The Role of Oral Contraceptives in Optic Neuritis: the Story Behind the Study, Initial Experiences, and Lessons Learned

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2013-07-15 The role of oral contraceptives in optic neuritis: the story behind the study, initial experiences, and lessons learned

Trufyn, Jessie J.

Trufyn, J. J. (2013). The role of oral contraceptives in optic neuritis: the story behind the study, initial experiences, and lessons learned (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/28340 http://hdl.handle.net/11023/817 master thesis

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The role of oral contraceptives in optic neuritis: the story behind the study, initial

experiences, and lessons learned

Jessie J. Trufyn

A THESIS

SUBMITTED TO THE FACULTY OF GRADUATE STUDIES

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

DEGREE OF MASTER OF SCIENCE

DEPARTMENT OF NEUROSCIENCE

CALGARY, ALBERTA

JULY, 2013

There is accumulating evidence of sex differences in multiple sclerosis, making hormones a possible research avenue for therapeutic agents. Oral contraceptives are a source of synthetic hormones, however, it is unclear whether hormone-based therapies help, hinder, or have no effect on the disease in women. In an attempt to elucidate the role of sex hormones, we are currently conducting an observational study of oral contraceptives in optic neuritis, a condition that often occurs in parallel with multiple sclerosis. The thesis describes the study rational and supporting evidence for the hypothesis that oral contraceptive use in our study population will be associated with beneficial outcomes. I also share experiences with study implementation and preliminary data. The final section of the thesis offers insight for researchers on the areas of optical coherence tomography, hormones, and human research.

ii Preface

My journey began in Fall 2008 when I started my position at the Calgary Multiple

Sclerosis Clinic as a health research coordinator under the supervision of Ms. Winona Wall and Dr. Luanne Metz. My experiences there, and interaction with Dr. Fiona Costello and

Dr. Jodie Burton led me on the pursuit of my Masters degree and this thesis.

iii Acknowledgements

There are many people I owe thanks to. First, my boyfriend. I met Michael Keough at the beginning of my graduate school adventure. His hard work has been an inspiration, his strength a pillar, and his friendship a source of happiness.

My parents for their unconditional support, positive thinking, and for making every trip home a mini vacation.

The staff at the MS Clinic and Eye Clinic for their smiling faces and smorgasbord of treats.

Dr. Michael Hill for recommending and lending me the book Epidemiology in Medicine.

My supervisory committee, Dr. Gordon Fick, Dr. Bernard Corenblum, and especially Dr. V

Wee Yong, for shaping my project and success. Also, Dr. Bill Fletcher for being my external examiner.

Above all, Dr. Fiona Costello and Dr. Jodie Burton, my supervisors. Dr. Costello, with her gumption and fortitude, has kept me moving forward, and instilled in me life lessons and good energy that I hope to always carry. Dr. Burton, with her commitment, brilliance, and instrumental feedback, has made this project and my experience something I am proud of.

Merged together, their support has enabled me to grow and and achieve. Jodie and Fiona, I will forever feel privileged to have worked with you.

iv Dedication

This thesis is dedicated to study patients, who selflessly contribute to science during stressful times of their lives.

v Table of Contents

Abstract ...... ii Preface ...... iii Acknowledgements ...... iv Dedication ...... v Table of Contents ...... vi List of Tables ...... x List of Figures and Illustrations ...... xi List of Symbols, Abbreviations and Nomenclature ...... xii

CHAPTER ONE: INTRODUCTION ...... 1 Chapter overview ...... 1 Background ...... 1 Multiple sclerosis ...... 1 Clinical course ...... 2 Etiology and pathogenesis ...... 3 Treatment ...... 4 Overview of sex differences ...... 5 Sex differences in multiple sclerosis ...... 7 Epidemiology and clinical course ...... 7 Pathological features ...... 8 Response to therapy ...... 9 Neuroendocrine axis ...... 10 Optic neuritis recovery ...... 12 Chapter summary and thesis objectives ...... 12

CHAPTER TWO: LITERATURE REVIEW ...... 14 Chapter overview ...... 14 Part I: Hormonal fluctuation studies in multiple sclerosis ...... 14 Pregnancy ...... 14 Clinical trials mimicking pregnancy ...... 15 Menstrual cycle ...... 16 Oral contraception ...... 18 Clinical trials of oral contraceptives ...... 21 Part I: Summary ...... 21 Part II: Impact and mechanisms of ethinylestradiol and progestin in female animal models of neuroinflammation and degeneration ...... 22 Ethinylestradiol ...... 22 Progestin ...... 25 Part II: Summary ...... 26 Part III: Another approach to study oral contraceptives in multiple sclerosis ...... 27 A model of multiple sclerosis ...... 27 Optic neuritis ...... 28 Evaluating optic neuritis ...... 29 High and low contrast acuity ...... 29 vi Optical coherence tomography ...... 30 Visual evoked potentials ...... 32 Magnetic resonance imaging ...... 33 Evidence that sex steroids may influence optic neuritis characteristics ...... 34 Sex differences in retinal nerve fiber layer thickness after optic neuritis ...... 34 Exogenous estradiol protects retinal ganglion cells in models of optic atrophy34 States of hormonal fluctuation influence retinal measures ...... 35 Part III: Summary ...... 36 Chapter summary ...... 36

