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. 74 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. 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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