomic tone as
determined by heart rate variability (HRV) (Low Frequency (LF), High Frequency (HF), LF:HF)
at baseline and HRV responses to time 30 min and time 60 min of graded Ang II infusion. These
models were further adjusted for age, BMI, cholesterol, renin, MAP, and baseline HRV (for the
responses). Additionally, we determined the differences in measures of cardiac autonomic tone at
baseline and HRV responses at time 30min and 60min of graded Ang II infusion between
subjects below 50th percentile and above 50th percentile of sex hormone levels. Parametric t-tests
and paired t-tests were used to determine significance. The effect of age as a confounding factor
was determined using analysis of covariance (ANCOVA) and repeated measures analysis of
covariance (ANCOVA) respectively for the baseline and autonomic response to Ang II infusion;
all assumptions for the two tests were met. Statistical analyses were performed using SPSS
(version 19, IBM, Armonk, NY) with two-sided statistical significance at p<0.05.
Results
Baseline Characteristics
All subjects were nonobese, normotensive, nondiabetic and had normal kidney function (table 1).
The majority of subjects were Caucasian and ethnic and racial proportions were not different
between men and women. Men were older, had higher blood pressure, lower high density
lipoprotein (HDL) and higher low density lipoprotein (LDL) cholesterol compared to women,
though all values were in the normal range for both sexes.
Sex hormone levels were within the sex-specific normal range for both men and women. As
anticipated, men had lower levels of estradiol, progesterone and sex hormone binding globulin
and higher levels of testosterone compared to women.
25
Sex Differences
Resting cardiac autonomic tone measures were within normal range for both men and women 174.
Men demonstrated higher baseline measures of cardiac sympathetic tone and lower measures of
cardioprotective vagal tone compared to women (figure 1). Consequently, overall
cardiosympathovagal balance was significantly higher in men compared to women.
Cardiac autonomic tone responses to AngII challenge, stratified by sex, are presented in figure 1.
While women maintained cardiosympathetic and cardiovagal tone in response to AngII, men
showed an unfavourable shift in cardiac autonomic tone (an increase in LF and a decrease in HF)
which was found to be significant after adjustment for age (LF(nu): p=0.05; HF(nu): p=0.04).
Testosterone
No association was observed between testosterone levels and measures of resting cardiac
autonomic tone in men or women.
Similarly, measures of baseline cardiac autonomic tone were similar in the low and high
testosterone group in both men and women even after adjustment for covariates.
In men, testosterone levels were negatively associated with the cardiosympathetic response and
positively associated with the cardiovagal response to 6ng/kg/min infusion of AngII (ΔLF (time
60): p=0.02, ΔHF (time 60) p=0.02). This lost significance after adjustment for age, estradiol and
progesterone (ΔLF (time 60): p=0.06, ΔHF (time 60) p=0.08) (figure 2).
Stratification of men by testosterone status revealed significant differences in the cardiac
autonomic tone response to the stressor (figure 3). Men with lower testosterone increased
cardiosympathetic (LF 6.53  2.81, p=0.045 vs baseline) and decreased cardiovagal (HF -5.57
26
 2.24, p=0.035 vs baseline) tone in response to the higher dose of AngII. These differences were
significant even after adjustment of covariates (LF p=0.02 vs baseline; HF p=0.01 vs
baseline). In contrast, men in the higher testosterone group maintained cardiac autonomic tone
(LF -0.28  2.0, p=0.89 vs baseline; HF 0.028  2.00, p=0.99 vs baseline) in response to the
stressor even after adjustment for covariates. As a result, there was a trend towards an overall
unfavourable shift in the cardiosympathovagal balance after 60min of AngII challenge (LF:HF
0.02  0.1, p= 0.088 vs baseline) in men with lower testosterone which was close to significance
after adjustment of covariates (p=0.06) that was not observed in the higher testosterone group
(LF:HF -0.016  0.15, p=0.92 vs baseline) even after adjustment for covariates.
Measures of cardiac autonomic tone were similar in women in the low and high testosterone
group even after adjustments for covariates.
All analyses of cardiac autonomic tone both at baseline and in response to AngII were repeated
using non-normalized units. Similar results were observed, though the difference in the vagal
response to AngII between the high and low testosterone male groups did not achieve statistical
significance.
Estradiol
No associations were observed between estradiol in men or women and the baseline
measurement of cardiac autonomic tone on univariate and multivariate analysis.
Cardiosympathetic tone, cardiovagal tone and cardiosympathovagal balance was not different
between groups of men stratified by above or below the median estradiol levels.
Similar to men, when women were stratified based low or high serum estradiol level, no
differences were observed in any measure of cardiosympathetic cardiovagal and
cardiosympathovagal balance.
27
There were no associations between the cardiac autonomic response to AngII and estradiol in
both men and women.
Similarly, when stratified to groups of high or low estradiol the cardiac autonomic response to
AngII was not different in both men and women.
Progesterone
No significant associations were found between progesterone levels and cardiac autonomic tone
in both men and women.
When stratified according to progesterone status, men in the high progesterone group
demonstrated significantly lower sympathetic tone and higher parasympathetic tone compared
with their lower progesterone counterparts (LF (nu): p =0.02; HF (nu): p = 0.02 vs. low group)
and consequently, men in the higher progesterone group had a significantly lower overall
cardiosympathovagal balance (LF:HF: p=0.02 vs. low group). These observed differences,
however, were no longer significant after adjustments for covariates.
In contrast to men, stratification based on progesterone status in women did not reveal any
differences in cardiac autonomic tone.
No associations were found between progesterone levels and the response to AngII in men or
women. Additionally, stratification based on low or high progesterone levels revealed no
significant associations between cardiac autonomic response to AngII in men and women.
Discussion
This is one of the first studies to examine the association between serum sex hormone levels and
cardiac autonomic tone at baseline and in response to a physiologic stressor in healthy men and
premenopausal women. The key findings in this study were: 1) No associations were found
between endogenous sex hormones and baseline cardiac autonomic tone in either men or women
28
and 2) lower testosterone levels were associated with an inability to maintain cardiosympathetic
and cardiovagal tone in response to AngII in men, but not women.
Animal studies have shown that cardiac autonomic tone is affected by the acute infusion of
angiotensin II 180, 181 and high CV risk populations, in a state of chronic RAS upregulation182,
demonstrate an acute change in cardiac autonomic tone in response to angiotensin converting
enzyme inhibitors183, therefore, the acute exposure to AngII in this study aimed to mimic the
clinical setting in a safe and controlled manner using a well-accepted protocol 184-187. A previous
study from our group found that in response to a stressor, women maintained cardiac autonomic
tone, while men showed an unfavourable shift in cardiosympathovagal balance 175. The results of
the present study suggest that testosterone is a factor in determining the cardiac response to a
stressor in men, with higher testosterone levels being protective.
Sex Hormones and Cardiac Autonomic Tone
Previous studies have supported a protective role for estradiol and cardiac autonomic tone in
animals188-190. Estradiol is a lipophilic hormone with the ability to cross plasma membranes as
well as the blood-brain barrier191 and estradiol receptors are found in the medulla oblongata,
which controls the autonomic nervous system 192. Systemic estradiol injections resulted in an
increase in overall vagal tone in both male and ovariectomized female Sprague-Dawley rats188,
190
; moreover, local estradiol injections in the localized parts of the brain involved with control of
autonomic tone resulted in a similar increase in vagal tone193. Additionally, central estrogen
injections in male and ovariectomized female rats led to a decrease in sympathetic activity189.
While we did not find an association between estradiol and any measure of cardiac autonomic
tone in either sex, this may simply reflect not only interspecies disparities and variation in the
29
response to acute vs. chronic exposure of the sex hormone, but also innate differences between
endogenous and exogenous ovarian hormones113.
Compared to estradiol, little is known about progesterone and its effect on the autonomic
nervous system. Progesterone has been found to increase systemic norepinephrine release
leading to neuronal sympathetic activation 194. Furthermore, receptors for progesterone have
been found in the hypothalamus of both male and female Sprague-Dawley rats195, suggesting a
means by which this sex hormone may contribute to control of autonomic tone. In the brainstem,
progesterone receptors were detected in the norepinephrine neurons of the nucleus tractus
solitarius in female Wistar rats, making it possible for progesterone to directly affect the
autonomic control of the heart 196. Of note, combined estrogen and progesterone treatment in
ovariectomized rats increased the density of beta adrenergic receptors in the heart 197, providing a
potential mechanism by which ovarian hormones may control cardiac tone. However, the lack of
association between progesterone and cardiac autonomic tone in our study population may, in
addition to the factors potentially contributing to disparities in the studies examining the effects
of estrogen on cardiac autonomic tone, highlight the differences between in vivo and in vitro
studies. There are very few published studies examining the effect of testosterone on cardiac
autonomic tone. In male and female rats, treatment with testosterone did not affect alpha
adrenergic receptor density198. Moreover, castration in rats decreases norepinephrine levels
which can be restored after testosterone replacement199, though whether these fluctuations in
norepinephrine levels result in changes in cardiac autonomic tone is unknown.
Sex steroids and cardiac autonomic tone in humans
Human studies examining the association between endogenous sex hormones and cardiac
autonomic tone are few and it is not possible to separate the independent effects of each
30
endogenous sex steroid on cardiac outcomes. We have previously shown that men have a higher
sympathetic and lower parasympathetic tone compared to women175, suggesting potential a role
for sex hormones in mediating these differences. Certainly, women of postmenopausal status, a
low estrogen and progesterone state, have decreased cardiac sympathovagal balance compared to
premenopausal women offering further support for a role for sex hormones in control of cardiac
autonomic tone154, 174. The menstrual cycle has been shown to influence cardiac autonomic tone
as assessed by resting heart rate variability though results are conflicting160, 164, 200. While some
studies have found that in the luteal (high estradiol and progesterone) phase women have a
higher sympathetic and lower parasympathetic tone compared to the follicular (low estrogen and
progesterone) phase 200, other studies have found no effect of the menstrual cycle on cardiac
autonomic tone160 or that the luteal phase is associated with a lower sympathetic and higher
parasympathetic tone 164. These conflicting reports likely reflect varying study population factors
such as oral contraceptive use, BMI, and dietary intake as well as different definitions of the
luteal and follicular phases, all of which may influence baseline cardiac autonomic tone in
women 140, 201.
While a recent systematic review and meta-analysis suggests a risk with exogenous testosterone
therapy and cardiac outcomes in men202, studies examining the association between endogenous
sex hormones and cardiac autonomic tone in men are limited. Similar to our study, Wranicz and
colleagues found no association between estradiol and heart rate variability parameters 177.
Dogru et al reported a positive association between serum estradiol levels and
cardiosympathovagal balance in men176, though progesterone levels were not reported. It is
therefore unknown if the effect of estrogen observed in the abovementioned study is independent
of progesterone levels. Additionally, the authors of this study highlight the influence of
31
differential aromatase activity on peripheral estradiol-testosterone conversion on the observed
results. This same study also reported a positive association between total testosterone and vagal
activity and a negative association with sympathetic activity, which is in contrast to a study of 22
male idiopathic hypogonadotropic hypogonadism patients in whom deficiency of the male
hypothalamo-pituitary-gonadal axis was associated with increased sympathetic and decreased
parasympathetic components of heart rate variability compared to age-matched healthy
controls203. The conflicting conclusions of these and our studies may be explained by
differences in fasting status and salt balance, which are known to affect measurement of cardiac
autonomic tone204, 205. Furthermore, progesterone levels were not reported in any of these studies
raising the possibility of residual confounding.
Sex Hormones and Response to a Physiologic Stressor
In this study, we found that men with lower testosterone were unable to maintain cardiac
autonomic tone in response to a physiologic stressor, indicating that the cardiac autonomic
response in men may be modulated by testosterone levels. Putting this into clinical context,
compared to men with higher levels of testosterone, the increase in sympathetic and decrease in
vagal activity observed in response to AngII challenge in the low testosterone group followed a
similar pattern, albeit to a lesser degree, to that observed in patients 2 weeks-post myocardial
infarction206.Similar results have been previously reported in populations with chronic
upregulation of the renin angiotensin system172, 177, 207. In a study of 88 men post myocardial
infarction, Wranicz and colleagues found that lower testosterone was associated with lower
cardioprotective parasympathetic tone, suggesting a protective role of testosterone in a high Ang
II state177. Similarly, in a multicenter study of 623 patients with end-stage kidney disease on
hemodialysis, a population known to have both considerable cardiac autonomic dysfunction 208
32
and significantly increased risk of sudden cardiac death22, lower testosterone was associated with
higher all-cause mortality 207. Thus, the limited available data supports a cardioprotective role
for testosterone in men.
Limitations
This study has strengths and limitations. First, while cardiac autonomic tone can be influenced
by lifestyle factors such as diet, obesity, and smoking 205, 209, 210 we included only healthy, nonsmoking and non-obese subjects in high-salt balance, a state reflective of the typical Western
diet211. Second, while 24 hour Holter ECG monitoring is considered the gold standard for
measurement of cardiac autonomic tone, the use of short term ECG recordings for heart rate
variability has been previously shown to have excellent correlation to 24h measurement and has
been validated in multiple studies143, 145, 165. Furthermore, we controlled for external factors such
as activity level212, diet204, 205 and time of day 213 that may influence measurement of HRV.
Third, our sample size was limited; however, the study of a homogenous population of healthy
individuals without comorbidities on a controlled protein and salt diet in a controlled lab
environment minimized the effect of confounders. Fourth, the participants in this study had sex
hormone levels in the normal range which may limit the power to detect a significant difference;
however, this eliminates the possibility of a spectrum bias within the study and makes it more
representative of a healthy population. Lastly, while this study focuses entirely on the role of
endogenous sex hormones on cardiac autonomic tone, it eliminates potential complications of
studying exogenous sex hormones such as differences in the route of administration, types of
hormones and the combinations of hormone therapy113, 202.
33
Conclusion
In this study of healthy men and premenopausal women, higher testosterone status was
associated with a more favourable cardiac autonomic tone profile in response to a physiological
stressor in men, but not women. While low-dose testosterone replacement in the elderly has not
been shown to have physiologically relevant beneficial effects 214, testosterone replacement
therapy in healthy older men in near physiological doses does not appear to incur serious adverse
events215. Certainly, a recent randomized controlled study examining the effects of gonadal
steroids demonstrated that the amount of testosterone required to maintain lean mass, fat mass,
strength, and sexual function varied widely in healthy men 216.Thus, while it remains unclear if
treatment with therapy mimicking endogenous testosterone would offer therapeutic benefit, the
potential role of testosterone in modulating cardiac autonomic tone merits attention.
34
Table 2-1 Baseline Characteristics
Baseline Characteristics
Men (n=21)
Women (n=27)
Age (yrs)
% Caucasian
BMI (kg/m2)
40 ± 14
80(17)
26 ± 4
33 ± 11*
85 (23)
24 ± 4
Mean Arterial Pressure (mmHg)
84 ± 13
75 ± 18
Fasting Glucose (mmol/L)
4.7 ± 0.4
4.5 ± 0.5
Total Cholesterol (mmol/L)
4.1 ± 0.7
4.0 ± 0.8
HDL Cholesterol (mmol/L)
1.2 ± 0.3
1.6 ± 0.3*
LDL Cholesterol (mmol/L)
2.5 ± 0.6
2.1 ± 0.6*
Estradiol (pmol/L)
Mean
100 ± 35
402 ± 340*
Median [IQR]
100 [76.5 – 123.5] 357 [25.6 – 531.0]
Progesterone (nmol/L)
Mean
1.7 ± 0.7
8.9 ± 15*
Median [IQR]
1.6 [1.1-2.3]
1.8 [1.1 – 8.4]
Mean
17 ± 5
1.0 ± 0.6*
Median [IQR]
16 [13.8 – 20.9]
0.9 [0.6 – 1.6]
Testosterone (nmol/L)
Sex Hormone Binding Globulin (nmol/L) 31 ± 19
63 ± 50*
Abbreviations: BMI, body mass index; HDL, High Density Lipoprotein; LDL, Low Density
Lipoprotein* P<0.05 vs men
35
Figure 2-1Cardiac autonomic tone at baseline and in response to AngII in men and women
36
Figure 2-2Association between cardiac sympathetic and vagal response to 6ng/kg/min of
Ang II infusion and serum testosterone in men
37
Figure 2-3: LF(nu) and HF(nu) response to 60min of Angiotensin II challenge in men, stratified by testosterone level.
* p<0.05 vs. baseline
38
Chapter Three: Cardiac autonomic response to angiotensin II in healthy premenopausal and
postmenopausal women
Ramesh S, Wilton SB, Holroyd-Leduc JM, James M, Sola DY, Ahmed SB Cardiac autonomic
response to angiotensin II in healthy premenopausal and postmenopausal women. (under peer
review)
39
Abstract
Objective: Women with chronic kidney disease have an abnormal sex hormone profile,
upregulated renin angiotensin system and a high risk of sudden cardiac death. As low estradiol
status is associated with poor cardiac autonomic tone, a marker of sudden cardiac death, in
healthy women, we sought to determine the association between menopausal status and
menstrual cycle in response to angiotensin II infusion in healthy women.
Methods: Forty-one healthy subjects (28 premenopausal, 13 postmenopausal women) were
studied in high–salt balance. Eleven premenopausal women were studied in the luteal and
follicular phase of the menstrual cycle. Cardiac autonomic tone was assessed by heart rate
variability, calculated by spectral power analysis (Low Frequency (LF), high frequency (HF) and
LF:HF) at baseline and in response to graded Angiotensin II (AngII) infusion
(3ng/kg/min×30min, 6ng/kg/min×30min) were measured. The primary outcomes were
differences in cardiac autonomic tone at baseline and in response to AngII in premenopausal
versus postmenopausal women, and in premenopausal women in the luteal versus the follicular
phase of the menstrual cycle.
Results: All subjects had sex hormone levels in the normal range. At baseline, postmenopausal
women had a lower LF and HF tone in comparison to premenopausal women. No differences
were observed between the baseline autonomic tone at the follicular or luteal phase of the
menstrual cycle. In response to Ang II postmenopausal women were unable to maintain their
cardiac autonomic tone (mean ΔHF = -0.43 ± 0.46 ln ms2, p=0.005 response vs. baseline) while
premenopausal women did. Premenopausal women in the luteal phase, however, failed to
maintain cardiac autonomic tone (ΔLF = -0.07 ± 0.46 ln ms2, p=0.048 response vs. baseline,
ΔHF = -0.33 ± 0.74 ln ms2, p=0.048 response vs. baseline).