CHAPTER THREE: THE ROLE OF REPRODUCTIVE HORMONES AND ORAL CONTRACEPTIVES IN OPTIC NEURITIS STUDY- DESIGN AND OPERATION ...... 46 Chapter overview ...... 46 Study development ...... 46 The Role of Vitamin D Status on Optic Neuritis Recovery study as a prototype .... 46 Helping define the population ...... 47 How long must participants be oral contraceptive users or non-users? ...... 48 Should all participants have their blood tested for hormones? ...... 48 When should blood testing occur? ...... 49 Should recruitment be limited to a specific demyelinating disease subtype? ... 49 Logistics of the outcome variables ...... 50 Communication between multiple sites ...... 50 Access to results ...... 51 Database development ...... 52 Tailoring the questionnaire ...... 52 Funding ...... 53 Process of submitting to the Conjoint Health Research Ethics Board ...... 53 Application overview ...... 53 Informed consent forms ...... 54 Approval and modifications ...... 54 Study implementation ...... 55 Recruitment ...... 55 Ongoing involvement ...... 55 Chapter summary ...... 56

CHAPTER FOUR: PRELIMINARY DATA ...... 57 Chapter overview ...... 57 Sources of data ...... 57 The Role of Reproductive Hormones and Oral Contraceptives in Optic Neuritis study ...... 57 The Role of Vitamin D Status on Optic Neuritis Recovery study ...... 57 Preliminary data ...... 58 Baseline characteristics ...... 58 Neurological outcomes ...... 58 Recovery of visual acuity after optic neuritis ...... 58 Optical coherence tomography outcomes ...... 59

vii Visual evoked potential outcomes ...... 59 Discussion ...... 59 Discussion topic 1: normality of data and outliers ...... 60 Discussion topic 2: potential confounders ...... 60 Age as a confounding variable ...... 61 Disease subtype as a confounding variable ...... 62 Vitamin D status as a confounding variable ...... 63 Discussion topic 3: addressing the screening process for optic neuritis mimics ...... 64 Discussion topic 4: study limitations ...... 64 Oral contraceptive behaviour bias ...... 64 Variability in oral contraceptive regimes ...... 65 Variability in serum hormone levels ...... 65 Sample size calculation ...... 66 Clinical significance of retinal nerve fiber layer thickening ...... 67 Retinal nerve fiber layer is not completely axonal in nature ...... 68 Chapter summary ...... 68

CHAPTER FIVE: LESSONS LEARNED ...... 77 Chapter overview ...... 77 Lessons learned from optical coherence tomography research ...... 77 Lesson 1: optical coherence tomography compliments current techniques ...... 77 Benefits ...... 78 Pitfalls ...... 79 Lesson 2: retinal nerve fiber layer interpretation is complicated by various factors 79 Optic neuritis history ...... 80 Multiple sclerosis history ...... 81 Demographics ...... 82 Comorbidities ...... 83 Lesson 3: optical coherence tomography technology and interpretation continues to evolve ...... 83 Lessons learned from reproductive hormone research ...... 85 Lesson 4: reproductive hormone research is topical and data must be communicated with caution ...... 85 Lesson 5: hormone type, dose, and route of administration matter ...... 86 Hormone type ...... 86 Dosage ...... 87 Route of administration ...... 87 Lessons learned from human research ...... 88 Lesson 6: study design ...... 88 Observational studies ...... 88 Recruitment and retention strategies ...... 89 Lesson 7: ethical framework ...... 90 Discussion of thesis objectives ...... 91 Conclusions ...... 93 Objective 1: the story behind the study ...... 93 Objective 2: initial experiences ...... 94 Objective 3: lessons learned ...... 95 viii Future directions ...... 96

REFERENCES ...... 101

APPENDIX A: EXPANDED DISABILITY STATUS SCALE SOURCE DOCUMENT ...... 149

APPENDIX B: LIST OF COMBINED ORAL CONTRACEPTIVES ...... 160

APPENDIX C: MENSTRUAL DIARY ...... 163

APPENDIX D: CASE REPORT FORMS ...... 164

APPENDIX E: PATIENT QUESTIONNAIRE ...... 168

APPENDIX F: THE ROLE OF REPRODUCTIVE HORMONES AND ORAL CONTRACEPTIVES IN OPTIC NEURITIS (ROC-ON) STUDY PROTOCOL . 174 Study overview ...... 174 Inclusion and exclusion criteria ...... 174 Inclusion criteria ...... 174 Exclusion criteria ...... 175 Study procedures ...... 175 Optical coherence tomography ...... 175 Visual evoked potentials ...... 176 Visual acuity ...... 176 Expanded Disability Status Scale ...... 176 Magnetic resonance imaging ...... 177 Serum hormone testing ...... 177 Menstrual/parity history ...... 178 Schedule of study activities and evaluations ...... 178 Outcome variables ...... 178 Primary outcomes ...... 178 Secondary/exploratory outcomes ...... 179 Planned analyses ...... 179 Primary analysis ...... 179 Secondary/exploratory analyses ...... 180 Ethical considerations ...... 180 Ethical conduct of the study ...... 180 Patient information and consent ...... 180 Patient confidentiality ...... 181

APPENDIX G: HORMONE REFERENCE INTERVALS ...... 182

ix List of Tables

Table 1 Categorical variables at baseline ...... 70

Table 2 Continuous variables at baseline ...... 70

Table 3 Normality of optical coherence tomography and visual evoked potential outcome variables at baseline and month six ...... 71

x List of Figures and Illustrations

Figure 1 Changes in maternal serum concentrations of sex hormones during pregnancy . 38

Figure 2 Changes in serum concentrations of sex hormones during the natural and oral contraceptive-treated menstrual cycle ...... 39

Figure 3 Relationship between the serum concentrations of 1,25-dihydroxyvitamin D3 and vitamin D binding protein in oral contraceptive users and non-users ...... 40

Figure 4 High-resolution images of the internal retinal structure taken with optical coherence tomography, demonstrating the processes involved in using this technology ...... 41

Figure 5 The human optic nerve ...... 42

Figure 6 Optical coherence tomography measured peripapillary retinal nerve fiber layer and macular volume reports ...... 44

Figure 7 Visual evoked potential setup and recordings ...... 45

Figure 8 Scattor plot showing the relationship between retinal nerve fiber layer thickness and age ...... 72

Figure 9 Retinal nerve fiber layer thickness in clinically isolated optic neuritis and multiple sclerosis patients ...... 73

Figure 10 Retinal nerve fiber layer thickness in patients with sufficient and low vitamin D status ...... 74

Figure 11 Scattor plot showing the relationship between retinal nerve fiber layer thickness and the delay in testing from optic neuritis onset ...... 75

Figure 12 Total retinal nerve fiber layer has a neuronal and non-neuronal component ..... 76

Figure 13 Time domain versus spectral domain optic coherence tomography ...... 99

Figure 14 Mean serum ethinylestradiol concentrations for subjects treated with contraceptive agents through various routes of administration ...... 100

xi List of Symbols, Abbreviations and Nomenclature

Symbol Definition

CHREB Conjoint Health Research Ethics Board CI confidence interval CIS clinical isolated syndrome EAE experimental allergic encephalomyelitis EDSS Expanded Disability Status Scale ETDRS Early Treatment for Diabetic Retinopathy Study GPER/ GPR30 G-protein coupled estrogen receptor DMT disease modifying therapy IQR interquartile range IVF in vitro fertilization MRI magnetic resonance imaging MS multiple sclerosis NMO neuromyelitis optica OC oral contraceptive OCT optical coherence tomography PLP proteolipid protein ROC-ON Role of Reproductive Hormones and Oral Contraception in Optic Neuritis RNFL retinal nerve fiber layer SD spectral domain TCPS-2 Tri-Council Policy Statement: Ethical Conduct for Research Involving Humans TD time domain Th1 T helper 1 Th2 T helper 2 VEP visual evoked potential VitaDON Role of Vitamin D Status on Optic Neuritis Recovery

xii

CHAPTER ONE: INTRODUCTION

Chapter overview

Canada has one of the highest prevalence rates of multiple sclerosis (MS) worldwide (Koch-Henriksen and Sørensen, 2010), with particularly high prevalence in

Alberta (Beck et al., 2005). Every day, new patients are assessed at the Calgary MS Clinic because of a high suspicion of demyelinating disease. As a demyelinating disease of the central nervous system, MS affects signal transmission in brain and spinal cord, and results in a broad spectrum of disability. Despite decades of MS research, the exact etiology remains unknown, the prognosis remains uncertain and variable, and treatment options are limited in their impact on long-term disability (Noseworthy et al., 2000). One prominent observation, however, is the presence of sex differences in MS. My first thesis chapter introduces the clinical and pathological features of MS, highlights relevant physiological differences between men and women, and emphasizes sex differences detected in the disease. The chapter also describes common nomenclature used throughout my thesis. To conclude, I outline the three objectives for my thesis.

Background

Multiple sclerosis

Multiple sclerosis is an inflammatory and degenerative disease of the central nervous system. Manifestations of MS commence primarily in young adulthood and include vision loss, paralysis, sensory loss, cognitive impairment, depression, pain, sphincter incontinence, and sexual dysfunction (Noseworthy et al., 2000). The individual

variation of disease susceptibility and disease course indicates that there is likely an interaction between environmental factors in a genetically susceptible host. Indeed, smoking, Epstein Barr virus infection, and vitamin D deficiency have been identified as risk factors for developing MS (Disanto et al., 2012). The role of the non-heritable factors is further supported by the only 30% concordance rate of MS in monozygotic twins

(Sadovnick et al., 1993). Epigenetic changes, or changes in gene regulation but not the genetic code itself, may help explain the complex connection between partial heritability and the influence of environmental factors (Koch et al., 2013). Overall, the participation of modifiable factors encourages the development of prevention strategies or innovative therapies to interfere with disease progression.

Clinical course

While the disease course in MS is unpredictable, it is traditionally categorized as relapsing-remitting, secondary progressive, primary progressive, or progressive-relapsing

(Lublin and Reingold, 1996). Relapsing-remitting MS is the most common form, and consists of episodes of neurological impairment (relapses) followed by partial or complete recovery (remission) and a stable course between attacks. A large percentage of relapsing- remitting patients convert to a secondary progressive disease course at a mean age of 54 years, after which time their condition declines independent of relapses (Koch et al., 2010).

Approximately 15 to 20% of MS patients experience a gradual worsening from onset and are defined as primary progressive, or the more rare progressive-relapsing if there are accompanying relapses and minor remissions (Confavreux et al., 1980; Cottrell et al.,

1999). The Expanded Disability Status Scale (EDSS) is a standard measure of neurological

impairment in MS patients, ranging from zero (no signs) to 10 (death due to MS) (Kurtzke,

1983) (Appendix A). Patients frequently undergo EDSS testing by their neurologists in an effort to monitor MS disease course and progression.

Etiology and pathogenesis

Inflammation and degeneration are the main pathogenic processes in MS, but the sequence and contribution of each is controversial. The traditional view holds that MS is an autoimmune disease targeting components of the myelin sheath. An inflammatory attack by the immune system causes demyelination and the subsequent axonal injury and pathology

(Dutta and Trapp, 2011). These active sites of inflammation involve a break in the blood brain barrier and are visible as gadolinium-enhancing lesions on magnetic resonance imaging (MRI) after intravenous administration of a contrast agent (Grossman et al., 1986).