40
Conclusion: An unfavourable shift in the cardiac autonomic tone in postmenopausal women and
premenopausal women in the luteal phase was observed in response to a stressor. Understanding
the role of sex hormones in modulation of cardiac autonomic tone may help guide risk reduction
strategies in women.
41
Introduction
Chronic kidney disease (CKD) is a prevalent condition, and patients with chronic kidney disease
have an upregulated renin angiotensin system1, 172. Patients with CKD also have a high burden of
cardiovascular disease and in particular sudden cardiac death3, 4, with up to 25% of the
cardiovascular deaths being due to sudden cardiac death217; however the etiology of sudden
cardiac death in this population is not well understood. Moreover, traditional prevention methods
for sudden cardiac deaths are not effective in the end stage kidney disease population indicating
the need for studies that aim to characterize the risk factors of sudden cardiac death146.
Premenopausal women have a low risk of sudden arrhythmic death compared to postmenopausal
women, suggesting a role for sex hormones in mediating these differences170. Despite sudden
arrhythmic death having a complex etiology impaired resting and dynamic changes in autonomic
tone appear to be indicative risk of sudden arrhythmic death in various at-risk populations174.
The contribution sex hormones, in particular estradiol, in the risk of sudden arrhythmic death
remains unclear.
Previous studies have shown that postmenopausal women have a high sympathetic and lower
vagal tone in comparison to premenopausal women; furthermore, cardiac autonomic tone
changes based on the menstrual cycle, and limited studies have suggested a role for estradiol176,
177
in control of cardiac autonomic tone. However, whether these difference exist in a high
angiotensin II state is currently unknown. As such, we sought to determine difference in cardiac
autonomic tone in a postmenopausal versus premenopausal women (permanent high/low
estradiol state) and in premenopausal women the follicular and luteal stage of the menstrual
cycle (acute high/low estradiol state) at baseline and in response to a stressor in the form of an
angiotensin (Ang) II challenge in healthy women.
42
Methods
Subjects
Forty-one women (28 premenopausal, 13 postmenopausal) healthy, normotensive, nonsmoking
subjects were enrolled. The University of Calgary Conjoint Health Research Ethics Board
approved the study and each subject provided written informed consent. Participants underwent a
detailed history and physical examination. None were on regular medications including oral
contraceptive medications. To account for variations in sex hormone levels and renin angiotensin
system (RAS) activity through the menstrual cycle178, premenopausal women were studied 14
days after the 1st day of the menstrual cycle (luteal phase) and 11 participants were studied again
on the 1st day of the menstrual cycle (follicular phase). Both phases were verified by measuring
sex-hormone levels.
All subjects were counselled to adhere to a diet that maintained their normal daily caloric intake,
while maintaining a high-salt state (150 mmol/day) for 3 days prior to the study day to ensure
maximal RAS suppression179 . Compliance with the high-salt diet was verified by urine
collection. Each study commenced at 0800h following an overnight fast in a quiet, temperaturecontrolled room with subjects in a supine position.
Study protocol
An 18-gauge peripheral venous cannula was inserted into each antecubital vein for infusion and
blood sampling. Systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean
arterial pressure (MAP) were collected via an automated sphygmomanometer (Dinamap, GE
Healthcare, Waukesha WI) every 15 minutes. Subjects were infused with graded, incremental
43
doses of Ang II (3 ng·kg-1·min-1 × 30 min; 6 ng·kg-1·min-1 × 30 min), followed by a 30-min
recovery period.
Heart Rate Variability Measurements
Ambulatory ECG data were collected with a commercially available Holter monitor (SEER MC
Recorder, GE Healthcare). Holter data were collected continuously for 180 min (baseline, each
dose of Ang II (time 30min, 3ng/kg/min and time 60min, 6ng/kg/min), and recovery). Frequency
domain measures of heart rate variability (HRV) were calculated using standard methods
(MARS version 7, GE Healthcare) (3). Frequency domain parameters were derived using power
spectral analysis, where cardiosympathetic power was partially represented by the low-frequency
(LF; ms2) band within the range of 0.04–0.15 Hz, and cardiovagal power was represented by the
high-frequency (HF; ms2) band within the range of 0.15- 0.40 Hz. The LF:HF ratio component
was calculated by comparing non-normalized, absolute LF and HF parameters, representing total
cardiosympathovagal balance.
Analytical methods
Plasma estradiol, progesterone and testosterone levels were determined using radioimmunoassay
(Roche E170 Modular, Roche Diagnostics, Indianapolis, In). The lower limit of detection for the
measurement of plasma progesterone is <1nmol/L. As such, we assigned a value of 0.99 nmol/L
for these data points. Plasma renin activity (PRA) was determined by the quantification of
plasma Angiotensin I, a surrogate measure of PRA, by radioimmunoassay (PRA 125I, DiaSorin,
Stillwater, MN). Ang II plasma levels were measured by standard laboratory immunoassay
techniques (Quest Diagnostics, San Juan Capistrano, CA). Serum cholesterol assays were
44
quantified by enzymatic colorimetric assay techniques (Roche/Hitachi Creatinine Plus; CHODPAP, HDL-C Plus, and TG GPO-PAP kits, Roche Diagnostics, Indianapolis, IN, respectively).
Statistical methods
Values are presented as mean ± SD, unless otherwise indicated. The primary outcome were to
compare cardiac autonomic tone as determined by heart rate variability (HRV) (Low Frequency
(LF), High Frequency (HF), LF:HF) at baseline and HRV responses to 6 ng/kg per min Ang II
infusion between 1) premenopausal women in the luteal phase and postmenopausal women and
2) premenopausal women in the luteal phase and the follicular phase. Parametric t-tests and
paired t-tests were used to determine significance. HRV measures were log transformed to obtain
normalized distributions. Statistical analyses were performed using SPSS (version 19, IBM,
Armonk, NY) with two-sided statistical significance at p<0.05.
Results
Baseline Characteristics
All subjects were nonobese, normotensive and nondiabetic (Table 1). The majority of subjects
were Caucasian and there was a higher proportion of Caucasian premenopausal women than
postmenopausal women. Postmenopausal women were older, had higher blood pressure and
higher total cholesterol compared to premenopausal women, though all values were in the
normal range for both groups.
Sex hormone levels were within the normal range for both premenopausal and postmenopausal
women (Table 1). As anticipated, postmenopausal women had lower levels of estradiol,
45
progesterone, testosterone and sex hormone binding globulin compared to premenopausal
women. Similarly, premenopausal women had higher estradiol levels in the luteal phase
compared to the follicular phase (Table 2).
Baseline Cardiac Autonomic Tone
At baseline, postmenopausal women, in comparison to premenopausal women, had a
significantly lower LF tone (6.0 ± 0.6 ln ms2 vs. 6.81 ± 6.0 ln ms2, p=0.01) and HF tone (5.13 ±
0.63 ln ms2 vs. 6.31 ± 1.05 ln ms2). LF:HF tone did not differ between postmenopausal and
premenopausal women. Table 3, Figure 1A.
No differences were found in baseline LF, HF, and LF:HF between premenopausal women in the
follicular and luteal phase of the menstrual cycle (Table 4, Figure 1B).
Cardiac Autonomic Response to Ang II
There were no significant differences in the response to 3ng/kg per min infusion of AngII
between postmenopausal and premenopausal women (Table 3). However, in response to 6ng/kg
per min of AngII postmenopausal women failed to maintain cardiac autonomic tone and
responded with an decrease in cardioprotectice in HF tone (mean ΔHF = -0.43 ± 0.46 ln ms2,
p=0.005 response vs. baseline) (Table 3, Figure 2A). Premenopausal women maintained cardiac
autonomic tone in response to AngII (Table 3, Figure 2B). No differences were observed in the
LF:HF response to the stressors (Table 3).
No significant differences in the response to 3ng/kg/min were observed in premenopausal
women in the follicular and luteal phase (Table 4). However, in response to 6ng/kg/min of AngII
premenopausal women in the luteal phase were unable to maintain cardiac autonomic tone and
responded with a decrease in LF tone mean (ΔLF = -0.07 ± 0.46 ln ms2, p=0.048 response vs.
baseline) and an decrease in HF tone (ΔHF = -0.33 ± 0.74 ln ms2, p=0.048 response vs. baseline)
46
(Table 4, Figure 3A) Premenopausal women in the follicular phase maintained cardiac
autonomic tone in response to AngII (Table 4, Figure 3B). No differences were observed in the
LF:HF response to the stressors (Table 4,).
Discussion
This cross-sectional study aimed to examine the differences in response to angiotensin II
between premenopausal women and postmenopausal women, and premenopausal women in the
follicular and luteal phase of the menstrual cycle. We found that postmenopausal women had a
lower baseline LF and HF tone compared to premenopausal women and in response to AngII
failed to maintain cardiac autonomic tone. While there were no differences in baseline cardiac
autonomic tone in the follicular and luteal phase of the menstrual cycle, premenopausal women
failed to maintain cardiac autonomic tone in the high estrogen luteal phase compared to the
follicular phase.
While previous studies have examined the difference in cardiac autonomic tone based on
menopausal status154-159 and the menstrual cycle160-164, this is the first study to determine
differences in the cardiac autonomic tone in response to a physiological stressor. The differences
in baseline HRV in this study between premenopausal and postmenopausal women is in keeping
with results from previous studies154-156, 158 which have found a decrease in LF and HF tone with
menopause. Additionally, in a study of 133 peri- and postmenopausal women, Akiyoshi et al
found a significant decrease in LF and HF ratio with increasing age218. Few studies have reported
an increase in LF tone with menopause157, 219. The observed differences in results can be
attributed to differences in postmenopausal hormone therapy and oral contraceptive use, dietary
intake, exercise and BMI, which have been found to modulate baseline LF tone 220, 221 222-224.
47
The association between different stages of the menstrual cycle and cardiac autonomic tone has
been studied previously, however, the results are conflicting. Some studies found a decrease in
vagal and increase in sympathetic tone during the luteal (high estradiol) phase in comparison to
the follicular phase160, 161, some studies found no change in autonomic tone across the menstrual
cycle162, 163, and one study found increased vagal and decreased sympathetic tone during the
luteal phase164. The varying results could be due to different definitions of the luteal and
follicular phases, presence of premenstrual symptoms, BMI and dietary intake222-224. A few
reasons we did not see a difference between the follicular and the luteal phase could include a
difference in short term versus long term exposure to low estradiol levels or due to a threshold
effect in the autonomic modulation of estradiol.
Molecular studies have supported a protective role for estradiol and cardiac autonomic tone in
animals189, 190, 225. Estradiol is a steroid hormone that has the ability cross the blood-brain
barrier191, furthermore estradiol receptors are found in the medulla oblongata, which is a
potential mechanism through with estradiol could control the autonomic nervous system192.
Studies have shown that control female Sprague-Dawley rats have a lower vagal tone in
comparison to female Sprague-Dawley rats that have been given systemic estradiol injections190,
225
; moreover, local estradiol injections in the localized parts of the brain involved with control of
autonomic tone resulted in a similar increase in vagal tone compared to control193. Additionally,
estrogen injections in the autonomic regulatory parts of the brain in ovariectomized female rats
led to a decrease in sympathetic activity190, highlighting possible mechanisms for the role of
estradiol in the maintenance of cardiac autonomic tone.
We found that postmenopausal women were unable to maintain cardiac autonomic tone in
response to a stressor, in particular, we observed withdrawal of the cardioprotective vagal tone in
48
response to a high angiotensin II state. A similar suppression of HRV is common in patients with
end stage kidney disease is indicative of poor cardiac autonomic control and sudden cardiac
death 226-228. As end stage kidney disease is characterized by a chronic high angiotensin II state
172
and women with end stage kidney disease experience premature menopause, the observed
menopause mediated suppression of heart rate variability in response to AngII could indicate a
potential mechanism for increased SCD risk in the kidney disease population.
In contrast, premenopausal women were unable to maintain cardiac autonomic tone in response
to AngII in the high estradiol, luteal, stage of the menstrual cycle. The observed suppression of
cardiac autonomic tone could be due to the presence of premenstrual symptoms (PMS) which
have found to alter cardiac autonomic tone in premenopausal women in the luteal phase229, 230.
PMS refers to a range of physical and emotional symptoms that are experienced during the
premenstrual week of the menstrual cycle231. As our study day for the luteal phase could have
been during the premenstrual week for some subjects, it may explain the cardiac autonomic
response observed in this study.
This study has strengths and limitations. First, while cardiac autonomic tone can be influenced
by lifestyle factors such as diet, obesity, and smoking205, 209, 210 we included only healthy, nonsmoking and non-obese subjects in high-salt balance, a state reflective of the typical Western
diet211. Second, while 24 hour Holter ECG monitoring is considered the gold standard for
measurement of cardiac autonomic tone, the use of short term ECG recordings for heart rate
variability has been previously shown to have excellent correlation to 24h measurement145.
Third, our sample size was limited; however, the study of a homogenous population of healthy
individuals without comorbidities on a controlled protein and salt diet in a controlled lab
environment minimized the effect of confounders. Fourth, the participants in this study had sex
49
hormone levels in the normal range which may limit the power to detect a significant difference;
however, this eliminates the possibility of a spectrum bias within the study and makes it more
representative of a healthy population.
Conclusion
In this study of premenopausal women and postmenopausal women, postmenopausal women
were found to have lower baseline cardiac autonomic tone (sympathetic and vagal) and respond
to a stressor, Ang II unfavourably. Baseline cardiac autonomic tone was not different between
premenopausal women in the follicular and luteal phase; however, in response to a stressor
women in the luteal phase failed to maintain cardiac autonomic tone. As kidney disease
populations are in a state of chronic RAS upregulation, are at a high risk of SCD, and
demonstrate a suppressed cardiac autonomic tone, the acute exposure to AngII in this study,
which aimed to mimic the clinical setting in a safe and controlled manner, helps us better
understand the pathophysiology of SCD in this population.
50
Table 3-1 Baseline characteristics of premeopausal and postmenopausal women
Baseline
Characteristics
Age (yrs)
% Caucasian
BMI (kg/m2)
Systolic Blood Pressure
(mmHg)
Diastoloc Blood Pressure
(mmHg)
Fasting Glucose
(mmol/L)
Total Cholesterol
(mmol/L)
HDL Cholesterol
(mmol/L)
LDL Cholesterol
(mmol/L)
Estradiol (pmol/L)
Mean
Median [IQR]
Progesterone (nmol/L)
Mean
Median [IQR]
Testosterone (nmol/L)
Mean
Median [IQR]
Sex Hormone Binding
Globulin (nmol/L)
All (n=41)
40 ± 14
76%
25.5 ± 4.2
114 ± 14
Premenopausal
(n=28)
33 ± 11
86%
24.3 ± 3.7
109.8 ± 9.2
Postmenopausal
p
(n=13)
55 ± 4
<0.0001
54% 0.03
28.0 ± 5.0
0.01
123.3 ± 16.9
0.002
65.6 ± 10.7
63.1 ± 70.8
70.8 ± 11.5
0.03
4.5 ± 0.5
4.5 ± 0.5
4.5 ± 0.5
0.6
4.2 ± 0.9
3.9 ± 0.75
4.7 ± 0.96
0.005
1.6 ± 0.3
1.6 ± 0.3
1.6 ± 0.4
0.6
2.3 ± 0.8
2.1 ± 0.64
2.6 ± 1.0
0.06
314.7 ± 329.1
162 [415]
397.0 ± 335.3
341 [382]
137.7 ± 241.5
42 [60]
0.02
6.65 ± 13.2
1.3 [1.51]
9.5 ± 15.5
1.95 [7.2]
1.0 ± 0.4
0.9 [0.3]
0.06
0.96 ± 0.56
0.8 [0.6]
1.1 ± 0.62
0.5 [1.1]
0.68 ± 0.26
0.7 [0.6]
0.03
59.2 ± 44.6
62.3 ± 49.4
51.5 ± 30.7
0.5
51
Table 3-2 Sex hormone levels in premenopausal women (follicular vs. luteal phase)
Follicular Phase (n=11) Luteal Phase
Estradiol
Mean
298 ± 210
Median [IQR] 252 [105]
555 ± 328*
447 [535]
Progesterone
Mean
6.7 ± 9.2
Median [IQR] 2 [6.11]
11.0 ± 15.8
4.55 [13]
Testosterone
Mean
1.04 ± 0.74
Median [IQR] 1.5 [0.9]
p<0.05 versus follicular phase
0.88 ± 0.51
1[0.4]
52
(n=11)
Table 3-3 Baseline cardiac autonomic tone and autonomic response to AngII infusion in
premenopausal versus postmenopausal women
Premenopausal (n=28) Postmenopausal (n=13)
HF (ln ms2)
6.31 ± 1.05
5.13 ± 0.63*
Response to 3ng/kg per min Ang II 6.38 ± 1.11
5.02 ± 0.62*
Response to 6ng/kg per min Ang II 6.26 ± 1.26
LF (ln ms2)
4.69 ± 0.63*‡
Baseline
6.81 ± 1.06
6.00 ± 0.62*
Response to 3ng/kg per min Ang II 6.94 ± 1.04
5.83 ± 0.71*
Response to 6ng/kg per min Ang II 6.85 ± 1.00
LF:HF
5.60 ± 0.63*
Baseline
1.35 ± 0.40
1.61 ± 0.49
Response to 3ng/kg per min Ang II 1.40 ± 0.42
1.56 ± 0.71
Response to 6ng/kg per min Ang II 1.40 ± 0.38
1.64 ± 0.47
Baseline
53
Table 3-4 Baseline cardiac autonomic tone and autonomic response to AngII infusion in
premenopausal women in the follicular vs luteal phase
Follicular Phase (n=11) Luteal Phase (n=11)
HF (ln ms2)
6.69 ± 1.00
6.51 ± 0.83
Response to 3ng/kg per min Ang II 6.60 ± 0.85
6.56 ± 1.04
Response to 6ng/kg per min Ang II 6.35 ± 0.94
LF (ln ms2)
6.13 ± 1.08‡
Baseline
7.18 ± 0.82
7.08 ± 0.75
Response to 3ng/kg per min Ang II 7.05 ± 0.93
7.16 ± 0.85
Response to 6ng/kg per min Ang II 7.11 ± 1.04
LF:HF
6.66 ± 0.77‡
Baseline
1.39 ± 0.43
1.41 ± 0.41
Response to 3ng/kg per min Ang II 1.41 ± 0.45
1.41 ± 0.42
Response to 6ng/kg per min Ang II 1.57 ± 0.56
1.44 ± 0.43
Baseline
54
Figure 3-1 Baseline HRV in premenopausal and postmenopausal women
55
Figure 3-2 : Response to 6ng/kg/min AngII infusion in A) premenopausal and B)Postmenopausal women
56
Figure 3-3 Baseline HRV in premenopausal follicular and luteal phase
57
Figure 3-4Response to 6ng/kg/min AngII infusion in A) Follicular and Luteal phase
58
Nephrologist survey on Sex Hormone Status
Chapter Four: Sex hormone status in women with chronic kidney disease: Survey of
nephrologists’ and renal allied health care providers’ perceptions.
Ramesh S, James MT, Holroyd-Leduc JM, Wilton SB, Seely EW., Wheeler DC, Ahmed SB.
Chapter Four: Sex hormone status in women with chronic kidney disease: Survey of
nephrologists’ and renal allied health care providers’ perception. Clinical Journal of the
American Society of Nephrology (under peer-review)
59
Nephrologist survey on Sex Hormone Status
Abstract
Background: Chronic kidney disease (CKD) in women is accompanied by menstrual, fertility
disorders and premature menopause. We sought to determine nephrologists’ and allied healthcare
providers’ perceptions on management of sex hormone status in women with CKD.
Study Design: Cross sectional study.
Setting and participants: An anonymous, internet-based survey was sent to members of the
Canadian, Australia and New Zealand Societies of Nephrology, the UK Renal Association and
the Canadian Association of Nephrology Nurses and Technologists (February-November 2015).
Outcomes: Reported perceptions and management of sex hormone status in women with CKD.
Results: One hundred seventy-five nephrologists (21% response rate) and 123 allied healthcare
workers (30%) responded. Sixty-eight percent of nephrologists and 46% of allied workers were
30-50 years and 38% of nephrologists and 89% of allied workers were female. Ninety-five
percent of nephrologists agreed that kidney function has an impact on sex hormones, although
only a minority of nephrologists reported often discussing fertility (35%, female vs male
nephrologists, p=0.06) and menstrual irregularities with their patients (15%, female vs male
nephrologists, p=0.03). Transplant nephrologists reported discussing fertility more often than did
non-transplant nephrologists (53% vs 30%, p=0.03). Physicians were more likely to report
discussing fertility (33% vs 7.5%, p<0.001) and menstrual irregularities (15% vs 9%, p=0.04)
with patients than were allied workers. Forty-three percent of physicians and 57% of allied
workers reported they did not know if there is a role for postmenopausal hormone therapy.
60
Nephrologist survey on Sex Hormone Status
Limitations: The study is limited by the self-reported nature of the survey. The sample was
derived from membership to nephrology societies which may limit generalizability.
Conclusion: Nephrologists recognize an impact of CKD on sex hormones in women but report
not frequently discussing sex hormone related issues with patients. Our international survey
highlights an important knowledge gap in nephrology.
Key words: Chronic kidney disease, nephrologist, survey, estradiol, sex hormone, hormone
therapy, women
61
Nephrologist survey on Sex Hormone Status
Introduction
Chronic kidney disease (CKD) affects 8-16% of individuals globally 232. In women with CKD,
disruptions in gonadotropin releasing hormone production result in an abnormal sex hormone
profile, ultimately resulting in low levels of estradiol 122 123, 124 and commonly leading to early
menopause, menstrual disorders and infertility in women with both dialysis and non-dialysisdependent CKD 14. Fertility rates among women of child-bearing age with CKD are low 16-18, 137,
with only 2% of female patients of childbearing age becoming pregnant over a 4 year period18,
and an estimated incidence of a pregnancy going beyond the first trimester to be only 0.3 per 100
patient years16. Moreover, complications to both mother and fetus are high when pregnancy
occurs19, 137 highlighting the importance of discussion of the potential risks of contraception233-235
compared to the risks of pregnancy. The largest study to date examining menstrual history in
women <55 years of age with end-stage kidney disease on dialysis reported more than a third of
women were amenorrheic at the start of dialysis236, with a mean age at menopause 4 years earlier
than in the general population237. The increased cardiovascular and decreased quality of life
associated with menopause33-36 make this change of sex hormone status an important time point
in the care of the female patient.
International guidelines suggest that women with premature menopause in the general population
use hormone therapy (HT) until the median age of natural menopause for symptom management
238-241
, although this has never been addressed in guidelines relevant to the CKD population. Data
62
Nephrologist survey on Sex Hormone Status
suggests that initiation of HT in women below the age of 45 years with surgical menopause
decreases cardiovascular mortality risk 54, 242. There is much debate about whether HT provides
any cardioprotective benefit with a recent report suggesting a potential benefit in healthy women
initiating HT within 10 years of menopause onset243. Studies on the role of HT in the CKD
population are limited244-250. Given the high prevalence of menstrual, pregnancy and fertility
disorders couples with the early menopause observed in the CKD population, we sought to
determine how health care providers in nephrology approach management of sex hormone status
in women with CKD.
Methods
Survey Development
A survey instrument (Appendix 1) was developed with input from a group of 15 physicians
(experts in the field of nephrology, endocrinology and cardiology) and renal allied healthcare
workers. Item generation was developed through a combination of literature review and
consultation with experts in the field as outlined above. An assessment of the face validity,
clarity, length and completeness of the survey was performed through semi-structured interviews
(pre-testing) with nephrologists. The instrument was pilot tested on 6 nephrologists. Test-retest
reliability was performed twice, approximately 4 weeks apart, and demonstrated an overall kappa
score of 0.38 for the survey responses251. Internal consistency was measured by using two
questions assessing similar domains with an overall kappa score of 0.51251.
Survey Administration
An e-mail cover letter explaining the purpose of the study and a link providing access to the
secure, web-based survey was sent to 2,441 members of the Canadian (n=285) and Australia and
63
Nephrologist survey on Sex Hormone Status
New Zealand (n=850) Nephrology Societies and the United Kingdom Renal Association
(n=1306) as well as to the 456 nurse members of the Canadian Association of Nephrology
Nurses and Technologists between February and November 2015. The study remained open 2
months after the initial e-mail contact. Reminder emails were sent up to 3 times, each at least 2
weeks apart. Additionally, the survey was included in the monthly International Society of
Nephrology (n=9000) electronic newsletter for July and September 2015. The survey was also
disseminated via Twitter by the International Society of Nephrology once in June and once in
August 2015.
Information was collected on demographics, type of nephrology practice, perceptions on
discussion of fertility, menstrual cycle irregularities and menopause with patients, and opinions
on use of postmenopausal hormone therapy in women with CKD. A copy of the survey can be
found as a Supplementary Appendix (appendix 2). The responses for questions assessed for
frequency (never, rarely, sometimes, often or always) or agreement (strongly disagree, disagree,
neither agree nor disagree, agree, strongly agree or I do not know). Additionally, each question
included an optional free form comment section. Clinical sensibility was assessed by reviewing
the comments from the survey sent to the Canadian Society of Nephrology, and the survey was
modified to include a question to further assess type of practice (adult, pediatric, or both) such
that the survey link to the United Kingdom Renal Association, Australia and New Zealand
Society of Nephrology, the International Society of Nephrology and the Canadian Association of
Nephrology Nurses and Technologists included a question regarding pediatric versus adult
nephrology practice.
64
Nephrologist survey on Sex Hormone Status
Ethics approval for the study was obtained from the University of Calgary Conjoint Health
Research Ethics Board. Participation in the study was voluntary and informed consent was
obtained electronically as part of the survey.
Data Analysis
The goal of the primary analysis was to describe the perceptions of nephrologists and nephrology
nurses on the importance and frequency of discussing sex hormones status with their female
patients with CKD of childbearing age, and their opinions on use of postmenopausal hormone
therapy in women with CKD. Furthermore, we compared the responses from female and male
nephrologists, as it has been reported that physicians use fewer resources to treat the genitalspecific conditions of same-sex patients 252, and physicians to allied renal healthcare workers. All
data was used to conduct stratified statistical analyses. Comparisons were made between female
and male nephrologists, transplant and non-transplant nephrologists, adult and pediatric practice,
physicians and allied healthcare workers and geographical location using ordinal logistic and
logistic regressions. For the qualitative analysis, each free form response was analyzed by two
reviewers (SR and SA), comments were reduced to broader themes by inductively developing
categories, and recurrent ideas were grouped into themes which were then reported. There was
consensus between the two reviewers.
Results
Demographics
65
Nephrologist survey on Sex Hormone Status
There were 296 respondents (157 nephrologists, 17 medical trainees in nephrology, 122
nephrology allied health care providers) to the survey. The average response rate was 21.2%
(47%, 8.4%, 1.3% and 28% respectively for the Canadian Society of Nephrology, United
Kingdom Renal Association, Australia and New Zealand Society of Nephrology, and Canadian
Association of Nephrology Nurses and Technologists). Additionally, 0.2% of the membership
from the International Society of Nephrology (ISN) responded to the survey, though the low
response rate was expected due to the fact that the Canadian and Australia and New Zealand
Societies of Nephrology also form part of the ISN.
Fifty eight percent of the respondents were nephrologists (n=157) and medical trainees (n=17) in
nephrology and 34% were nurses or nurse practitioners. Other respondents included pharmacists
(2%).
Table 1 summarizes the demographic characteristics of the nephrologist respondents. Sixty-eight
percent of the nephrologists were between the ages of 30-50 years and 38% of nephrologists
were female. There was a broad range in terms of length of practice with the majority practising
nephrology in a clinical and academic setting. Twenty percent of the nephrologists and medical
trainees in nephrology respondents were transplant nephrologists. Of the 55 nephrologists and
trainees in nephrology respondents where this information was available, 4% of nephrologist
respondents were primarily involved in pediatric practice.
In contrast, 46% of the allied healthcare workers (nurses and pharmacists) were between the ages
of 30-50 years and 89% of them were female. 56% of the respondents reported being in
nephrology practice for greater than 25 years. None of the allied healthcare respondents worked
66
Nephrologist survey on Sex Hormone Status
primarily with a transplant population and 2% of the allied healthcare respondents reported being
primarily involved in pediatric nephrology practice.
Impact of kidney disease on sex hormones and discussion of symptoms with patients
The perceptions of nephrologists on sex hormone status and kidney function are summarized in
Figures 1-4. Ninety-five percent of the respondents strongly agreed or agreed that kidney
function has an important effect on sex hormone status, but only a third of respondents reported
often or always discussing fertility with their female patients of childbearing age and even fewer
(15%) reported always or often discussing menstrual irregularities with their female patients of
childbearing age. Though there were no differences between female and male nephrologists in
how often they reported discussing fertility (p=0.06), female nephrologists reported more
frequently discussing menstrual irregularities with their patients compared to male nephrologists
(p=0.02) (Figure 1). Moreover, transplant nephrologists were more likely to report discussing
fertility with their female patients (53% transplant nephrologist vs. 30% non-transplant
nephrologist p=0.03). There were no differences based on geographical location of the
respondents. As only 4% of the nephrologists reported having a mainly pediatric practice, we
were unable to analyze the difference between adult and pediatric nephrologists.
Thirteen percent of respondents provided comments. The main themes that emerged from
qualitative analyses of the optional comments were that discussions regarding fertility were
mainly focused on either contraception counselling in the setting of use of teratogenic drugs or
pre-kidney transplant, or when prescribing drugs associated with the potential to cause ovarian
failure. Additionally, some physicians outlined that discussion of fertility with their patients was
67
Nephrologist survey on Sex Hormone Status
not warranted because the female patients in their practice were mainly postmenopausal older
women.
In contrast, 48% of their female patients of childbearing age, as reported by nephrologists,
wished to discuss fertility and 15% wished to discuss menstrual status and menopause. Female
nephrologists stated that their female patients of childbearing age brought up issues about
fertility with them more frequently than did male nephrologists (p=0.03); however, this gender
difference was not observed for patients discussing menstrual status or menopause (p=0.14)
(Figure 2). Female patients were reported to bring up issues about fertility more often by
transplant nephrologists than non-transplant nephrologists (67% transplant nephrologist vs. 45%
non-transplant nephrologist p=0.01). Despite respondents reporting that patients wished to
discuss concerns associated with fertility and menstrual irregularities, only 35% of the
respondents asked patients to address these concerns with their family physician or another
physician (endocrinologist or gynecologist), though this may reflect that nephrologists were
managing these issues themselves. This was not different between female and male nephrologists
(p=0.53).
Similarly, among allied healthcare workers, 87% agreed or strongly agreed that kidney function
has an important impact on sex hormone status; however, very few reported always or often
discussing fertility (7.5%) or menstrual irregularities (9%) with their female patients. This was
significantly different from that reported by physicians (fertility: p<0.001 menstrual
irregularities: p=0.04) Twenty-seven percent of allied healthcare workers agreed or strongly
agreed that their female patients of childbearing age wished to discuss fertility often and 30%
wished to discuss menstrual abnormalities or menopause. Allied healthcare workers were less
68
Nephrologist survey on Sex Hormone Status
likely to report that their female patients wished to discuss fertility (p<0.001) but more likely to
report that female patients wished to discuss menstrual irregularities (p=0.003) compared to
physicians.
Postmenopausal hormone therapy
While most physicians (43%) reported not knowing whether there is a role for postmenopausal
hormone therapy (HT) in women with CKD, 17% agreed or strongly agreed that there is a role
for HT in women with CKD. When asked whether the benefits of HT outweigh the risks in
patients with CKD, 46% of the physicians reported they did not know (Figure 3). Furthermore,
47% reported they did not know if HT should be considered in perimenopausal women with
CKD and 45% reported they did not know if HT should be considered in postmenopausal
women with CKD.
Fifty-three percent of the respondents agreed or strongly agreed that the formulation of HT and
whether it is used in conjunction with progesterone are important in women with a uterus and
35% reported they did not know. Similarly, 37% agreed or strongly agreed that the route of HT
administration is important and 37% reported they did not know. Thirty-four percent of the
physicians agreed or strongly agreed that the stage of CKD should be considered for HT use
while 39% reported they did not know. When asked at what stage of CKD the benefits of HT
outweigh the risks, 75% reported they did not know. (Figure 4)
Among allied healthcare professionals 57% reported they did not know whether there is a role
for HT in women with CKD and 58% reported they did not know if the formulation of HT
should be taken into consideration for women with CKD. Forty-one percent reported they did not
know if the route of administration is important to take into consideration for women with CKD.
69
Nephrologist survey on Sex Hormone Status
When asked about HT prescription practice prior to the publication of the Women’s Health
Initiative trials (WHI) in 2002253, 47% of the physicians reported they were not in nephrology
practice prior to 2002, 5.8% reported they often prescribed HT, and 37% reported they did not
prescribe HT. In contrast, 83% stated they did not prescribe HT after the WHI trials were
published. Among the 5% of physicians who provided written comments on HT prescription
practice, most suggested that they were likely to recommend HT for symptom relief in their
female patients with CKD.
Discussion
This cross-sectional survey examined the views of nephrologists and nephrology allied
healthcare professionals on the impact of kidney disease on sex hormone status. We found that
while nephrologists and allied healthcare professionals recognize the important effects of kidney
disease on sex hormone status in women with CKD, reported discussion of the sequelae of low
estradiol levels such as infertility, menstrual disorders or premature menopause with their
patients was limited.
There was significant variability in the responses stratified by gender, transplant and nontransplant nephrologists, and physicians and allied healthcare workers. Female nephrologists
were more likely to report discussing menstrual irregularities with their patients and patients
were reported to more frequently bring up issues about fertility by female nephrologists.
Transplant nephrologists reported discussing fertility was more often than did non-transplant
70
Nephrologist survey on Sex Hormone Status
nephrologists. Physicians were more likely to report discussing fertility and menstrual
irregularities with their female patients than allied healthcare workers.
Despite awareness that kidney-disease mediated abnormalities in sex hormone levels are
common, nephrologists and allied healthcare workers reported not knowing if there was a role
for HT in the CKD population, which is likely a reflection of the paucity of data on the effects of
HT in this group. Taken together, the results of this study suggest that while the nephrology
community is aware of the impact of CKD on fertility, menstrual disorders and menopause with
patients, incorporation of this knowledge into clinical practice is more limited.
In women with CKD, disruptions in GnRH production result in an abnormal sex hormone profile
ultimately leading to low levels of estradiol, though the pathophysiology remains unclear 122 123,
124 128, 133
. Low levels of estradiol result in physical and psychological symptoms such as
decrease in fertility and sexual desire and satisfaction, menstrual abnormalities, hot flashes, sleep
disturbances, and mood changes which are associated with a decrease in quality of life in
women, at least those without CKD 34-36. Fertility rates among women of child-bearing age with
CKD are low 16-18, however complications to both mother and fetus are high when pregnancy
occurs, highlighting the importance of discussion of the potential risks of different forms of
contraception compared to the risks of pregnancy 19. A study of 100 women with CKD reported
that 88% had menstrual problems or were menopausal, with 20% of menopausal women being <
40 years of age 14. A previous cross-sectional study examining gynecological issues in 76
women on dialysis showed that 59% of women reported irregular menses15. Additionally, a
meta-analysis of women with CKD found that the prevalence of sexual dysfunction as defined by
decreased sexual desire and satisfaction ranged from 30-80%20. There is therefore evidence to
71
Nephrologist survey on Sex Hormone Status
suggest that women with CKD demonstrate have a significant burden of signs and symptoms
associated with low estradiol levels.
There may be, however, several reasons why nephrologists reported not often discussing fertility,
menstrual irregularities and menopause with their female patients. First, they may feel this falls
outside their domain of expertise or the primary purpose of their clinical encounter with the
patient. Second, it has been shown that in patients with chronic conditions with multiple
comorbidities and disease states that are not prominently related to the primary condition are
often not addressed254. Given the disease burden of the average CKD patient, there may be
inadequate time to address issues related to sex hormone status 255, 256. Third, as suggested by
Cochrane et al. 14, although nephrologists are often considered the primary care provider by their
patients 257, women with CKD may not often raise gynaecological issues for discussion with
their nephrologists as these concerns may appear less important in comparison to other kidneyrelated health issues. Transplant nephrologists reported discussing fertility with their female
patients of childbearing age more often than non-transplant nephrologists. This could be due to
the younger age of patients receiving transplant258 and the recommendation to avoid pregnancy
within the first 1-2 years of transplant, coupled with the necessary prescription of potentially
teratogenic medications in this setting
Women with kidney disease experience cessation of amenorrhea significantly earlier than do the
general population 236 and while there are no CKD-specific recommendations, international
guidelines suggest that women with premature menopause initiate hormone therapy until the
natural age of menopause 238-241. However, our survey highlights that nephrologists and other
allied healthcare providers are not confident discussing or prescribing HT in women with CKD.