Serial MRI studies suggest gadolinium-enhancing lesions are transient (Miller et al., 1988;

Bastianello et al., 1990) and may even precede symptoms and other MRI pathology

(Kermode et al., 1990), consistent with the traditional view. Other MRI pathology includes

T2 (hyperintensities) and T1 (hypointensities)-weighted lesions. The number and volume of T2-weighted lesions are used to quantify total disease burden, however, only mildly correlate with clinical disability (Filippi et al., 1995). This clinical-radiological paradox can be partly explained by the heterogeneity of the T2-weighted lesions (Truyen et al.,

1996). Edema, gliosis, demyelination, remyelination, and axonal injury are not easily distinguishable changes on T2-weighted images (Noseworthy et al., 2000; Martinelli

Boneschi et al., 2004). T1-weighted lesions are more characterized in that they are associated with severe tissue destruction (extensive demyelination and axonal loss) and are

often referred to as “black holes" (Truyen et al., 1996; van Walderveen et al., 1998).

Nonetheless, the range of hypointensities (i.e. voxel intensity) prevents any clear correlation between clinical measures and T1-weighted lesion volume as well (Tam et al.,

2011).

There has been increasing focus on the neuronal and axonal loss independent of the inflammatory response (Barnett and Prineas, 2004; DeLuca et al., 2006; Kooi et al., 2009), which supports an alternate pathogenic mechanism. In this philosophy, neurodegeneration is primarily responsible and inflammation is one of the consequences which further perpetuates disease (Stys et al., 2012). There are several reasons to speculate why MS may be the result of oligodendrocyte degeneration or a myelinopathy, including that early myelin abnormalities have been detected at the inner side of intact myelin sheaths, away from sites of inflammation (Rodriguez and Scheithauer, 1994). Moreover, while disease- modifying therapies (DMTs) are effective at reducing relapses and haematopoietic stem cell transplantation is capable of halting neuroinflammation, both fail to stop disease progression (Molyneux et al., 2000; Inglese et al., 2004; Metz et al., 2007).

Treatment

There are currently six DMTs for MS available in Canada—interferon beta-1a

(Rebif and Avonex), interferon beta-1b (Betaseron or Extavia), glatiramer acetate

(Copaxone), natalizumab (Tysabri), and fingolimod (Gilenya). They are known as immune modulators. Interferons are a family of proteins that exist naturally to regulate the immune response. When given as a therapy, interferon boosts the anti-inflammatory response by

decreasing antigen presentation and T helper 1 (Th1; cellular immunity) cell production, and increasing interleukin 10 levels (Castro-Borrero et al., 2012). The mechanism of action for glatiramer acetate is less understood, but as a polymer found in myelin basic protein, this therapy is centered on the idea of creating a decoy for the immune attack (Kala et al.,

2011), which expands T helper 2 (Th2; humoral immunity) polarized cells (Yong, 2002).

Natalizumab is a selective adhesion molecule (α4 integrin) inhibitor that prevents inflammatory cells from crossing the blood brain barrier (Chataway and Miller, 2013).

Finally, fingolimod acts by sequestering lymphocytes in lymph nodes by down-regulating chemoattractant receptors for sphingosine-1-phosphate (Mandala et al., 2002) and it is the first approved DMT taken orally rather than by injection.

Overview of sex differences

The presence or absence of the Y chromosome initiates a cascade of events that ultimately produces a male or female phenotype. Two fundamental hormones in this process are testosterone and estradiol. Exposure to dissimilar concentrations of these sex hormones during fetal development also leads to more inconspicuous sex differences found in the nervous system (McCarthy et al., 2012). On average, healthy men have larger brains than women even after controlling for body weight (Sacher et al., 2012). Most studies agree that the grey to white matter ratio is greater in women, but disagree whether this is due to the less white matter, more gray matter, or both (Gur et al., 1999; Allen et al., 2003;

Cosgrove et al., 2007). Volume differences have been noted in specific brain regions and may be related to the distribution of sex hormone receptors (Kawata, 1995; Goldstein et al.,

2001; Sacher et al., 2012). For example, the sexually dimorphic nucleus of the preoptic

area (in the hypothalamus) is larger in males than in females and the neurons highly express estrogen receptors (Kawata, 1995). Administration of an estrogen receptor antagonist, or elimination of testosterone or aromatase (an enzyme that converts testosterone to estradiol), during development prevents sexual differentiation of this region

(Kawata, 1995). Several small scale studies also suggest women have increased neurotransmission and cerebral blood flow compared to men (Gur et al., 1982; Cosgrove et al., 2007).

Sex differences are likewise prominent in the immune system and these discrepancies are evident in a variety of clinical settings. Men are more susceptible to bacterial infections after trauma and surgery (Schröder et al., 1998; Offner et al., 1999;

Wichmann et al., 2000). Women have a greater susceptibility to autoimmune diseases, including rheumatoid arthritis (Th1 mediated) and systemic lupus erythematosus (Th2 mediated) (Whitacre et al., 1999). A large evaluation of graft and patient survival after renal transplant revealed male donor tissue was superior irrespective of whether the recipient was male or female (Zeier et al., 2002). This may be reflective of the increased antigenicity in female organs. Overall, women have a more robust immune response, which is at least in part due to the aforementioned sex hormones (Whitacre et al., 1999).

Sex differences typically encompass one of the following definitions: 1) sexual dimorphisms, or characteristics present in one sex but not the other (e.g. courtship behaviour); 2) dissimilar averages between groups of males and females (e.g. olfactory skills); and 3) sexual convergence or divergence, which are instances where the sexes reach

the same endpoint through different means or have opposing reactions to the same starting point (e.g. care-giving behaviour) (McCarthy et al., 2012). In this chapter, I will almost exclusively refer to the second definition whereby differences in averages are noted with varying degree of overlap.