72
Nephrologist survey on Sex Hormone Status
The knowledge gap on this issue likely reflects the paucity of literature on hormone therapy in
this population as very few studies have examined the role of HT specifically in women with
kidney disease 244-250. As such, it is possible that discussion of clinical sequelae of low estrogen
levels is limited due to lack of evidence for a viable treatment option in this population.
Limitations
The limitations of our survey include the self-reported nature of the data, as well as the
possibility of differences in practice between respondents and non-respondents. The physician
sample population self-identified as mainly nephrologists in academic practice and was derived
through membership to international nephrology societies of high-income countries, which may
not accurately capture global practice and thus may limit the generalizability of the results.
However, our average response rate of 21% is comparable to the typical 20% response rate to
online surveys of clinicians 259, 260. Of note, 43% of the nephrologists who responded were
women, which is higher than recent estimates of female nephrologists (34%) 261 which may also
limit generalizability. Lastly, previous studies have suggested that social desirability bias results
in the tendency of survey respondents to answer questions in a manner that will be viewed
favourably by others262, thus suggesting that actual discussion rates of issues related to sex
hormone status in women with CKD are likely in reality lower than reported. However, a recent
meta-analysis has suggested that computer-based survey may limit social desirability bias263,
though this has been disputed262.
73
Nephrologist survey on Sex Hormone Status
Conclusion
In summary, in an international survey of nephrologists and renal allied health care providers, we
found that while physicians and allied health care providers were aware of the impact of CKD on
sex hormone levels, discussion and management of menstrual irregularities, fertility and
menopausal status were reported to be relatively infrequent in women of childbearing age. In
identifying this important knowledge and practice gap, we have highlighted the need for further
study of this common clinical issue in women with CKD.
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Nephrologist survey on Sex Hormone Status
Table 4-1 Baseline Characteristics of Nephrologists and medical trainees in nephrology
%Female
(n=67)
%Total (n=175)
Age
< 30 Years
30-40 Years
41-50 Years
51-60 Years
61-70 Years
>70 Years
8
32.57
36
17.71
3.43
2.29
%Male
(n=108)
4.63
34.33
35.82
10.45
5.97
0
P value
(Χ2)
0.04
13.43
31.48
36.11
22.22
1.85
3.7
0.528
Years of Practice
<5 Years
5-10 Years
10-15 Years
15-20 Years
20-25 Years
> 25 Years
23.43
22.29
24
10.86
6.86
12.57
29.85
20.9
23.88
11.94
4.48
8.96
19.44
23.15
24.07
10.19
8.33
14.81
Type of Practice*
Clinical
Academic
Community
Research
Education
Administrative
62.86
61.71
12.57
25.14
25.71
13.14
61.19
50.75
11.94
32.84
31.34
14.93
63.89
68.52
12.96
20.37
22.22
12.04
Transplant Nephrologist
Yes
No
Patient Population
Adult
Pediatric
Both
0.351
20
80
16.42
83.58
22.22
77.78
(n=57)
91.23
5.26
3.51
(n=26)
92.31
0
7.69
(n=31)
90.32
9.68
0
0.086
75
Nephrologist survey on Sex Hormone Status
Figure 4-1 Nephrologist impression of sex hormone status in CKD
76
Nephrologist survey on Sex Hormone Status
Figure 4-2 Nephrologist impression on patient discussion of sex hormone status in CKD
77
Nephrologist survey on Sex Hormone Status
Figure 4-3 : Nephrologist impression on hormone therapy in CKD
78
Nephrologist survey on Sex Hormone Status
Figure 4-4 Nephrologist impression on factors to consider for hormone therapy in CKD
79
Chapter Five: Hormone therapy and clinical and surrogate cardiovascular endpoints in
women with chronic kidney disease: a systematic review and meta-analysis
Ramesh S, Mann MC, Holroyd-Leduc JM, Wilton SB, James MT, Seely EW, Ahmed SB. Chapter
Five: Hormone therapy and clinical and surrogate cardiovascular endpoints in women with
chronic kidney disease: a systematic review and meta-analysis. Menopause (In Print)
80
Abstract
Objectives: Women with chronic kidney disease (CKD) experience kidney dysfunctionmediated premature menopause. The role of postmenopausal hormone therapy in this population
is unclear. We sought to summarize current knowledge regarding use of postmenopausal
hormone therapy and (1) cardiovascular outcomes, and (2) established surrogate measures of
cardiovascular risk in women with CKD.
Methods: This is a systematic review and meta-analysis of adult women with CKD. We
searched electronic bibliographic databases (MEDLINE, EMBASE, Cochrane Central Register
of Controlled Trials) (inception to 2014 December), relevant conference proceedings, tables of
contents of journals and review articles. Randomized clinical trials and observational studies
examining postmenopausal hormone therapy compared to either placebo or untreated control
groups were included. The intervention of interest was postmenopausal hormone therapy and the
outcome measure were all-cause and cardiovascular mortality, non-fatal cardiovascular event
(myocardial infarction, stroke), and surrogate measures of cardiovascular risk (serum lipids,
blood pressure).
Results: Of 12,482 references retrieved, 4 RCTs and 2 cohort studies (N=1666 patients) were
identified. No studies reported on cardiovascular outcomes or mortality. Compared with placebo,
postmenopausal hormone therapy was associated with decreased low-density lipoprotein (LDL)
cholesterol (-13.2 mg/dL (95% CI: -23.32, -3.00 mg/dL)), and increased HDL (8.73 mg/dL (95%
CI: 4.72, 12.73)) and total cholesterol (7.96 mg/dL (95% CI: 0.07, 15.84 mg/dL). No
associations were observed between postmenopausal hormone therapy triglyceride levels or
blood pressure.
81
Conclusion: Studies examining the effect of postmenopausal hormone therapy on cardiovascular
outcomes in women with CKD are lacking. Further prospective study of the role of
postmenopausal hormone therapy in this high-risk group is required.
Keywords: Hormone therapy, Chronic kidney disease, Cardiovascular disease, Lipids, Blood
pressure, Systematic Review
82
Introduction
Women with chronic kidney disease (CKD) carry a high burden of all-cause and cardiovascular
(CV) disease 6, 264. Menopause at a younger age is associated with increased CV risk 53 in the
general population. As a consequence of kidney-mediated hypothalamic-pituitary-gonadal
dysfunction, 236, 265, 266 the average age of menopause in women with CKD is 46.8 years, 5 years
earlier than that of the general population 267. There is strong evidence against treatment with
hormone therapy (HT) for prevention of CV disease in postmenopausal women overall 253, 268, 269.
However, the pathophysiology of cessation of menses in women with CKD differs markedly
from primary ovarian insufficiency or natural menopause 129 and thus the role of HT in this
population is unclear.
Recent reports have suggested that there may still be cardioprotective benefit in healthy women
who initiate HT within 10 years of the onset of menopause 243 and initiation of estrogen therapy
in young women <45 years with surgical menopause decreases CV mortality risk 54. This
suggests that sex hormones could play a protective role in terms of CV risk in select populations,
including women with CKD who experience early menopause. International guidelines have
suggested that women with premature menopause use HT until the median age of natural
menopause 238-241, though no guidelines exist in the case of the woman with CKD-mediated
menopause. Furthermore, although large trials of healthy women have shown no CV benefit after
oral HT 253, 268, 269, other important factors play a role in determining risk and benefit such as
timing of initiation of HT with respect to onset of menopause, and dose, formulation, type of
progestin and route of administration of postmenopausal HT 112, 270, 271. These demographic and
83
biologic differences can influence baseline CV risks and may modify the overall observed effects
of HT on cardiovascular risk.
Given the potentially increased CV risk associated with low estrogen status in women with CKD
and the uncertainties pertaining to the role of HT in these patients, the objective of this
systematic review and meta-analysis was to determine the associations with and effects of HT on
1) all-cause mortality and CV mortality and morbidity and 2) and surrogate measures of CV risk
factors in women with CKD.
Methods
Protocol
Our systematic review protocol was drafted using guidance from the Preferred Reporting Items
for Systematic reviews and Meta-analyses for Protocols (PRISMA-P)272. This protocol was
registered with the international prospective systematic review register PROSPERO
(CRD42014014566) and published in an open access journal 273. Our methods are described
briefly here. We originally intended to examine the effect of HT on all-cause mortality and CV
mortality and morbidity in women with CKD; however, because none of the studies reported on
these outcomes we expanded our outcomes of interest to include surrogate measures (lipid
profile and blood pressure) of CV disease and adverse events.
Information sources and literature search
Published and unpublished studies were identified through searching electronic databases
(MEDLINE, EMBASE ), from inception until December 2014 without language restrictions. All
relevant text words and Medical Subject Heading (MeSH)/Emtree terms for chronic kidney
disease (“Renal Insufficiency Chronic”, “Kidney Failure”, “Kidney Diseases”, “Renal
Replacement Therapy”, “Uremia”, “Dialysis”, “Hemodialysis”, “Peritoneal Dialysis” or
84
“Predialysis”) and hormone therapy (“Hormone Therapy”, “Hormone Replacement Therapy”,
“Estrogen Replacement Therapy”, “Estrogen”, “Estradiol”, “Conjugated Equine Estrogen”,
“Progesterone”, “Progestin”, “Medroxyprogesterone acetate” or “Dydrogesterone”) were
combined separately using the “OR” Boolean operator. These two search themes were then
combined using the Boolean “AND” operator. ). A second search was used to identify
randomized control trials studying the effect of hormone therapy on cardiovascular mortality and
morbidity. In addition to the abovementioned search terms for hormone therapy, all relevant
search terms for cardiovascular disease (“Cardiovascular Diseases”, “Stroke”, “Hypertension”,
“Hyperlipidemias”, “Arteriosclerosis”, “Cholesterol” “Blood Pressure”, “Coronary Heart
Disease”, “Mortality”, “Survival Rate”, “All Cause Mortality”, “Cause of Death”, or “Death”)
and randomized control trial were included.
The literature search was drafted by an experienced information specialist and peer reviewed by
a second information specialist using the Peer Review of Electronic Search Strategies (PRESS)
checklist274. This search was supplemented with a grey literature search using various methods,
including scanning the reference lists of included studies, relevant reviews and published
conference proceedings, and asking experts in the field.
Study selection and data extraction
After conducting a calibration exercise, we reviewed each title and abstract from the literature
search independently using the inclusion and exclusion criteria. We documented the
categorization of abstracts of each reviewer and quantified the chance-corrected agreement
between the two reviewers. We resolved any disagreement in study inclusion by discussion and
inclusion of a third reviewer (SA). The same process was followed for screening potentially
85
relevant full-text articles. The data abstraction forms are available from the authors on request.
When data were missing or clarification of published data was needed we contacted the authors.
Inclusion criteria were: 1) study population was women with CKD (as defined by authors); 2)
intervention was postmenopausal hormone therapy; 3) comparator was placebo or no treatment;
4) outcome was CV mortality or morbidity, all-cause mortality or surrogate markers of CV risk;
5) study design was or randomized controlled trial or observational study.
Appraisal of methodological quality and risk of bias
Each study was appraised for quality and risk of bias by each reviewer using the Cochrane risk
of bias tool for randomized controlled trials for RCTs or the Cochrane risk of bias tool for cohort
studies 275. Study quality was assessed for different categories: for RCTs, randomization process,
allocation concealment, blinding, intention-to-treat analysis were used and for cohort studies,
description of loss to follow-up, selection bias, measurement bias and proper accounting of
confounders and effect modifiers(assessed by use of co-interventions such as statins and antihypertensives) were used.
Outcome
The outcomes of interest were: 1) cardiovascular mortality, all-cause mortality and
cardiovascular morbidity, 2) changes in surrogate measures of cardiovascular risk (lipid profile
and blood pressure), and 3) adverse events.
Data synthesis and analysis
Summary estimates of the weighted mean differences were obtained using a DerSimonian and
Laird random effects model276. The amount of variability across studies due to heterogeneity was
estimated using I2 statistic277 and Cochran’s Q tests of homogeneity. A two-sided p-value < 0.05
86
was considered statistically significant for all analyses. All analyses were performed using Stata
(version 12, StataCorp LP, College Station, TX).
Results
Study Selection
Of 12,482 potentially relevant citations screened, 11,550 were excluded and 932 full-text
publications were retrieved for further review (Figure 1). After full-text review, 927 studies were
excluded, leaving two parallel randomized controlled trials 278, 279, one crossover randomized
controlled trial 265 and two cohort studies that met inclusion criteria 280, 281. An additional parallel
randomized controlled trial was included from the grey literature search 279. Chance-corrected
agreement between the 2 independent reviewers who evaluated study eligibility was excellent
with 90% agreement between the two reviewers.
Study Characteristics
A total of 1666 patients were included from the six studies (Table 1) 265, 278-280, 282. All studies
were designed to test the effect or association of hormone therapy in women with CKD and
reported outcomes relevant to this investigation.
The mean age of study participants in the RCTs ranged from 38.5 years to 62.5 years and all
were defined by the authors as postmenopausal. The mean age of the study participants in the
observational studies was 51.9 years. Patients in five of the included studies (4 RCTs and 2
cohort) had end-stage kidney disease and were on dialysis (4 on hemodialysis and 1 unspecified)
265, 278-280, 282
, while one cohort study did not specify the stage of kidney disease 281. None of the
studies included patients with a kidney transplant.
None of the studies reported all-cause mortality or cardiovascular morbidity and mortality as an
outcome. Among the RCTs, all four studies reported lipid levels (100%). The data was
87
abstracted from all the studies for meta-analyses. A transdermal route of administration was
examined in one RCT. The other three RCT examined an oral route of postmenopausal hormone
administration. The types of hormone therapy used in RCTs included oral estradiol265, the
selective estrogen receptor modulator raloxifene278, conjugated equine estrogen282, and
transdermal estradiol279. In all but one RCT, which examined the effect of raloxifene278, estrogen
treatment was used in combination with a progestin. The type of progestins used in RCTs
included oral medroxyprogesterone acetate265, 282 and transdermal norethindrone acetate279. The
duration of treatment ranged from 8 weeks to 1 year.
Both observational studies examined an oral route of administration of postmenopausal hormone
therapy280, 281. The hormone therapy used in both studies included oral estradiol with the
progestin norgestrel. One observational study reported total cholesterol and triglyceride levels280
and one observational study reported blood pressure outcome281.
Study Quality
The risk of bias is highlighted in table 2. None of the RCTs described how the randomization
was achieved or whether the investigators were aware of the allocation sequence in the study.
Two of the four studies were blinded265, 278. All of the studies had a low risk of bias for
incomplete data, selective outcome reporting and other sources of bias. The crossover RCT
included a washout period to eliminate any carryover effect.
The included observational studies had low risk of selection and measurement biases. Both
studies accounted for possible confounders and effect modifiers.
88
Outcomes
The primary outcome of this study was to determine the effect of postmenopausal HT on allcause and CV mortality and morbidity in women with CKD. However, none of the studies
reported this outcome.
The four RCTs each reported on the effect of postmenopausal hormone therapy on LDL and
HDL cholesterol 265, 278, 279, 282 in women with CKD. Neither of the observational studies reported
LDL or HDL cholesterol levels.
The overall pooled estimate of the weighted mean difference in LDL cholesterol in RCTs
between hormone treatment and placebo in women on hemodialysis was -13.2 mg/dL (95% CI: 23.3, -3.00 mg/dL) or -0.34 mmol/L (95% CI: -0.60, -0.08 mmol/L) (Figure 2). The statistical
heterogeneity among these studies was not significant (I2=7.2%).
The pooled estimate of the mean difference in HDL cholesterol between hormone therapy and
placebo in RCTs was 8.73 mg/dL (95% CI: 4.72, 12.73) or 0.23 mmol/L (95% CI: 0.12, 0.33
mmol/L). The statistical heterogeneity among these studies was not significant (I2=14.4%).
Of the six studies, four RCTs and one observational study reported on total cholesterol levels and
triglyceride levels. When stratified according to study design, the pooled estimate of the
weighted mean difference in total cholesterol between hormone treatment and placebo was 2.94
mg/dL (95% CI: -9.90, 15.78 mg/dL) or -0.08 mmol/L (95% CI: -0.26, 0.41 mmol/L) in RCTs
and 11.00 mg/dL (95% CI: 1.01, 20.99 mg/dL) or 0.28 mmol/L (95% CI: 0.03, 0.54 mmol/L) in
one cohort study The statistical heterogeneity among these studies was not significant (I2<1%).
The pooled estimate of the weighted mean difference in total cholesterol between hormone
therapy and placebo or no treatment in women with CKD was 7.96 mg/dL (95% CI: 0.07, 15.84
mg/dL) or 0.21 mmol/L (95% CI: 0.002, 0.41 mmol/L) (Figure 4).
89
When stratified according to study design, the pooled estimate of the weighted mean difference
in triglyceride levels between hormone treatment and placebo was -5.59 mg/dL (95% CI: -30.92,
19.75 mg/dL) or -0.06 mmol/L (95% CI: -0.34, 0.22 mmol/L) in RCTs and 47 mg/dL (95% CI:
21.66, 72.24 mg/dL) or 0.53 mmol/L (95% CI: 0.24, 0.82 mmol/L) in one cohort study. There
was significant heterogeneity among these studies (I2=74.9%). The pooled estimate of the mean
difference in triglyceride levels between estrogen treatment and placebo or no treatment was 9.77
mg/dL (95% CI: -23.72, 43.26) or 0.11 mmol/L (95% CI: -0.27, 0.49 mmol/L) (Figure 5).
No RCTs examined the effect of hormone therapy on blood pressure and only one of the two
cohort studies investigated the effect of hormone therapy on blood pressure281. This study
reported no association between oral postmenopausal hormone therapy and systolic and diastolic
blood pressure.
Reporting of adverse events was inconsistent, and those reported had unclear relevance to the use
of postmenopausal hormone therapy. Only two of the four RCTs and one of the two
observational studies reported on adverse outcomes (n=101 patients) 278, 279, 281. In one of the
RCTs two women in the treatment group developed persistent headaches and dizziness and
recurrent vascular access thrombosis, but the authors did not report if this was statistically
significant 279, whereas in the other RCT there were no observed adverse events in either group
278
. No adverse outcomes were reported in the cohort study 281.
Discussion
We performed a comprehensive review of studies examining the use of postmenopausal hormone
therapy conducted among over 1600 women with CKD. Of note, the majority of women
(n=1499) were from one prospective cohort study280. Our main findings were that in women with
CKD: 1) no studies have examined the effect of postmenopausal hormone therapy on all-cause
90
mortality and CV mortality and morbidity, 2) hormone therapy was associated with a decrease in
levels of LDL cholesterol, increases in levels of HDL cholesterol and total cholesterol, and was
not associated with triglyceride levels, and 3) though reporting of adverse events was limited to
only three of the six studies, complications related to postmenopausal hormone treatment were
not common. It is important to note, however, that the included studies had different study
designs (treatment regimens, dose, and route of therapy) resulting in high heterogeneity among
these studies, and this should be taken into consideration when interpreting the results.
The pathophysiology by which women with CKD develop cessation of menses differs markedly
from that of natural menopause. The generally accepted definition of menopause is amenorrhea
for 12 months or more and an FSH level of greater than 25 IU/L283 Women with CKD have an
abnormal sex hormone profile and abnormal hypothalamic-pituitary-gonadal activity
characterized by the lack of pulsatile GnRH secretion and low estradiol 122, 127, 128, 265, resulting in
absence of menses, infertility and premature menopause which can be reversed in the setting of
kidney transplantation or quotidian hemodialysis284, 285. This is a distinct contrast from natural
menopause whereby atresia, the continuous depletion of oocytes, ultimately results in
irreversible cessation of the production of estradiol and progesterone286. Early menopause in
healthy women is associated with a 2% increase in cardiovascular mortality54, but initiation of
postmenopausal hormone therapy in younger women with surgical menopause 55 and those
within 10 years of the menopausal period 287 decreases the risk (hazard ratio: 1.93 (95% CI: 1.252.96) in women without hormone therapy vs. 1.27 (95%CI: 0.67-2.39) in women with hormone
therapy). As women with CKD routinely experience menopause almost five years earlier than
the general population 236, 266, 267, 288, this suggests a potential therapeutic role for short-term
estrogen treatment in this high cardiovascular risk population 289.
91
Our analysis, demonstrating a small improvement in lipid profile with hormone treatment, is
similar to previous studies on this topic; however, the clinical significance of this improvement is
unclear. Previous investigations in healthy women have demonstrated a favorable effect of oral
estradiol on lipid profile. In the Postmenopausal Estrogen/Progestin Interventions trial of 875
healthy postmenopausal women, both oral conjugated equine estrogen (CEE) alone and CEE
plus progestin (medroxyprogesterone acetate or micronized progesterone) treatment resulted in
an increase in HDL cholesterol and a decrease in LDL cholesterol 65. This is further supported by
studies that have shown that postmenopausal hormone therapy increases the production of HDL
cholesterol and decreases clearance of HDL cholesterol290, 291. However, caution should be
exercised when interpreting these results as previous studies of lipid-lowering therapy in CKD
populations have not always translated into improved clinical outcomes 27, 292, 293.
Although a recent meta-analysis of postmenopausal hormone therapy in healthy postmenopausal
women found no evidence of cardioprotection294, the results varied among the included studies
due to the different hormone regimens, timing of hormone therapy initiation with respect to onset
of menopause, and the type of estradiol and progestin used294, 295. Furthermore, 8 of the 13 trials
included in the meta-analysis were examining the effect of hormone therapy as secondary, rather
than primary, prevention in a high CV risk population 294.
There are limitations to this systematic review. First, baseline characteristics of patients varied
with respect to age, type of hormone therapy and route of administration and the paucity of
studies did not allow for analyses stratified by these factors. Second, there is evidence that serum
estradiol levels are variable, depending on the route of administration 296 and degree of CKD 129.
It is important to note that most of the participants in the included studies in this systematic
review were women with end stage kidney disease treated with dialysis. As there is evidence to
92
suggest that hormonal irregularities are seen as early as stage 3 in women with non-dialysis
dependent CKD129, the effect of postmenopausal hormone therapy at an earlier stage of CKD
may differ compared to later stages of CKD. However, our results suggest that at least in the
population with end-stage kidney disease treated with dialysis, hormone therapy is associated
with an improved lipid profile. Third, all studies included a modest number of participants with
short duration of postmenopausal hormone treatment and reported surrogate markers of
cardiovascular risk rather than clinical outcomes. Fourth, reporting of adverse events was limited
across studies and most studies were not powered sufficiently to determine the rate of adverse
events associated with the use of hormone therapy in this population. Moreover, the incidence of
malignancy is higher in patients with end-stage kidney disease on dialysis297; as PHT has been
associated with an increase in breast cancer risk 287, this would be an important consideration
with the use of this therapy in the CKD population.
Conclusion
In conclusion, this meta-analysis suggests potential short-term efficacy of postmenopausal
hormone therapy in improving lipid profiles in younger women with CKD. However, no
conclusions can be drawn related to cardiovascular morbidity or mortality. The North
American Menopause Society endorses the use of hormone therapy for short-term symptom
management in women with symptoms of menopause, and highlights the need for further
research with respect to the long term outcomes of HT 298. Currently there are no Kidney
Disease Improving Global Outcomes (KDIGO) guidelines on the use of hormone therapy in
women with CKD. A large, prospective trial in women with variable hormone status and
stages of CKD, which includes clinical outcomes, is needed to guide clinical practice in terms
of the role for postmenopausal hormone therapy among this high risk population.
93
Table 5-1 Study Characteristics
Authors
Study Inclusion Criteria
Randomized Controlled Trials
Women ≥45 years old who
were undergoing HD for ≥6
Ginsburg et al.
months and had no
(1998) (5)
menstrual periods for ≥ 1
year
Women >50 years old who
have been menopausal for
Hernandez et al.
>2 years with severe
(2003) (27)
osteopenia or osteoporosis
and who were treated with
dialysis
Women who had secondary
Matuszkiewiczamenorrhoea, with serum
Rowinska et
estradiol levels <30pg/mL
al.(1999) (28)
and were on HD for ≥6
months
Women who were on HD
for ≥ 6 months, had no
Park et al. (2000)
menstrual periods for ≥ 1
(31)
year and had serum estradiol
levels <25pg/mL
Observational Studies
Women in the USRDS
Stehman-Breen et Dialysis Morbidity and
al. (1999) (29)
Mortality Study (DMMS)
(Prospective
Wave 2, which is a database
Cohort)
of chronic hemodialysis
patients
Szekacs et al.
Postmenopausal women
(2000) (30) (Prewith nephropathy and type 2
post intervention
diabetes and hypertension
study)
Country
Study
Size
Mean Age
in
Treatment
Mean Age
in Placebo
Follow up
Route of
Administration
of HRT/Prior
use of HRT
Dose of HRT
Study Related Outcomes
10mg
medroxyprogesterone
acetate for one week
followed by 1mg
estradiol for 8 weeks
LDL-c, HDL-c, total
cholesterol, triglycerides
USA
11
61.4
61.4
8 weeks
Oral/ No HRT
use 6 weeks
before study
Venezuela
50
63.1
61.9
52 weeks
Oral/ No
previous use of
HRT
Oral raloxifene 60 mg per
day
LDL-c, HDL-c, total
cholesterol, triglycerides,
adverse events
52 weeks
Transdermal/
No HRT use 5
years before
study
Transdermal estradiol
50mg with 0.25 mg
norethindrone acetate 2
times a week
LDL-c, HDL-c, total
cholesterol, triglycerides,
adverse events
LDL-c, HDL-c, triglycerides
Poland
25
.
.
Japan
65
57
(median)
61
(median)
12 weeks
Oral/ Unknown
Oral conjugated equine
estogen 0.625 mg with
medroxyprogesterone
acetate 2.5mg daily for 12
weeks
USA
1499
.
.
Crosssectional
study
Oral/ Unknown
.
Total cholesterol and
triglycerides
Hungary
16
51.9
51.9
14 weeks
Oral/ No
previous use of
HRT
Oral estradiol 2mg/day
and oral norgestrel
0.5mg/day
Blood pressure
Abbreviations: LDL-c: low density lipoprotein cholesterol HDL-c: high density lipoprotein choleste
94
Table 5-2 Randomized controlled trial and observational studies risk of bias assessment
Sequence
generation
Allocation
concealment
Blinding of
participants,
personnel and
outcome
assessors
Unclear
Unclear
Low
Low
Low
Low
Hernandez et al Unclear
MatuszkiewiczRowinska et al. Unclear
Park et al.
Unclear
Unclear
Low
Low
Low
Low
Unclear
Unclear
Unclear
High
Low
Low
Low
Low
Low
Low
Ginsburg et al.
Authors
Stehmanbreen et
al.
Szekacs
et al.
Incomplete Selective
outcome
outcome
data
reporting
Other
sources
of bias
Did the study match
exposed and
unexposed for all
variables that are
associated with the
outcome of interest
or did the statistical
analysis adjust for
these prognostic
variables?
Can we be
confident in the
assessment of
the presence or
absence of
prognostic
factors?
Can we be
confident in
the
assessment
of outcome?
Was the
follow up
of cohorts
adequate?
Were co‐
Interventions
similar
between
groups?
Was selection
of exposed and
non‐exposed
cohorts drawn
from the same
population?
Can we be
confident in
the
assessment
of exposure?
Can we be
confident that
the outcome
of interest
was not
present at
start of
study?
DY
PY
DN
DY
DY
DY
DN
PY
DY
DY
DY
DY
DY
DY
PY
DY
Abbreviations: DY: definitely yes, PY: probably yes; PN: probably no DN: Definitely no
95
Figure 5-1 PRISMA flow diagram showing the identification process for eligible studies
96
Figure 5-2Forest Plot of the effect of hormone therapy on LDL cholesterol
97
Figure 5-3Forest plot of the effect of hormone therapy on HDL cholesterol
98
Figure 5-4 Forest plot of the effect of hormone therapy on total cholesterol
99
Figure 5-5 Forest plot of the effect of hormone therapy on triglyceride levels
100
Chapter Six: Menopause status is associated with mortality in women on hemodialysis in
Canada
Ramesh S, Wilton SB, James MT, Holroyd-Leduc JM, Seely EW, Tonelli M, Hemmelgarn BR
and Ahmed SB. Menopause status is associated with mortality in women on hemodialysis in
Canada (to be submitted)
101
Abstract
Introduction: Young women with end-stage kidney disease (ESKD) have low survival
compared to age-matched men. Women with ESKD have an abnormal sex hormone profile
characterized by low estradiol levels, which in turn are associated with increased mortality in the
healthy population. We sought to determine if menopausal status and estradiol levels were each
associated with death in women with ESKD.
Methods: In this multi-centered study all women who initiated hemodialysis since February,
2005 in 3 centres were classified as premenopausal, perimenopausal and postmenopausal using
the Women’s Ischemia Syndrome Evaluation criteria. The associations between menopausal
status and all cause, cardiovascular (CV) and non-cardiovascular (non-CV) mortality were
determined by survival curves and Cox regression.
Results: Four hundred eighty-two women (60±16years, 53% diabetic, estradiol 116±161pmol/L)
were followed for 2.9 years (IQR=3.47 years) with 241 deaths (31% CV). Thirty percent of
women in dialysis had a premenopausal sex hormone profile, while 7% and 58% had a
perimenopausal and postmenopausal sex hormone profile respectively. In comparison to
postmenopausal women, perimenopausal and premenopausal women had higher all-cause
mortality after adjustment for covariates (HR:1.87, p=0.009 and HR: 1.27, p<0.001,
respectively), and higher CV mortality after adjustment for co-variates (HR: 2.72 p=0.008 and
1.81, p=0.04, respectively). Moreover, in comparison to women classified as postmenopausal,
premenopausal women had a higher risk of non-CV mortality after adjustment for co-variates
(HR: 1.71, p=0.02). Low estradiol was associated with higher all-cause and non-cardiovascular
risk only in women over the age of 51 years.
102
Conclusion: Peri- and premenopausal women with ESKD on hemodialysis each have a higher
risk of all-cause, cardiovascular, and non-cardiovascular mortality compared to postmenopausal
women, suggesting that factors other than estradiol are associated with the increased risk of
death in younger women. Further studies are required to determine the cause of death in young
women with ESKD.
103
Introduction
Young women with end stage kidney disease have the highest mortality compared to all other
groups299. The reasons for this high risk of death are unclear, though low estrogen levels have
been postulated to contribute to increased mortality 300 Women with end stage kidney disease
(ESKD) have an abnormal sex hormone profile characterized by hypothalamic pituitary
disturbances and low estradiol levels236, 265, 266. The average age of menopause in the female
dialysis population is 46.8 years267, which is 5 years earlier than the general population 288. In the
healthy population menopause, and in particular early menopause, is associated with an increase
in the incidence of cardiovascular disease and mortality301-306. Hypothalamic hypoestrogenemia
in primates is related to premenopausal atherosclerosis307-309, and premenopausal women with
coronary artery disease are more likely to have hypothalamic hypoestrogenemia310.
Given the association between ESKD and an abnormal sex hormone profile characterized by
hypothalamic pituitary abnormalities leading to hypoestrogenemia, we sought to determine the
associations between menopausal status and all-cause, cardiovascular, and non-cardiovascular
mortality in a cohort of women with ESKD initiating hemodialysis. We hypothesized that
postmenopausal women would have higher mortality in comparison to premenopausal and
perimenopausal women.
Methods
Study Design
This is a prospective multicenter cohort study using data from the Canadian Kidney Disease
Cohort Study (CKDCS), whose methodology has been discussed in detail previously 311.
Briefly, patients initiating hemodialysis in 4 dialysis centers in Calgary, Edmonton, Ottawa and
Vancouver from February 2005 to March 2016 were enrolled and followed prospectively. A
104
principal investigator was responsible for recruitment, data collection, and follow-up for each
site. Information on patient demographic, medical history, and mortality was collected. Ethics
approval for the study was obtained from the institutional review boards of all participating
centers and was conducted according to the Declaration of Helsinki of medical research in
humans311.
Study Participants
The inclusion criteria were all female hemodialysis patients over the age of 18 years from the
four participating hemodialysis centers. All eligible patients were approached by study staff
within 8 weeks of hemodialysis initiation. The exclusion criteria included subjects who were not
competent to provide consent (n=42), had limited life expectancy (n=10), difficult to locate
(n=9), or with acute kidney injury(n=7) (Figure 1).
Data Collection
All consenting participants underwent a structured interview at baseline to collect information on
demographic characteristics and medical history. Information from the patients’ clinical record
was used to supplement the medical history. Follow-up visits were conducted at 6 months and 1,
2, 3, 4 and 5 years. After the fifth year, follow-up visits were conducted every 5 years.
Sera from blood samples were collected within 3 months of initiation of hemodialysis and sera
were processed and frozen in 0.5-mL cryovials at −85°C within 72 hours of sample collection.
The frozen serum samples of all women over the age of 18 years were analysed for serum
estradiol, follicular stimulating hormone (FSH) and prolactin levels at a central laboratory
Outcome Measures
105
The primary outcome of this study was all-cause mortality, cardiovascular mortality and noncardiovascular mortality. The date of death of study participants was identified through regular
follow-up by study coordinators. Cause of deaths were ascertained by two physicians
independently using medical records, discharge summaries, and autopsy reports (if available);
any disagreement was resolved by consensus. If independent assessment of cause of death was
not available, data was obtained from Canadian Vital Statistics.
Deaths due to acute myocardial infarction, atherosclerotic cardiovascular disease, circulatory
complications of diabetes, ischemic heart disease, hypertrophic cardiomyopathy, stroke,
aneurysm, vascular disorder, and peripheral vascular disease were classified as cardiovascular.
All other deaths were classified as non-cardiovascular.
Statistical analysis
Baseline characteristics are presented as means ± standard deviation or proportions unless
otherwise stated. The women were stratified based on serum estradiol levels using the Women’s
Ischemia Syndrome Evaluation algorithm for menopausal status116. Briefly, women were
classified as follows: 1) premenopausal if (a) FSH<10 IU/L or (b) FSH between 10-20IU/L and
estradiol>184pmol/L, 2) perimenopausal if (a) FSH 10-20 IU/L and estradiol <184 pmol/L (b)
FSH 20-30 IU/L (c) FSH>30 IU/L and estradiol ≥ 184 pmol/L or (d) age ≥ 45 years and estradiol
≥ 734.2 pmol/L, and 3) postmenopausal if FSH >30 IU/L and estradiol <184 pmol/L. Cox
proportional hazards models were used for time-to-event analyses for all-cause, cardiovascular
and, non-cardiovascular mortality. Censoring occurred due to switching of modality, emigration
to another province, and loss to follow-up or end of study (March 31st 2016). Postmenopausal
women were used as the referent group for all analyses as previous studies have shown that older
women on dialysis have a survival advantage over younger women299. Additionally, to determine
106
the association between serum estradiol levels and mortality, subjects were stratified based on
estradiol levels (above and below the median), and age (above and below 51 years, the average
age of menopause in the general population288. For cardiovascular mortality, all noncardiovascular deaths were censored and vice versa for non-cardiovascular mortality. All Cox
regression models were adjusted for potential confounders including age, body mass index
(BMI), diabetes, hypertension, coronary artery disease, and prolactin312. Additionally, interaction
by age was assessed in all Cox regression models using age as both a categorical (above and
below 51 years) and continuous variable. A two-sided p-value < 0.05 was considered statistically
significant for all analyses.
For sensitivity analyses, we explored differences in adjudicated deaths versus cause of deaths
defined by vital statistics data as well as the effect of storage on serum estradiol measurement by
plotting estradiol levels versus time since collection.
All analyses were performed using Stata (version 12, StataCorp LP, College Station, TX).