Sex differences in multiple sclerosis

Epidemiology and clinical course

During the second half of the twentieth century, support gradually grew for the view that there was a female preponderance in MS (Kurland, 1952; Schumacher, 1960;

Acheson, 1977; Confavreux et al., 1980; Noseworthy et al., 2000). In Canada, the sex ratio has reached three to one for women (Orton et al., 2006). Theorized reasons include: a decline in smoking among men (Palacios et al., 2011), stronger proinflammatory effects of vitamin D deficiency in women, possibly associated with sun avoidance (Kragt et al.,

2009), and earlier diagnosis of milder forms in women (Marrie et al., 2005). Also initially debated, the majority of studies now suggest women appear to have a better MS prognosis than men (Detels et al., 1982; Weinshenker et al., 1991; Confavreux et al., 2003;

Confavreux and Vukusic, 2006; Glad et al., 2009; Koch et al., 2010; Koutsis et al., 2010;

Gholipour et al., 2011; Damasceno et al., 2013). This idea is based on the observations that male patients are less likely to remain benign (Glad et al., 2009), reach disability landmarks more rapidly (Detels et al., 1982; Koutsis et al., 2010; Damasceno et al., 2013), and convert to the secondary progressive subtype after a shorter time and at younger age (Confavreux and Vukusic, 2006; Koch et al., 2010). There is a higher proportion of male patients diagnosed as primary progressive (Runmarker and Andersen, 1993), but recent studies

suggest the initial course, relapsing or progressive, does not substantially affect the age at disability landmarks (Confavreux and Vukusic, 2006; Scalfari et al., 2011).

Pathological features

The majority of MRI investigations comparing men and women with MS have found similar lesion burden when clinical disease factors are matched (Fazekas et al.,

2009). Some studies have reported a greater number of gadolinium-enhancing lesions in women, which may speak to their more inflammatory MS course (Weatherby et al., 2000;

Pozzilli et al., 2003; Tomassini et al., 2006). Advanced MRI techniques have drawn our attention to the importance of grey matter atrophy in MS (Fisher et al., 2008; Geurts and

Barkhof, 2008). Grey matter atrophy, particularly cortical damage, is felt to be the substrate of cognitive decline and fixed disability (Calabrese et al., 2012; Geurts et al., 2012;

Calabrese et al., 2013). No sex effects in grey matter changes were detected in cohorts of clinically isolated syndromes (CIS, a single inflammatory or demyelinating episode of the central nervous system) and early relapsing-remitting patients (Chard et al., 2002;

Bergsland et al., 2012), however studies of patients with longer disease duration found central and subcortical regional grey matter atrophy more pronounced in men compared to women (Antulov et al., 2009; Schoonheim et al., 2012).

Histopathological experiments of the affected brain areas have left many unanswered questions with regard to sex differences in MS. Kuhlmann et al. compared brain biopsies from male and female MS patients for markers of inflammation, axonal damage, and remyelination (a sign of recovery). In limited tissue samples, no sex

differences were noted in the number of microglia or T cells in MS lesions, the axonal density of active and inactive lesions, or the number of oligodendroglial progenitors or oligodendrocytes (Kuhlmann et al., 2009). Studies focused on the functioning of immune cells have found mixed results (Pelfrey et al., 2002; Greer et al., 2004; Moldovan et al.,

2008). Given the wide range of peptides used to compare cytokine responses, this inconsistency is not surprising. For instance, Moldovan et al. investigated cytokine responses of peripheral blood mononuclear cells to various antigens. The authors found interferon gamma was highly elevated in MS women compared to MS men, control women, and control men when presented with proteolipid protein (PLP) 40–60, but not with other relevant myelin peptides, including PLP 103–120 and PLP 139–158 (Moldovan et al., 2008). Because cytokines are unlikely to work in isolation, it is also difficult to extrapolate from these studies. Diverging Th cell responses and cytokine environments to develop gender specific drugs remains a challenging area of interest for some (Contasta et al., 2012).

Response to therapy

Certain studies suggest gender affects treatment response, however, the sex differences already mentioned make this inherently challenging to discern. A favourable response in either sex may be attributed to differences in progression rates. In a clinical trial of glatiramer acetate in primary progressive MS, treatment appeared to delay deterioration in men but not women. A closer look revealed the favourable response was likely not a gender related treatment effect but rather a reflection of the higher progression rates in men compared to women (Wolinsky et al., 2009). A similar explanation was given

in a fingolimod versus placebo trial where men displayed a higher relative reduction in annualized relapse rates compared to women (Devonshire et al., 2012). Alternatively, in randomized trials of interferon beta, women with secondary progressive MS demonstrated more of a robust response according to MRI outcomes when each sex was compared to placebo (Li et al., 2001) and women with relapsing-remitting MS had a reduced probability of disease progression as measured by a decrease in EDSS (Tomassini et al., 2006). For both of these trials, the favourable response in women may be attributed to the more favourable disease course. Pooled data from five clinical studies of interferon beta in relapsing-remitting patients failed to find significant sex differences in various outcome measures, including relapse rate and adverse events (Rudick et al., 2011).

Neuroendocrine axis

There is evidence suggesting serum levels of sex hormones may be altered in MS patients. Testosterone concentrations have been reported to be reduced in MS women

(Tomassini et al., 2005) and men (Safarinejad, 2008) compared to controls. Furthermore, the relatively older age of MS onset in men compared to women coincides with a decline in testosterone levels, suggesting testosterone, at least in young men, may have a protective effect (Gold and Voskuhl, 2009). One year treatment of testosterone in a pilot trial of 10 male MS patients, whereby testosterone levels increased by 50%, failed to reduce gadolinium-enhancing lesions but was associated with reduced brain atrophy and an improvement in cognitive performance (Sicotte et al., 2007). Testosterone treatment decreased the percentage of CD4+ T cells and reduced delayed-type hypersensitivity skin

response, suggesting testosterone also had anti-inflammatory effects in the male MS patients (Gold et al., 2008).