Results
Study Participants
Of 1955 patients with ESKD treated with hemodialysis in the 4 centers, 1650 were approached
and 1454 were enrolled (Figure 1). Of these, 553 women were eligible women. Of the 553
women, 482 plasma samples were collected. However, all collected plasma was used for
alternative analysis with no plasma remaining in two subjects, one subject withdrew consent, and
three samples were mislabelled and therefore not included in analysis. As such, 476 plasma
samples were available for analysis. In the 10 years of follow up there were 170 withdrawals
107
(kidney transplant =51, switched dialysis modality=73, moved away from region=15, recovered
kidney function=17, withdrew consent=11, other=3).
Baseline characteristics and menopausal status
The baseline characteristics of the 482 women are listed in Table 1. The mean age of the women
was 60±16 years and the participants were predominantly Caucasian (75%). A majority of the
women had clinically documented hypertension (87%) and 53% of the women were diabetic.
The participants were followed up for an average of 2.9±2.4 years (median: 2.45 interquartile
range: 3.47).
Baseline characteristics of participants stratified by menopausal status are listed in Table 1.
Serum sex hormones levels were available in 476 women, 35% (n=164) were classified as
premenopausal, 7% (n=34) were classified as perimenopausal, and 58% (n=278) were classified
as postmenopausal. Women classified as premenopausal were significantly younger than
perimenopausal and postmenopausal women (p<0.0001), have lower systolic blood pressure and
higher diastolic blood pressure compared to postmenopausal women (p=0.002 and p=0.01
respectively).
Two hundred and forty one participants died over a mean follow up of 2.9 years. Seventy-five
(31%) of the deaths were due to cardiovascular causes and 110 (46%) of the deaths were due to
non-cardiovascular causes). Of 241 deaths, the cause of death in 165 (68%) were adjudicated by
independent reviewers, and the cause of death was missing for 56 (23%) of the participants.
108
Sex Hormone Levels
The mean serum estradiol level for the cohort was 116.1 ± 161.5 pmol/L (median: 64 IQR: 94
pmol/L), the mean serum FSH level was 58 ± 55 IU/L (median: 49 IQR: 89 IU/L) and the mean
serum prolactin level was 68 ± 137 IU/L (median: 27 IQR: 31 IU/L) (Table 1).
Sex hormone levels stratified by menopausal status are presented in Table 1. As expected,
premenopausal and perimenopausal women had higher estradiol levels compared to
postmenopausal women (p<0.0001). Serum FSH levels were significantly different between the
three groups with premenopausal women having the lowest and postmenopausal women the
highest FSH levels (p<0.0001). Serum prolactin levels were significantly different between the
groups with serum prolactin being lowest in postmenopausal women (p=0.04).
Associations between menopausal status and all-cause mortality
A nonsignificant trend towards greater all-cause mortality was observed when comparing
premenopausal women to postmenopausal women (HR 1.27, p=0.09) (Table 2, Figure 2). This
risk was significant after adjustments for covariates (HR: 1.8, p<0.0001, Table 2).
In comparison to postmenopausal women, perimenopausal women had a significantly higher risk
of all-cause mortality (HR 1.63, p=0.03) (Table 2, Figure 2), a risk that increased after
adjustment for covariates (HR: 1.87, p=0.009) (Table 2). There was no interaction between age
and menopausal status.
Associations between menopausal status and cardiovascular (CV) mortality
The unadjusted risk of CV mortality was not significantly different between premenopausal
women and postmenopausal women (HR: 1.32, p=0.28, Table 2, Figure 3); however, after
adjustment for covariates the risk of CV death in premenopausal women was significantly higher
than postmenopausal women (HR: 1.81, p=0.04, Table 2).
109
In comparison to postmenopausal women, perimenopausal women had a significantly higher risk
of CV mortality (HR: 2.1 (p=0.045, Table 2, Figure 3). This association became stronger after
adjustment for covariates (HR: 2.72 p=0.008, Table 2). There was no interaction between age
and menopausal status.
Associations between menopausal status and non-cardiovascular (CV) mortality
The unadjusted risk of non-CV mortality was not significantly different between premenopausal
women and postmenopausal women (HR: 1.14, p=0.5, Table 2, Figure 4); however, after
adjustment for covariates the risk of non-CV death in premenopausal women was significantly
higher than postmenopausal women (HR: 1.71, p=0.02, Table 2).
In comparison to postmenopausal women, perimenopausal women did not have a higher risk of
non-CV mortality (HR: 1.46 (p=0.26, Table 2, Figure 4). This association was not significant
after adjustment for covariates (HR: 1.73 p=0.1, Table 2). There was no interaction between age
and menopausal status.
Associations between serum estradiol levels and mortality
A low serum estradiol was associated with higher all-cause mortality in women over the age of
51 years (HR: 1.58, p=0.001) even after adjustment for covariates (HR: 1.60, p=0.001, Table 3).
No association was found in women under the age of 51 (unadjusted HR: 0.69, p=0.3, adjusted:
HR 0.87, p=0.7, Table 3). In contrast, low serum estradiol was not associated with CV mortality
in women over the age of 51 years (unadjusted HR: 1.41, p=0.2 adjusted HR: 1.45, p=0.2, Table
3) and under the age of 51 years (unadjusted: 0.43, p=0.1 adjusted 0.63, p=0.2, Table 3). In
women over the age of 51, low serum estradiol was associated with a higher risk of non-CV
mortality even after adjustment for co-variates (unadjusted HR: 1.64, p=0.02, adjusted HR: 1.60,
110
p=0.03, Table 3). No association was found in women under the age of 51 (unadjusted HR: 0.88,
p=0.82 adjusted HR: 1.35, p=0.7, Table 3).
Sensitivity analysis
Similar results and trends were found after the exclusion of non-adjudicated causes of death. No
associations were found between estradiol levels and date of serum collection (p=0.2).
Discussion
In this multi-center prospective cohort study of 482 women on hemodialysis we aimed to
examine the associate ion between menopausal status and mortality. In contrast to our hypothesis
we found that in comparison to women classified as postmenopausal, premenopausal and
perimenopausal women had a significantly higher risk of all-cause mortality and cardiovascular
mortality after adjustment for covariates. Additionally, in comparison to postmenopausal
women, premenopausal women had a higher risk of non-cardiovascular mortality after
adjustment for covariates. However, low estradiol levels were associated with higher all-cause
and non-cardiovascular mortality only in women over the age of 51 years after adjustment for
covariates. Our study indicates that a premenopausal status in women with end stage kidney
disease is associated with higher mortality.
To our knowledge, this is the first study examining the associations between menopausal status
and mortality in women on hemodialysis. Our results are in keeping with a previous study that
examined the relationship between estradiol and mortality in 283 women on hemodialysis over
the age of 45300. Over a mean of 3 years of follow-up the high and low tertiles of estradiol were
associated higher all-cause and cardiovascular mortality in comparison to the middle tertile. In
contrast to this study, our study found that low estradiol levels were associated with lower
mortality in older women. These differences could be due to several reasons: first, it could be
111
attributed to different age cut offs in the two populations. In our study 51 years was used as a cut
off for age, based on the average age of menopause in the general North American population; in
comparison, Tarisev and colleagues only included women over the age of the age of 45 years.
Second, women over the age of 51 years in our study had lower mean estradiol levels as
compared to the cohort of women in the study by Tarisev et al. The observed difference in
estradiol levels could explain the varying results. Finally, our study included incident dialysis
patients, and serum estradiol levels were measured within the first 3 months of dialysis initiation.
In comparison, the women included in the study by Tarisev et al. were on dialysis for at least 6
months.
A chief finding in our study is that women classified as premenopausal and perimenopausal had
higher all-cause, cardiovascular and non-cardiovascular mortality in comparison to
postmenopausal women, which is the reverse of the trend observed in the general population306.
Limited knowledge about the hormone milieu in women starting dialysis, and the role of sex
hormones in the mediation of risk in women on dialysis, prevents us from determining the cause
of increased risk in premenopausal and perimenopausal women in this population; however,
there could be several possible reasons for the observed results. Although the estradiol levels
were higher in premenopausal and perimenopausal women compared to the postmenopausal
women, the distribution of estradiol levels in the premenopausal group was found to be narrower
than what is expected in the general population 116, 313, 314. Therefore, one potential reason for the
increase in mortality risk in the premenopausal and perimenopausal groups in the dialysis
population is the early loss of estradiol in premenopausal compared to postmenopausal women.
There is evidence to suggest the early loss of estradiol, through early menopause, in the general
population is associated with higher overall mortality. In a cohort of 12,134 healthy women, later
112
menopause was associated with a 2% decrease in risk per year delay in menopause onset 54.
Additionally, surgical menopause in women under the age of 45 years was associated with a 67%
increase in mortality compared to controls 55. As the premenopausal women in this study were
younger than postmenopausal women during dialysis initiation, we speculate the observed
difference in risk could be due to a longer exposure to higher estradiol levels in the older
postmenopausal women compared to the younger premenopausal women. Other factors that may
have played a role in the increase in risk, not considered in this study, include, a decrease in the
variability of estradiol levels in premenopausal women on dialysis as compared to
premenopausal women in the general population, and type of vascular access in premenopausal
compared to postmenopausal women.
This study has strengths and limitations. First, serum sex hormone levels were used to determine
menopausal status of women on dialysis, through the WISE algorithm for hysterectomized
women, instead of information on menstrual cycle. The reason for doing so was two-fold: first,
studies have shown that self-reported regularity of menstruation is unreliable, subjective and is
variable based on cultural background118-121; second, previous studies have shown that menstrual
disorders and amenorrhea is highly prevalent in the CKD population14, 236, therefore using
menstrual regularity and cycle length as a criteria for menopausal status may not apply to this
particular population. The WISE classification, however, may have limited applicability in the
CKD population as it was derived from a population of healthy postmenopausal women with a
hysterectomy, and validated in healthy postmenopausal women116. As a result, misclassification
due to the WISE classification should be considered. Previous studies have suggested the
observed hypothalamic pituitary gonadal abnormalities in CKD stem from a lack of pulsatile
GnRH secretion, resulting in low LH and FSH levels, amenorrhea and presumed menopause128.
113
In contrast natural menopause is associated with high levels of LH and FSH288. As a result, all
women with low FSH levels were classified as premenopausal in our study introducing a
potential misclassification bias. As an example, 13% of women classified as premenopausal in
our dataset were over the age of 60. Second, a one-time measurement of estradiol was used to
determine menopausal status; however, the WISE classification algorithm was developed base
on a one-time measurement of serum sex hormone levels116 and , single measurements have been
shown to be reliable measures of long term sex hormone levels in previous studies 315, 316. Third,
due to the nature of a cohort study residual confounding may not have been appropriately
addressed; however, we have included most known risk factors for menopause and mortality in
the multivariate models. Finally, this is an observational study and therefore whether the
associations assessed in this study are causal cannot be determined; however, given the paucity
of knowledge around the role of estradiol and menopausal status, cohort studies are important for
hypothesis generation for studies that would determine novel treatment in this population.
Conclusion
Our findings suggest that in women with end stage kidney disease on hemodialysis those
classified as premenopausal have a higher risk of all-cause, cardiovascular and noncardiovascular mortality compared to postmenopausal women, and those classified as
perimenopausal have a higher risk of all-cause and cardiovascular mortality compared to
postmenopausal women. While menopause status may be a novel risk factor for mortality in this
patient group, the mechanism for this modification of risk is not well understood and warrants
further studies.
114
Tables and figures
Table 6-1 Baseline Characteristics
All (n=482)‡
Premenopausal Perimenopausal Postmenopausal
(n=164)
(n=34)
(n=278)
p
35%
7%
58%
50 ± 17 *†
64±14
66±11 <0.0001
26.5±10
26.6±10
27.5±7.4
0.5
136.9±26.1 †
147.1±26.0
145.0±26.5
0.002
77.4±15.9†
71.1±16.1
73.6±13.8
0.01
48
56
56
0.28
85
85
89
0.51
25
24
30
0.48
16
14.7
12.7
0.6
2.6±2.4 †
2.7±2.4
3.2±2.5
0.046
Age (years)
BMI (kg/m2)
SBP
DBP
% Diabetes
%Hypertension
%CAD
%Glomerulonephritis
Follow up (years)
Smoking Status
% Never
% Current
%Past
60±16
27.1±8.6
142.0±26.4
73.6±13.8
53
87
28
14.1
2.9±2.4
49.8
15
35.2
46.7
22.1
31.1
51.7
10.3
37.9
Estradiol (pmol/L)
FSH
Prolactin
116±161
58.0±55.0
68.7±137.2
196±187†
3.8 ± 3.0*†
89.8±148†
230±335†
23.1±19.5†
61±109
‡ Sex hormone levels only available for 476 women
* Significantly different from perimenopausal
† Significantly different from postmenopausal
115
50.3
11.5
38.1
0.1
55±38 <0.0001
94.5±43.7 <0.0001
56±131
0.04
Table 6-2 Unadjusted and adjusted risk of all-cause mortality, cardiovascular mortality
and non-cardiovascular mortality by menopause status
Postmenopausal
n=278
All-cause Mortality
Number of Events
HR (95% CI) - Unadjusted
HR (95% CI) - Adjusted‡
Cardiovascular Mortality
Number of Events
HR (95% CI) - Unadjusted
HR (95% CI) - Adjusted‡
Perimenopausal
n=34
134
1.00 (reference)
1.00 (reference)
23
1.63(1.05-2.54)
1.87(1.17-2.99)
39
1.00 (reference)
1.00 (reference)
Premenopausal
n=164
80
1.27(0.96-1.68)
1.8 (1.32-2.46)
9
2.10(1.02-4.34)
2.72(1.30-5.70)
25
1.32(0.80-2.18)
1.81(1.03-3.17)
Non Cardiovascular Mortality
Number of Events
63
10
35
HR (95% CI) - Unadjusted
1.00 (reference)
1.46(0.75-2.85)
1.14(0.75-1.72)
HR (95% CI) - Adjusted‡
1.00 (reference)
1.73(0.88-3.43)
1.71(1.07-2.73)
‡Adjusted for age, body mass index (BMI), diabetes, hypertension, coronary artery disease, and
prolactin
116
Table 6-3 Unadjusted and adjusted risk of all-cause mortality, cardiovascular mortality and non-cardiovascular mortality by
median estradiol levels
All women
Estradiol ≤ 64
Estradiol >64
pmol/L
pmol/L
n=241
n=235
≤ 51 Years
Estradiol ≤ 64
Estradiol >64
pmol/L
pmol/L
n=32
n=94
> 51 Years
Estradiol ≤ 64
Estradiol >64
pmol/L
pmol/L
n=209
n=141
All Cause Mortality
Number of Events
117
120
13
26
HR (95% CI) – Unadjusted
1.00(Reference)
1.26(0.97-1.62)
1.00(Reference)
0.69(0.35-1.35)
HR (95% CI) - Adjusted‡
1.00(Reference)
1.48(1.13-1.94)
1.00(Reference)
0.87(0.41-1.84)
Cardiovascular Mortality
Number of Events
38
35
7
9
HR (95% CI) – Unadjusted
1.00(Reference)
1.07(0.68-1.69)
1.00(Reference)
0.43(0.16-1.16)
HR (95% CI) - Adjusted‡
1.00(Reference)
1.25(0.77-2.03)
1.00(Reference)
0.63(0.22-1.80)
Non Cardiovascular
Mortality
Number of Events
52
56
4
10
HR (95% CI) – Unadjusted
1.00(Reference)
1.28(0.88-1.88)
1.00(Reference)
0.88(0.27-2.81)
HR (95% CI) - Adjusted‡
1.00(Reference)
1.47(0.99-2.19)
1.00(Reference)
1.35(0.31-5.76)
‡Adjusted for age, body mass index (BMI), diabetes, hypertension, coronary artery disease, and prolactin
117
104
94
1.00(Reference)
1.58(1.2-2.09)
1.00(Reference)
1.60(1.20-2.15)
31
1.00(Reference)
1.00(Reference)
26
1.41(0.84-2.38)
1.45(0.84-2.50)
79
1.00(Reference)
1.00(Reference)
72
1.64(1.09-2.46)
1.60(1.06-2.43)
Figure 6-1 CONSORT Diagram
118
Figure 6-2 Kaplan Meier survival curve all-cause mortality stratified by menopause status
0.50
0.25
0.00
Survival
0.75
1.00
All Cause Mortality
0
2
4
6
Time (Years)
8
Perimenopausal
Postmenopausal
Premenopausal
119
10
Figure 6-3 Kaplan Meier survival curve cardiovascular mortality stratified by menopause
status
0.50
0.25
0.00
Survival
0.75
1.00
CV mortality
0
2
4
6
Time (Years)
Postmenopausal
Premenopausal
120
8
Perimenopausal
10
Figure 6-4 Kaplan Meier survival curve for non-cardiovascular mortality stratified by
menopause status
0.50
0.25
0.00
Survival
0.75
1.00
Non Cardiovascular Mortality
0
2
4
6
Time (Years)
Postmenopausal
Premenopausal
121
8
Perimenopausal
10
Chapter Seven: Characterization of sex hormone levels, menopausal status and menopausespecific quality of life in women with chronic kidney disease
Ramesh S, Holroyd-Leduc JM, Wilton SB, James MT, Seely EW, Ahmed SB. Characterization of
sex hormone levels, menopausal status and menopause-specific quality of life in women with
chronic kidney disease. (under peer-review)
122
Abstract
Introduction: Women with chronic kidney disease (CKD) have an abnormal hypothalamus
pituitary gonadal axis and poor quality of life. Symptoms of uremia including infertility,
menstrual disorders, sleep disorder and mood disorders are similar to symptoms of menopause.
We aimed to determine associations between kidney function, sex hormone levels and quality of
life in women with chronic kidney disease not on dialysis.
Methods: A cohort of female patients between the age of 18 and 55 with non-dialysis dependent
chronic kidney disease were recruited from the Southern Alberta Renal Program from May 2015
to March 2016. Estimated glomerular filtration rate (eGFR) was determined via chart review and
serum sex hormone levels were measured. Additionally, participants were asked to fill the
Menopause Specific Quality of Life- Intervention questionnaire (MENQOL). Furthermore,
participants were asked about their opinions on frequency of discussion of symptoms of low
estradiol in the clinical setting.
Results: Seventy-six women with CKD were recruited (mean age: 39.6 ±.8.6 years). The
majority of the women enrolled had CKD stage 1-3. Sex hormone levels were available for 36 of
the 78 participants. We found no difference in serum sex hormone levels between CKD stages,
and eGFR did not correlate with the somatic, physical, psychological, or sexual domains of the
MENQOL questionnaire. Furthermore, there was no correlation between the MENQOL score
and serum sex hormone levels in women with CKD. Women reported limited discussion of
fertility, menopause symptoms, and menstrual cycle with their nephrologists.
Conclusion: Serum estradiol levels did not differ based on stage of CKD, and menopause
specific quality of life was not associated with eGFR. Furthermore, MENQOL scores did not
correlate with estradiol levels in this population. Despite these findings women with CKD
123
continue to have reproductive and gynaecological abnormalities, and further studies are needed
to better understand the pathophysiology of CKD mediated endocrine changes.
124
Introduction
Patients with chronic kidney disease (CKD) have a poor quality of life2. Symptoms such as
fatigue, lack of stamina, cramps, restless legs, and sleep disturbances are highly prevalent, and
mood disorders such as anxiety and depression are common in the CKD population.10, 12, 13
Women with CKD report poorer health-related quality of life and greater sexual dysfunction in
comparison to men.9, 317 As health-related quality of life has been identified by patients with
CKD as an important research uncertainty318, there is an urgent need for novel investigations into
this area.
Many of the symptoms attributed to uremia are common to those of menopause in women8, 37, 319.
CKD adversely affects the hypothalamic-pituitary-gonadal axis function which commonly
manifests as menstrual abnormalities, impaired fertility, sexual dysfunction, and premature
cessation of menses and menopause in women14, 128, 136, 236, 266, 320.In women with CKD Abnormal
production of GnRH, LH and FSH cause low levels of estradiol and premature menopause 122 123,
124
. In the healthy population, the menopausal transition is associated with a decline in the quality
of life, including sleep disturbances and mood disorders37, 39, 41, 42, 44, 45, 321.
Although previous studies have characterized the menstrual status14, 128, 136, 236, 266, 320, fertility136,
236, 320
and endometrial morphology279 in women with end-stage kidney disease treated with
dialysis or kidney transplant, studies characterizing the sex hormone profile women with endstage kidney disease128, 322, 323 are limited and do not include data on quality of life. As such, our
primary study aim was to determine associations between kidney function, sex hormone levels
and quality of like in women with chronic kidney disease not on dialysis.
125
Methods
Study Objectives
The study objectives were three-fold: we sought to 1) characterize the sex hormone profile and
menopausal status of women across the spectrum non-dialysis-dependent CKD, 2) determine the
menopause specific quality of life and the prevalence of menopausal symptoms in women with
non-dialysis dependent CKD, and 3) determine the views of this population on the importance of
discussing menopausal signs and symptoms with their nephrologist.
Participants
A cohort of female patients between the age of 18 and 55 years with chronic kidney disease in
the Southern Alberta Renal Program, a tertiary referral center, were recruited consecutively for
this exploratory cross-sectional study from May 2015 to March 2016. Patients on dialysis, or
requiring a kidney transplant, and current users of hormone therapy were excluded from the
study.
Data collection
Chart review was used to determine serum creatinine measurements and glomerular filtration
rates for the participants. Study day involved a 15-minute survey during the patient’s clinic visit
followed by collection of blood samples through Calgary Laboratory Services within 6 months
of survey completion.
Ethics approval for the study was obtained from the University of Calgary Conjoint Health
Research Ethics Board. Participation in the study was voluntary and informed consent was
obtained for each participant. Research was conducted according to the Declaration of Helsinki
of medical research in humans311.
126
Measurement of exposure
The exposure of interest for the first sub-study was estimated glomerular filtration rate (eGFR).
eGFR was measured using the Chronic Kidney Disease Epidemiology Collaboration (CKD-Epi)
equation324.
The exposure of interest for the second sub-study was serum estradiol levels. Serum estradiol,
follicle stimulating hormone (FSH), luteinizing hormone (LH), progesterone, testosterone, and
prolactin levels were measured within 6 months of the study day. Sex hormone levels were
measured using chemiluminescence assay by Calgary Laboratory Services (Calgary, AB).
The final sub-study was descriptive in nature.
Definition of Outcomes
The outcomes of interest for the first sub-study were serum estradiol, follicle stimulating
hormone, luteinizing hormone, progesterone, testosterone, and prolactin.
The outcome of interest for the second sub-study was menopause specific quality of life.
Participants were administered The Menopause Specific Quality of Life- Intervention
Questionnaire (MENQOL-Intervention)168 on the study day. The MENQOL-intervention was
used to assess menopause related quality of life in this population. This questionnaire contains 32
questions divided into 4 different domains: vasomotor, physical, psychosocial and sexual
appendix 1. Briefly, the general format of each item is a binary question regarding whether the
subject has experienced a particular symptom in the past month. If the answer was yes, the
participant was asked to rate how bothersome the symptom is on a 7 point Likert scale. The
mean of the scores from each of the 4 domains is calculated and reported. As a validation
measure, the EuroQol (EQ) 5D quality of life survey was also administered to determine general
127
quality of life in women with CKD325. This survey has been validated in the CKD population 326,
327
.
The outcomes of interest in the third sub-study were patients’ perceptions on the importance of
discussions surrounding fertility, menstrual cycle irregularities and menopause with
nephrologists, and opinions on use of postmenopausal hormone therapy. Demographic
information was also collected as part of the survey. A copy of the survey can be found in
appendix 3. The questions assessed for frequency (never, rarely, sometimes, often or always) or
agreement (strongly disagree, disagree, neither agree nor disagree, agree, strongly agree or I do
not know).
Baseline data such as age, sex, estimated glomerular filtration rate (eGFR), proteinuria, current
medications, comorbidities (specifically hypertension, diabetes) were collected from a medical
chart review.
Statistical Analysis
Baseline characteristics are presented as means ± standard deviation or proportions unless
otherwise stated. The primary analyses included the assessment of the linear associations
between eGFR and serum sex hormone levels using linear regression models. Differences in sex
hormone profile and MENQOL-intervention score across the 5 stages of CKD were determined
using a one-way analysis of variance test (ANOVA) and post-hoc Tukey’s test to account for
multiple comparisons. Associations between sex hormone levels and MENQOL-intervention
score were tested using univariate and multivariate linear regressions. The MENQOLintervention score was log transformed to normalize the distribution. A stepwise linear
regression model was used to account for potential confounding by age, BMI, and diabetes. All
analyses were performed using Stata (version 12, StataCorp LP, College Station, TX).
128
Results
Baseline characteristics
Table 1 summarizes the baseline characteristics of the participants. Seventy-six women with
chronic kidney disease were enrolled in the study. The mean age of the participants was 39.6
±.8.6 years (Median: 40 years, IQR: 12 years). Women with CKD stage 1 were significantly
younger than women with CKD stage 3 (p=0.002), but all women were below the age of 55 and
they were predominantly Caucasian (67%). The mean eGFR for the women included in the study
was 69±35 mL/min per 1.73 m2 (Median: 69 IQR: 59) and 24 hour estimated proteinuria 6±8
g/day. The majority of the women enrolled in the study had stages 1-3 CKD. Of the 40
participants for whom details on etiology of CKD was available 22 (55%) had
glomerulonephritis, and five of these subjects underwent cyclophosphamide treatment.
All other comparisons between the stages of CKD were non-significant after post hoc analyses of
ANOVA.
Sex hormone levels
Blood samples were available for 36 of the 76 women, serum estradiol levels were available for
all 36 participants, serum FSH levels and prolactin levels were available for only 35 participants,
and serum progesterone, testosterone, and LH levels were available for 23 of the 36 participants.
Serum sex hormone levels are summarized in table 2, stratified by stage of CKD. The mean
serum estradiol level was 298±289 pmol/L, FSH level was 15.6 ± 29.4 IU/L, and prolactin level
was 21.7 ± 18.6 ug/L. Serum estradiol, FSH and prolactin levels did not differ based on CKD
stages (Table 2). The mean serum LH level was 12.8 ± 13.9.
129
Quality of life
The quality of life determined by the EQ5D was varied, with 73% having no problems walking
about, 93% having no problems with self-care, 77% with no problems performing usual
activities, 58% having no pain or discomfort, and 63% having no anxiety and/or depression. The
average score for a good health state was found to be 73±19 on a scale from 0 to 100, with 0
being the worst imaginable health state and 100 being the best imaginable health state.
The mean MENQOL score for the somatic domain was 2.3±1.7 (median: 1.7 IQR: 2), physical
domain was 3.5±1.6 (median: 3.25 IQR:2.5), psychological domain was 2.9±1.6 (median: 2.6
IQR:2.2) and sexual domain was 2.3±1.6 (median: 1.7 IQR: 1.8).
There were no significant associations between glomerular filtration rate and quality of life for
the total MENQOL score (univariate: p=0.64 multivariate p=0.40), somatic MENQOL score
(univariate: p=0.39, multivariate: p=0.98), physical MENQOL score (univariate: p=0.37,
multivariate: p=0.65) psychological MENQOL score (univariate p=0.93, multivariate=0.66) and
sexual score (univariate p=0.84, multivariate: p=0.12) (Figure 1)
Similarly, when stratified according to CKD stages, there were no differences between the log
transformed values of the total MENQOL score (p=0.96), somatic MENQOL score (p=0.82),
physical MENQOL score (p=0.82), psychological MENQOL score (p=0.83) and sexual
MENQOL score (p=0.69) (Figure 2)
There were no significant correlations between serum estradiol levels and total MENQOL score
(univariate: p=0.10 multivariate: p=0.72), somatic MENQOL score (univariate: p=0.38
multivariate: p=0.17), physical MENQOL score (univariate: p=0.13 multivariate: p=0.94),
psychological MENQOL score (univariate p=0.06, multivariate: p=0.68), and sexual score
(univariate: p=0.31, multivariate: p=0.3) Figure 3.
130
Patient survey
All 76 participants completed the patient survey. The results are presented in table 3. The
majority of the participants (64%) said they never discussed fertility with their nephrologist, and
53% said they never discussed their menstrual cycle with their nephrologist. Furthermore, 80%
of the participants reported to have never discussed menopause with their nephrologist. Twentythree percent of the participants strongly agreed or agreed with the statement that their
nephrologist often brings up issues about fertility in a clinical setting. A similar proportion of the
participants (24%) strongly agreed or agreed with the statement that their nephrologist often
brings up issues about their menstrual cycle in a clinical setting. In comparison only 12% of the
participants agreed with the statement that their nephrologist often brings up issues about
menopause in a clinical setting. Eighteen percent of the participants strongly agreed or agreed
that they would like to discuss birth control with their nephrologist, and 21% strongly agreed or
agreed that they would like to discuss postmenopausal hormone therapy with their nephrologist.
Thirty-nine percent of the participants strongly agreed or agreed that they would prefer to talk
about fertility and menopause with their family physician as opposed to their nephrologist.
Discussion
In this cross-sectional study that aimed to examine the associations between kidney function and
estradiol levels, and menopause specific quality of life in women under the age of 55 years, we
found that 1) there are no associations between eGFR and serum estradiol levels and no
associations between eGFR and menopause specific quality of life 2) there are no associations
between serum estradiol levels and menopause specific quality of life in patients with CKD and
3) discussion of fertility, menstrual irregularities and menopause are limited in the clinical
131
setting, and patients reported a preference towards discussing health issues concerning fertility
and menopause with their family physician as opposed to their nephrologist.
To the best of our knowledge, this is the first study to characterize the sex hormone profile in
women with chronic kidney disease, and to examine the association between chronic kidney
disease severity and menopause specific quality of life and symptoms.
We found a wide range of serum estradiol levels in women with non-dialysis dependent CKD.
This range is similar to the range reported in the healthy population (7.3pmol/L to 1071
pmol/L)116. Our results are in keeping with findings of previous studies in the dialysis
population128, 322. In a study of 24 patients on hemodialysis, Lim et al. found that serum sex
hormone levels were within the normal range of the general population; however, estradiol levels
did not reach the mid cycle peaks normally seen in healthy women128. Similarly, a study of 75
women under the age of 45 on hemodialysis found that only 33% of the women had low estrogen
levels. Despite our finding of normal sex hormone profile in the CKD population, it is important
to note that women with chronic kidney disease continue to have gynecological and reproductive
problems16-18, 236. A previous cross-sectional study examining gynecological issues in 76 women
on dialysis showed that 59% of women reported irregular menses236. This is further reflected in
low fertility rates among women with CKD of child-bearing age 16-18. Additionally, menstrual
abnormalities, poor fertility, and sexual dysfunction are common in uremic women with CKD1418
. A study of 100 women with CKD reported that 88% had menstrual problems or were
menopausal, with 20% of menopausal women being under the age of 40 years 14. Therefore,
further studies examining the specific differences between the sex hormone milieu in the healthy
versus CKD populations are warranted.
132
In addition to reproductive abnormalities, studies have also found that in patients with kidney
disease physical symptoms such as fatigue, lack of stamina, cramps, restless legs, and sleep
disturbances are common8. A study of 1,284 patients with CKD (40% women) found a higher
prevalence of fatigue and cramps with worsening kidney disease9, 10; additionally, women with
kidney disease were more likely to have worse quality of life associated with physical symptoms
of uremia compared to men9. In addition to fatigue, women on dialysis have also been found to
have a high prevalence of sleep disorders including insomnia, sleep apnea, and restless leg
syndrome11. Similarly, mood disorders such as anxiety and depression are prevalent in the
chronic kidney disease population10, 12, 13.
Symptoms associated with low estradiol levels in postmenopausal women are similar to those
observed in uremic patients8, 37, 319. Studies have found that the prevalence of sleep disturbance
ranges from 32 to 46 percent in postmenopausal women compared to 15 percent in the general
population 37 39. Additionally, postmenopausal women are 3.4 times more likely to report sleep
disorders compared to premenopausal women 40. Multiple studies have also reported a
significant increase in depression during menopausal transition compared to premenopausal
years, indicating an important role of estradiol in mood-related disorders 41-45. A recent study
reported a 2 to 4 fold higher risk of depression in perimenopausal and early postmenopausal
women 36.
Despite the similarities between menopause specific symptoms and symptoms of uremia, we
found no association between menopause specific quality of life and kidney function. This could
be due to several reasons. First, given the disease burden of CKD and CKD-related poor quality
of life, women with CKD may not consider some of the symptoms associated with low estradiol
as a major contributor to their quality of life9, 328. Second, although the MENQOL-intervention
133
questionnaire is a comprehensive and extensively validated tool to determine menopause specific
quality of life168, 329, it was developed for cohort of healthy postmenopausal women. This
questionnaire may not, therefore, be addressing issues unique to the CKD population as a result
of low estradiol levels. General quality of life as determined by EQ5D was in keeping with a
previous study in the CKD population326.
Finally, the survey of patients on their opinions on discussion of fertility and menopause with
their nephrologist reflected a trend towards patients preferring to discuss these issues with their
primary care provider as opposed to their nephrologist. One possible reason for this observation
is that the patient feels there may be inadequate time to address issues related to sex hormone
status with their nephrologist given the burden of disease of CKD and the comorbidities
associated with CKD255, 256.
This study has strengths and limitations. First, this is a cross-sectional study examining the
associations between menopause specific quality of life and kidney function and estradiol levels
in the CKD population, therefore no causal relationship can be established. However, this is the
largest study to date examining these associations in this population. There is a knowledge gap in
the understanding of the contribution of estradiol in CKD and this study will shed some light on
the complexity of this association. Second, since this study used a menopause specific quality of
life questionnaire developed in the healthy population, it may not be applicable in the CKD
population. However, the MENQOL-intervention questionnaire has been validated in several
different populations including patients with diabetes (78, 79). Third, this study is limited by its
sample size, however, this was aimed to be a descriptive, hypothesis generating study that would
lead to further studies with the aim of understanding the hormonal milieu and the role of
estradiol in the CKD population. Lastly, previous studies have suggested that social desirability
134
bias results in the tendency of survey respondents to answer questions in a manner that will be
viewed favourably by others262, in order to reduce the effect of social desirability bias, all
interviews were conducted in a private room and all participants were told their data would be
kept confidential and anonymous.
Conclusion
In summary, in this exploratory cross-sectional study we found that there is no association
between renal function and menopause specific quality of life in women with CKD under the age
of 55 years. Additionally, estradiol levels did not differ based on stage of CKD and there was no
correlation between estradiol levels and menopause specific quality of life in this population. The
study highlights the need to better understand the specific role of estradiol in this high risk
population.
135
Table 7-1 Baseline Characteristics
Age (years)
GFR (mL/min
per 1.73 m2)
Albumin (ug/L)
%Caucasian
% Diabetes
% Hypertension
%
Glomerulonephri
tis
% Never
Smokers
Mean Weight
(kg)
All
(n=76)
39.6 ±
8.6
68.7 ±
34.8
34.9 ±
4.4
67
20
66
CKD
Stage 1
(n=24)
36.0 ±
7.9*
109.2 ±
10.1
36.1 ±
3.7
50
13
58
CKD
Stage 2
(n=19)
38.5 ±
7.2
76.5 ±
8.5
34.3 ±
5.7
31
7
46
CKD
Stage 3
(n=20)
45.1 ±
8.2*
46.4 ±
8.4
34.7 ±
4.8
30
23
58
CKD
Stage 4
(n=7)
38.7 ±
8.5
22.7 ±
3.4
34.3 ±
1.7
14
43
100
CKD
Stage 5
(n=6)
40.5 ±
6.0
10.0 ±
2.0
32.3 ±
1.9
0
33
100
53
71
73
38
29
17
63
67
67.9 ±
14.2
24.8 ±
5.2
P
ANO
VA
0.006
<0.000
1
0.9
0.12
0.31
0.04‡
0.02‡
85
67
40
20
0.26
77.6 ±
78.2 ±
74.9 ±
95.2 ±
77 ± 20
19.9
20
11.1
33.2
0.11
28.9 ±
27.2 ±
29.7 ±
26.0 ±
48.5 ±
9.3
10.1
8.9
3.7
1.3
0.01
BMI (kg/m2)
127.5 ±
121.8 ± 122.4 ± 135 ±
139.5 ±
17.7
130 ± 25 14.4
7.7
17.8
18.2
0.13
SBP (mm Hg)
80.2 ±
81.3 ±
77.9 ±
77.6 ±
82.7 ±
86.5 ±
11.1
12.6
13.5
7.4
7.8
10.5
0.45
DBP (mm Hg)
* Significantly Different from Stage 1 CKD
‡ Comparison no longer significant after post hoc
Tukey's test
Abbreviation: GFR- Glomerular Filtration Rate, BMI - Body Mass Index, SBP - Systolic
Blood Pressure DBP- Diastolic Blood Pressure
136
Table 7-2 Serum sex hormone levels across CKD specturm
Estradiol (pmol/L)
FSH (IU/L)
All
n=36
298.2 ±
288.7
15.6 ±
29.4
12.8 ±
13.9
LH (IU/L)
Progesterone(nmol/L)
11 ± 17.3
*
0.61 ±
Testosterone
0.38
(nmol/L)*
21.7 ±
18.6
Prolactin (ug/L)*
* n=24
CKD
Stage 1
n=12
310 ±
316
6.25 ±
9.13
6.0 ± 4.4
17.6 ±
21.8
0.64 ±
0.31
17.8 ±
9.3
CKD
Stage 2
n=9
140 ±
203
15.3 ±
30.9
14.8 ±
16.8
2.1 ±
2.35
0.60 ±
0.5
24.3 ±
32.9
CKD
Stage 3
n=7
433 ±
356
35.4 ±
49.6
21.8 ±
18.3
15.2 ±
21.9
0.48 ±
0.35
23.7 ±
10.8
CKD
Stage 4
n=3
564 ±
157
CKD
Stage 5
n=5
206 ±
131
15.4 ±
4 22.7
7.7 ±
17 10.7
0.09 6.8 ± 6.3
0.7 ±
0.9 0.56
23.4 ±
23 ± 9.9
16.8
Abbreviations: FSH- follicular stimulating hormone LH- Luteinizing Hormone
137
p for
trend
0.7
0.4
0.4
0.4
0.9
0.6
Table 7-3 Impressions of patients on discussion of symptoms associated with low sex hormone with nephrologist
Never
How often do you discuss fertility with your
nephrologist?