Mean prolactin levels are often similar between MS patients and controls, although a comprehensive review found hyperprolactinemia to be more common in MS men and women (Zhornitsky et al., 2012). A trial of bromocriptine to suppress serum prolactin failed to slow disease progression in 14 out of 15 MS patients, despite being well tolerated

(Bissay et al., 1994). On the other hand, hyperprolactinemia may be a response and repair mechanism rather than a cause of disease activity. While there has yet to be a trial of prolactin treatment in MS, prolactin infusions in animal models of MS reduced the volume of the demyelinating lesions by half compared to untreated mice (Gregg et al., 2007).

The three naturally occurring estrogens are: estrone, estradiol, and estriol. Estrone and estriol are associated with menopause and pregnancy, respectively, while estradiol is the predominant estrogen during reproductive years. Lower estradiol levels have also been reported in female MS patients compared a healthy population (Wei and Lightman, 1997).

In two recent studies, 56 to 60% of women with MS had abnormal sex hormone profiles, which were supposedly related to disease activity (Zakrzewska-Pniewska et al., 2011;

Trenova et al., 2013). There have been no trials attempting to increase serum estradiol levels in MS, likely because the risk of breast and uterine cancer is too high. Estriol treatment and administering synthetic estrogen derivatives, as well as exploiting natural hormonal changes, have been ways to overcome this.

Optic neuritis recovery

One specific example in which sex differences in MS have been noted is in optic neuritis. Optic neuritis is an inflammatory injury to the optic nerve that frequently occurs in

MS. This type of injury often causes a sudden, painful onset of decreased vision. Visual acuity is often restored but some deficits may remain, including contrast sensitivity (Trobe et al., 1996) and the speed at which visual information is transmitted to the brain (Halliday et al., 1972). Non-MS patients can also experience optic neuritis but face an increased risk for developing MS in the future (Optic Neuritis Study Group, 2008). A recent study by

Costello et al. found that women appear to sustain less damage from optic neuritis than men, at least after six months (Costello et al., 2012). Damage was measured by the loss of the retinal nerve fiber layer (RNFL), which shares axons with the optic nerve. This finding was interesting to us; it raised exciting questions about the influence of gender in MS and the potential of sex hormones therapies.

Chapter summary and thesis objectives

The female preponderance in MS, differences in disease course between men and women, and the endocrine-immune partnership has led to the belief that hormones influence MS progression. Exogenous hormones have become a research avenue for symptom management (Goldsmith, 1988) and disease modification (Gold and Voskuhl,

2009), as clinical observations and scientific experiments have accumulated. A large contribution to the literature came from understanding the effects of pregnancy in MS.

More can be learned about the therapeutic potential of sex hormones by focusing on the

disease during predictable patterns of hormonal variations experienced by women.

Therefore, the objectives of this thesis are to:

1. Review the literature on the effects of hormonal variations in women with MS.

Explore the rational for sex hormones as anti-inflammatory and/or neuroprotective therapies in women. Analyze the current approaches to studying hormones in MS.

2. Assist in designing an observational research study that offers new insight to the potential of sex hormone therapy in demyelinating conditions. Through implementing and managing this observational study, as well as analyzing preliminary data, address any foreseeable issues.

3. Share persistent themes and lessons learned.

CHAPTER TWO: LITERATURE REVIEW

Chapter overview

This chapter covers my literature review, organized into three sections. Part I highlights the effect of hormonal fluctuations in women with MS by reviewing studies of pregnancy, menstruation, and oral contraception. The effect of oral contraceptives (OCs) in

MS was variable and interesting, consequently directing Part II of my review towards experiments of OCs in animal models of neuroinflammation and degeneration. Part III describes optic neuritis and how it is evaluated, and demonstrates that by using optic neuritis to study axonal degeneration, we can further elucidate the role of oral contraception in MS.

Part I: Hormonal fluctuation studies in multiple sclerosis

Pregnancy

Pregnancy in MS has always been an area of interest as disease onset peaks during the second and third decade of life, a time when many women are in their childbearing years (Noseworthy et al., 2000). However, opinions have drastically evolved from recommendations of “therapeutic abortion” (Beck, 1913) to declaring no overall effect

(Tillman, 1950). Largely a result of the Pregnancy in Multiple Sclerosis study, pregnancy is now associated with fewer MS related symptoms and relapses coupled with an increased risk of MS and MS related exacerbations in the first six months post-partum (Confavreux et al., 1998; Alonso et al., 2005). There is speculation that pregnancy may even have long- term benefits including delaying wheelchair dependency (Verdru et al., 1994), reaching an

EDSS of 6.0 (the need for a cane) (D'hooghe et al., 2010), and the change from a relapsing- remitting course to a chronic progressive course (Runmarker and Andersen, 1995).

Clinical trials mimicking pregnancy

As a physiological mechanism to prevent the mother from eliciting an immune response against the fetus, pregnancy induces a general state of immunotolerance. From week five to week 40 of gestation, estradiol, progesterone, and prolactin levels rise considerably (Figure 1) (O'Leary et al., 1991). It is believed that high levels of these hormones shift the immune response from a pro-inflammatory to anti-inflammatory state

(Wegmann et al., 1993; Whitacre et al., 1999), and consequently provide temporary protection from MS activity. One hormone specific to pregnancy is estriol, as it is produced in large quantities by the placenta (Hay and Lorscheider, 1976). Studies have attempted to replicate the protective effects of pregnancy by administering oral estriol. Specifically, these trials treat with oral estriol 8.0 mg/day to reach levels comparable to women during their sixth month of pregnancy (Sicotte et al., 2002).

In the pilot trial of oral estriol, there were six women with relapsing-remitting MS and four with secondary progressive MS (Sicotte et al., 2002). The crossover design involved a six-month pre-treatment period, six-month treatment period, and six-month post-treatment period in which MRI and other clinical measures (e.g. number of relapses and EDSS) were collected. Only the relapsing-remitting patients experienced a decrease in the total volume and number of gadolinium-enhancing lesions during the treatment period

(Sicotte et al., 2002). The effect disappeared in the post-treatment period and was visible

for a second time when estriol treatment was reinstituted (Sicotte et al., 2002). The number of relapses and EDSS scores did not change for either MS subtype, however, an improvement in cognitive testing was evident in the relapsing-remitting patients only

(Sicotte et al., 2002). Overall, the treatment was safe and well tolerated. A phase II trial, with oral estriol as an add-on therapy to glatiramer acetate has been initiated in 150 women with relapsing-remitting MS to better show efficacy and results are pending

(http://clinicaltrials.gov/ct2/show/NCT00451204).

A phase III trial examined the ability of high-dose progestin (10 mg/day) and low- dose estradiol (0.075 mg/week) treatment to prevent post-partum relapses. Recruitment was stopped early due to the lack of pregnant patients unwilling to forgo breastfeeding, as lactation was an exclusion criterion (http://clinicaltrials.gov/ct2/show/NCT00127075).

When presenting results at the 28th Congress of the European Committee for Treatment and Research in Multiple Sclerosis (Lyon, France), Vukusic acknowledged the insufficient statistical power but attributed the lack of treatment effects to inadequate sex steroid doses.

It is equally probably that the therapeutic potential of progesterone may not be generalized to progestin, the synthetic form. This is discussed further in Part II (Impact of ethinylestradiol and progestin in female animal models of neuroinflammation and degeneration).

Menstrual cycle

The arrival of menarche follows a yearlong increase in estradiol (Legro et al.,

2000). Studies of menarche history and MS have failed to find a protective role of

estradiol. Earlier age at menarche has been linked to an increased MS risk (Ramagopalan et al., 2009), earlier age of first MS symptoms (Sloka et al., 2006), and accelerated MS progression (D'hooghe et al., 2012). However, increased estradiol is merely one characteristic of female puberty. Other features include a rise in gonadotropins, testosterone and insulin-like growth factor-1 levels (Legro et al., 2000; Christoforidis et al.,

2005).

Hormones fluctuate to a much lesser extent during the menstrual cycle compared to puberty. Nonetheless, studies have found 42 to 82% of women experience MS worsening during the premenstrual period, when the estradiol and progesterone concentrations are falling (Smith and Studd, 1992; Zorgdrager and De Keyser, 1997; 2002). The hormonal variation within a normal female cohort and daily fluctuations make these investigations inherently difficult. Two such MRI studies have been attempted and obtained conflicting results (Bansil et al., 1999; Pozzilli et al., 1999). Bansil et al. divided 30 MS women into groups based on hormone levels—low estradiol, high estradiol with low progesterone, and high progesterone. The high estradiol with low progesterone group had more active MRI lesions than low estradiol group, however, this group also had a higher proportion of relapsing-progressive patients (Bansil et al., 1999). Pozzilli et al. imaged eight women with relapsing-remitting MS in different stages of consecutive menstrual cycles and reported that a high progesterone to estradiol ratio correlated to a larger number and size of enhancing lesions (Pozzilli et al., 1999). A third study found no differences in lesions between the follicular and luteal phases, but stated that serum testosterone levels were significantly lower in women with MS compared to controls (Tomassini et al., 2005).

Surprisingly, there is a shortage of imagining studies examining lesions based on hormonal contraception status.

Menopause provides another opportunity to explore the role of estrogens in MS.

Through questionnaires, studies have found that 39 to 54% of postmenopausal patients experienced MS worsening during menopause (Smith and Studd, 1992; Holmqvist et al.,

2006). Studies of menopause in MS patients are also inherently challenging because there are symptoms common to both MS and menopause, such as fatigue. Moreover, many patients will have entered the progressive phase in which disease worsening is more apparent than in the early stages. In these small study populations, seven to 75% of women reported that their MS improved with hormonal replacement therapy and less than eight percent reported worsening (Smith and Studd, 1992; Holmqvist et al., 2006). It is difficult to speculate if hormonal replacement therapy may lessen MS exacerbations because these studies were small, retrospective, and lacked of specification regarding the start or duration of hormonal therapy.

Oral contraception

Oral contraceptives are typically a combination of a progesterone derivative

(progestin) and an estrogen derivative (ethinylestradiol). The Food and Drug

Administration approved the first combined OC, Enovid, in June 1960. The original formula was 9.85 mg of norethynodrel and 0.15 mg ethinylestradiol 3-methyl ether (Tyler,

1961). Over the decades, OCs have undergone dramatic changes to improve safety and tolerability, including modified and reduced progestin components, reduced

ethinylestradiol dosages, and new multiphasic dosages and various cycle regimes

(Burkman et al., 2011). Today, the ethinylestradiol dosage typically ranges from 0.02 to

0.05 mg, a 67 to 87% dose reduction from Enovid, and most OC users ingest a third- generation progestin (desogestrel, gestodene, or norgestimate) (Hall and Trussell, 2012).