How
do you discuss menstrual cycle with your
nephrologist?
How
often do you discuss menopause with your
nephrologist?
My nephrologist often brings up issues about fertility
My nephrologist often brings up issues about menstrual
cyclenephrologist often asks about menopausal
My
Isymptoms
would like to discuss birth control with my
Inephrologist
would like to discuss postmenopausal hormone
therapy with my nephrologist
I have concerns about taking hormone replacement
Itherapy
prefer to talk about fertility and postmenopausal
hormone therapy with my family physician
Sometime
s
22.70%
28%
9.30%
Often
Strongly
Disagree
Disagree
32%
34.70%
43.20%
18.40%
16%
7.90%
9.20%
64%
53.30%
80%
138
Alway
s
2.70%
2.70%
1.30%
Not
Applicable
5.30%
8%
8%
Neither
agree nor
disagree
Agree
Strongly
agree
Not
Applicable
9.30%
14.70%
18.50%
19.70%
18.70%
6.70%
9.30%
6.80%
5.30%
12%
4%
1.30%
0.00%
6.60%
2.70%
29.30%
17.30%
24.30%
38.20%
32%
13.20%
9.20%
22.40%
31.60%
18.70
%
22.70
%
12.20
%
11.80
%
18.70
%
13.20
%
14.50
%
22.40%
25%
21.10%
10.50%
5.30%
8%
1.30%
Figure 7-1 MENQOL score stratified by CKD Stage
139
Figure 7-2 Correlations between GFR and MENQOL scores
140
Figure 7-3 Correlations between estradiol and MENQOL scores
141
Chapter Eight: Conclusions
8.1 Summary of Findings
In healthy men and women, there was no correlation between serum sex hormone levels and
baseline cardiac autonomic tone; however, men with lower testosterone levels were unable to
maintain cardiovagal and cardiosympathetic tone in response AngII. Postmenopausal women
demonstrated a lower baseline cardiosympathetic and cardiovagal tone compared to
premenopausal women. There were no differences in cardiosympathetic and cardiovagal tone in
premenopausal women in the follicular versus luteal phase of the menstrual cycle. In response to
AngII, postmenopausal women were unable to maintain cardiac autonomic tone and responded
with a decrease in cardiovagal tone, and premenopausal women in the luteal phase failed to
maintain cardiac autonomic tone
While studying low estradiol status in the context of CKD, we found that although nephrologists
and allied healthcare professionals recognize the impact of kidney disease on sex hormone status
in women with CKD, reported discussion of the sequelae of low estradiol levels such as
infertility, menstrual disorders or premature menopause with their patients was limited. To
determine the role of hormone therapy in the CKD population, we performed a systematic
review of studies examining the use of postmenopausal hormone therapy. The systematic review
included over1600 women with CKD, though the majority of the populations studied were
women with ESKD on dialysis. Our main findings were that in women with CKD: 1) no studies
have examined the effect of postmenopausal hormone therapy on all-cause mortality and CV
mortality and morbidity, 2) hormone therapy was associated with a decrease in levels of LDL
cholesterol, increases in levels of HDL cholesterol and total cholesterol, and was not associated
with triglyceride levels, and 3) though reporting of adverse events was limited to only three of
142
the six studies, complications related to postmenopausal hormone treatment were not common.
In a multi-center, prospective cohort study of women with ESKD on hemodialysis, we found
that, based on the Women’s Ischemia Syndrome Evaluation (WISE) classification, in
comparison to postmenopausal women, premenopausal and perimenopausal women had a
significantly higher risk of all-cause mortality and cardiovascular mortality after adjustment for
covariates. Additionally, in comparison to postmenopausal women, premenopausal women had a
higher risk of non-cardiovascular mortality after adjustment for covariates. These results,
however, should be interpreted with caution and misclassification due to the WISE classification
should be considered. Previous studies have suggested the observed hypothalamic pituitary
gonadal abnormalities in CKD stem from a lack of pulsatile GnRH secretion, resulting in low LH
and FSH levels, and amenorrhea and presumed menopause128. In contrast natural menopause is
associated with high levels of LH and FSH288. As a result, all women with low FSH levels were
classified as premenopausal in our study introducing a potential misclassification bias. As an
example, 13% of women classified as premenopausal being over the age of 60. However, high
estradiol levels were associated with higher all-cause and non-cardiovascular mortality only in
women over the age of 51 years after adjustment for covariates. Finally, in a cross-sectional
study that aimed to examine the associations between kidney function and estradiol levels and
menopause specific quality of life in women with CKD under the age of 55 years, we found that
1) there were no associations between glomerular filtration rate (GFR) and serum estradiol levels
and no associations between glomerular filtration rate and menopause specific quality of life 2)
there are no associations between serum estradiol levels and menopause specific quality of life in
patients with CKD and 3) discussion of fertility, menstrual irregularities and menopause are
143
limited in the clinical setting, and patients reported a preference towards discussing health issues
concerning fertility and menopause with their family physician as opposed to their nephrologist.
8.2 Implications
These results indicate a complex association between estradiol and risk of cardiovascular disease
and mortality, and quality of life in this population. To our knowledge, this is the largest program
of research to characterize the sex hormone profile in the CKD population, and the first study to
examine menopause specific quality of life in this population.
In our studies mimicking the high AngII state observed in the CKD population, menopausal
status was associated with cardiac autonomic changes, and therefore may indicate a potential role
for sex hormones in the mediation of cardiovascular, in particular sudden cardiac death, risk in
the CKD population.
This thesis examines several relevant outcomes of abnormal sex hormone profile in women with
CKD. We first examined the impressions of nephrologists on the role of sex hormone status on
kidney disease, through which we identified a gap in care with respect to discussion of infertility,
menstrual irregularities, and menopause with female patients who have CKD. Further studies are
therefore required to appropriately highlight the burden of low sex hormones and menopausal
symptoms in this population, and identify barriers to care for women with CKD. Through this
study we found that majority of nephrologists reported not knowing whether hormone therapy
was a potential treatment option for women with CKD, and being unaware of the risk and
benefits of hormone therapy in the CKD population. This led to a systematic review with the aim
of determining the effect of postmenopausal hormone therapy on cardiovascular outcomes in
women with CKD. There was an overall paucity of studies examining the association and effect
144
of hormone therapy in women with CKD, with no studies reporting all-cause mortality,
cardiovascular mortality, and cardiovascular disease. This study highlights the need for clinical
trials with the aim of determining the potential role, risks, and benefits of hormone therapy and
other treatments for the abnormal hypothalamus pituitary gonadal axis in women with CKD.
In order to assess the potential associations between menopausal status and mortality in women
with ESKD, we used the CKDCS database of women initiating hemodialysis with a mean follow
up of 3 years. Similar to previous studies our study found that the average estradiol levels in
women of reproductive age with ESKD was within the normal range; however, the estradiol
levels, while within the normal range, were lower than what is observed in the general
population128, 322. Contrary to our hypothesis, however, a premenopausal state was associated
with a higher risk of mortality in the dialysis population, suggesting that the sex hormone milieu
in the CKD population may modulate risk differently from the general population. Possible
reasons for this paradox may include the early exposure to lower than normal levels of estradiol
in premenopausal women compared to the postmenopausal women 116, 313, 314, lack of cyclicity of
sex hormone secretion in women with ESKD128, different etiology of cardiovascular and noncardiovascular disease in the general versus ESKD population23, and type of vascular access330.
Classification of menopausal status was challenging in this population. The commonly used
Stages of Reproductive Aging Workshop (STRAW) guidelines, derived from a group of nonobese, non-smoking women without chronic menstrual irregularities, and with a uterus, use
menstrual cycle regularity as the only principal criteria to determine menopausal status14, 15 but
information regarding menstrual status was not available in the Canadian Kidney Disease Cohort
Study (CKDCS) population331. Additionally, previous studies have outlined that irregular menses
are common among women with CKD 14, 136 and thus we believed categorization of menstrual
145
status based on sex hormone levels to be more applicable in this population. The WISE
classification, however, may have limited applicability in the CKD population as it was derived
from a population of healthy postmenopausal women with a hysterectomy, and validated in
healthy postmenopausal women116. Studies suggest that menopause in women with CKD is
characterized by a decrease in estradiol and progesterone levels, a decrease in LH and FSH, and
is reversible with kidney transplant and intensive dialysis136. This is in stark contrast to natural
irreversible menopause where serum FSH and LH levels are high288. The findings from this
study highlight the importance of further studies aiming to characterize the hypothalamic
pituitary gonadal axis in women with CKD, and determination of a method of classifying
hypothalamic pituitary abnormalities and “true” menopause in this population.
Finally, contrary to previously published literature, we did not observe an association between
kidney function and serum estradiol levels129, 136, 332; furthermore, unlike previous published
work14, 15, 133 most women included in this study would not be classified as postmenopausal
according to the STRAW guidelines14, 15. Additionally, estradiol levels in women with CKD fell
within the normal range observed in healthy women. Perhaps then not surprisingly, we found
that the menopause specific quality of life (MenQoL) survey was not valid in the young female
CKD population as there was no correlation between estradiol levels and quality of life. The
menopause specific quality of life questionnaire was developed in healthy menopausal women,
therefore, may not apply to the young female CKD population given that the mean estradiol level
in our study population was within the normal range observed in the non-CKD population.
Overall, these exploratory studies have set the stage for further research in determining the
differences in the role of sex hormones in healthy women versus women with CKD.
146
8.3 Recommendations for future research
This program of research has led to several possible future studies. First, a study to determine
whether women with CKD have similar cyclical production of LH, FSH, estradiol, and
progesterone as healthy women is required in order to better understand etiology of the menstrual
and reproductive abnormalities observed in the women with CKD. Additionally, a study
examining the difference in the hypothalamus pituitary gonadal axis across the stages of CKD,
different modality of dialysis, and before and after transplantation is required to elucidate the
factors that play a role in the hypothalamic pituitary gonadal abnormalities in women with CKD.
Second, a study of the differences in the association between hypothalamic pituitary gonadal axis
function and menstrual cycle and fertility in healthy women compared to women with CKD will
help better understand the pathology of the previously observed menstrual irregularities and
infertility in women with ESKD14, 15. Third, a study to determine if women with CKD who have
low estradiol levels have similar symptoms of low estradiol as postmenopausal women in the
general population, and whether the determinants of quality of life in women who are
postmenopausal due to CKD are similar to those of women who have had a natural menopause.
This study would identify symptoms associated with low estradiol in the CKD population and
whether these symptoms are associated with poor quality of life in women with CKD thereby
guiding treatment options in the future. Fourth, a study to determine an appropriate menopausal
classification system for women with CKD which would help identify if hormone therapy would
be beneficial to women with CKD of a particular menopausal status. Finally, a randomized
controlled trial examining the effect of hormone therapy on heart rate variability, all-cause
mortality and cause specific mortality, fracture risk, thrombolytic events, quality of life and
147
cancer in women with CKD would help determine if hormone therapy is beneficial in this
population.
In conclusion, this body of work aimed to examine the associations between serum estradiol
levels and menopause status in women with CKD. Through this project we have shown that the
association between estradiol in women CKD is complex and not fully understood. These
exploratory studies have, therefore, provided for further research in this field.
148
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170
Appendices
Appendix 1: Menopause Specific Quality of Life Questionnaire
171
172
Appendix 2: Nephrologist survey
I understand the above stated purpose of this survey and consent to the use of my
responses for the purpose of academic research:
Yes
No
Demographic Information
1. Age
30-40 Years
41-50 Years
51-60 Years
61-70 Years
>70 Years
2. Sex
Male
Female
3. Years of Practice
5-10 Years
10-15 Years
15-20 Years
20-25 Years
> 25 Years
4. How are you involved in nephrology practice?
Nephrologist
Medical trainee in nephrology
Nurse practitioner
173
Nurse
Other (please specify) ______________________
5. Type of Practice (check all that apply)
Academic
Community
Clinical
Education
Administrative
Research
Other (Please Specify) ______________________
6. Are you a transplant nephrologist?
Yes
No
7. What type of patients are in your practice?
Pediatric
Adult
Both
Please answer the following based on your personal clinical practice
1. How often do you discuss fertility with your female patients of childbearing age?
Never
Rarely
Sometimes
Often
Always
Comments (optional)
2. How often do you discuss menstrual irregularities with your female patients of
childbearing age?
174
Never
Rarely
Sometimes
Often
Always
Comments (optional)
3. How often do you ask patients to address concerns associated with fertility and
menstrual irregularities with their family doctor or refer them to another physician
(endocrinologists or gynecologists)?
Never
Rarely
Sometimes
Often
Always
Comments (optional)
To what extent do you agree or disagree with the following statements:
4. Kidney function has an important impact on sex hormone levels, menstrual status, and
fertility.
Strongly Agree
Agree
Neutral
Disagree
Strongly Disagree
I Do Not Know
Comments (optional)
175
5. My female patients of childbearing age often wish to discuss fertility.
Strongly Agree
Agree
Neutral
Disagree
Strongly Disagree
Comments (optional)
6. My female patients under the age of 55 often wish to discuss menstrual status and
symptoms or treatment of menopause.
Strongly Agree
Agree
Neutral
Disagree
Strongly Disagree
Comments (optional)
7. There is a role for postmenopausal hormone replacement therapy (HRT) in patients with
CKD.
Strongly Agree
Agree
Neutral
Disagree
Strongly Disagree
I Do Not Know
Comments (optional)
176
8. The benefits of HRT far outweigh the risks in patients with CKD.
Strongly Agree
Agree
Neutral
Disagree
Strongly Disagree
I Do Not Know
Comments (optional)
9. In women with a uterus, the formulation of the hormone therapy and whether it used in
conjunction with progesterone is important.
Strongly Agree
Agree
Neutral
Disagree
Strongly Disagree
I Do Not Know
Comments (optional)
10. The route of administration of HRT (oral, transdermal, transvaginal) is important.
Strongly Agree
Agree
Neutral
Disagree
Strongly Disagree
177
I Do Not Know
Comments (optional)
11. I often prescribed HRT PRIOR to the Women's Health Initiative (WHI) trials (JAMA
2002).
Strongly Agree
Agree
Neutral
Disagree
Strongly Disagree
I Do Not Know
I was not in nephrology practice prior to 2002
Comments (optional)
12. I often prescribe HRT AFTER the Women's Health Initiative (WHI) trials (JAMA
2002).
Strongly Agree
Agree
Neutral
Disagree
Strongly Disagree
I Do Not Know
Comments (optional)
13. Stage of CKD is important when considering HRT use.
Strongly Agree
Agree
178
Neutral
Disagree
Strongly Disagree
I Do Not Know
Comments (optional)
14. The benefits of HRT outweigh the risks in the following stages of CKD (check all that
apply).
Stage 1 CKD
Stage 2 CKD
Stage 3a CKD
Stage 3b CKD
Stage 4 CKD
Stage 5 CKD
Stage 5 D CKD
I do not know
HRT should never be prescribed to CKD patients
Comments (optional)
15. HRT use should be considered in perimenopausal women with CKD.
Strongly Agree
Agree
Neutral
Disagree
Strongly Disagree
I Do Not Know
Comments (optional)
179
16. HRT use should be considered in postmenopausal women with CKD.
Strongly Agree
Agree
Neutral
Disagree
Strongly Disagree
I Do Not Know
Comments (optional)
180
Appendix 3: Patient Survey
Patient Questionnaire
Demographic information
Age
Gender of Nephrologist
How long have you been a
Nephrology patient?
□ <30 yrs □ 30-40 yrs □ 41-50 yrs □ 51-60 yrs □ 61-70 yrs □ >70 yrs
□ Male □Female
□<5 yrs □5-10 yrs □10-15 yrs □15-20 yrs □20-25 yrs □>25 yrs
Never
Sometimes
Strongly
Disagree
Disagree
Often
Always
Not
Applicable
Agree
Strongly
agree
How often do you discuss
fertility with your nephrologist?
How do you discuss menstrual
cycle with your nephrologist?
How often do you discuss
menopause with your
nephrologist?
Neither
agree nor
disagree
My nephrologist often brings up
issues about fertility
My nephrologist often brings up
issues about menstrual cycle
My nephrologist often asks
about menopausal symptoms
I would like to discuss birth
control with my nephrologist
I would like to discuss
postmenopausal hormone
therapy with my nephrologist
I have concerns about taking
hormone replacement therapy
I prefer to talk about fertility
and postmenopausal hormone
therapy with my family
physician
Are you postmenopausal? □ Yes □ No
If yes what was the cause of menopause? □ Surgical (hysterectomy etc.) □ Non-surgical (aging)
181
Not
Applicable
Appendix 4: Permissions to submit thesis prior to journal submission
182
183
184
185
186
187
188