Combined OC brands and compositions are listed in Appendix B.

The increasing MS prevalence and incidence among women but not men seems to predate the approval and increasing popularity of OCs, however the overlap unquestionably required investigations. There have been four large epidemiological studies investigating

MS risk and OC use (Villard-Mackintosh and Vessey, 1993; Thorogood and Hannaford,

1998; Hernán et al., 2000; Alonso et al., 2005). The most recent study, conducted from

1993 to 2000, found a beneficial relationship. The General Practice Research Database collected prospective data on over three million Britons. Over 100 MS cases were recorded with at least three years of preceding electronic medical records and up to 10 controls were matched per patient. In this report by Alonso et al., recent OC users had a 40% lower MS risk than non-users (Alonso et al., 2005). Compared to the earlier epidemiology studies, which did not find any association between OC use and MS risk, this study focused on more recent OC use prior to MS diagnosis. Together, these four studies suggest OC use does not increase MS risk and a beneficial effect remains possible.

Since the report by Alonso et al., research has focused on the possibility that hormonal contraception may delay MS onset or influence severity. Holmqvist et al. reported the average age of MS onset was 25 years for women with less than one year of

OC use versus 20 years for “never-users” (Holmqvist et al., 2010). Recently, Sena et al. studied 132 women with relapsing-remitting MS before disease modifying therapy initiation. Compared to women who never used OCs or discontinued before MS onset, OC users had more of a benign disease course (Sena et al., 2012). D’hooghe et al. collected questionnaires from almost 1000 female MS patients, 70% of which were self characterized as relapsing-remitting onset. Oral contraceptive use in the progressive onset group was associated with an increased risk of reaching EDSS of 6.0 (D'hooghe et al.,

2012). It is possible that hormonal contraception may have opposing roles in the different

MS subtypes, or rather the interaction of hormonal contraception and MS pathology may be heterogeneous. Overall, the influence of OCs on MS severity is not well understood.

Although hormones are absorbed into the bloodstream with OC use, it is a common misconception that OC users have an increase in mean serum concentrations of sex steroids. Exogenous estrogen and progesterone cause suppression of their natural forms

(Figure 2) (Johansson, 1975; Jung-Hoffmann et al., 1988; Aden et al., 1998). The average serum concentration of ethinylestradiol during OC treatment is approximately 30 to 44 pg/mL (Kuhnz et al., 1992; van den Heuvel et al., 2005), which fails to negate the 70% estradiol reduction visible by the sixth day of treatment (Aden et al., 1998). Similarly, progestin exposure during OC treatment does not compensate for the progesterone peak that occurs only in natural luteal phase (Johansson, 1975; Lunell et al., 1979; Kuhnz et al.,

1992; 1993a).

Clinical trials of oral contraceptives

Currently, there is one clinical trial of ethinylestradiol plus progestin in female MS patients. The trial is investigating safety and tolerability of estroprogestins in two different doses as an add-on to interferon beta-1a (http://clinicaltrials.gov/ct2/show/NCT00151801).

Promising preliminary results were presented at the 28th Congress of the European

Committee for Treatment and Research in Multiple Sclerosis (Lyon, France). The number of enhancing lesions was lower in both treatment groups compared to the interferon beta-1a only group, significantly in those treated with 0.04 mg ethinylestradiol and 0.125 mg desogestril (the higher ethinylestradiol and lower desogestril dose of the two treatments)

(Pozzilli et al., 2012). Differences in secondary endpoints, including annualized relapse rate and EDSS progression, were not detected among groups and adverse events causing

DMT discontinuation were equally distributed (Pozzilli et al., 2012). Tolerability to the ethinylestradiol plus progestin treatment has yet to be reported in detail.

Part I: Summary

Temporary relapse relief during late pregnancy and relapse worsening during early post-partum are the hallmark of pregnancy in MS. On the other hand, studies of the menstrual cycle are difficult to interpret because of the normal variability and excess of confounding variables, and are very onerous to execute. Oral contraceptive use is presumably not responsible for the increasing MS incidence among women, however, its impact on disease severity warrants further investigation. Thus far, the effect of OCs on

MS disease course is limited to global scores of disability and interpretation is hampered by the heterogeneity of the disease subtypes in the study population. Much basic science

research has been devoted to studying the effects of these exogenous steroids more closely in animal models of MS and unearthing possible mechanisms of action to explain the clinical observations.

Part II: Impact and mechanisms of ethinylestradiol and progestin in female animal models of neuroinflammation and degeneration

Ethinylestradiol

Experimental autoimmune encephalomyelitis (EAE) has been used to model human demyelinating disease since the 1940s (Wolf et al., 1947). The EAE model is an immune- mediated disease induced by subcutaneous injection of an adjuvant and myelin peptides, including fragments of myelin oligodendrocyte glycoprotein, myelin basic protein, or proteolipid protein (Denic et al., 2011). Depending on the method of induction and strain of species, the disease course can exhibit chronic inflammation or spontaneously resolve to mimic progressive or relapsing-remitting disease, respectively (Constantinescu et al.,

2011). A sex bias causing greater disease in females is detected in half of the murine strains

(SLJ, ASW, and NZW/LACJ but not B10.PL, PL/J, C57BL/6, or NOD strains) (Papenfuss et al., 2004).

Feeding female rodents oral ethinylestradiol delays EAE onset and reduces disease severity (Arnason and Richman, 1969; Trooster et al., 1993; Subramanian et al., 2003;

Yates et al., 2010b). Ethinylestradiol treatment down-regulated Th1 cytokines (interferon- gamma and tumor necrosis factor-alpha) but had no influence on Th2 cytokine levels

(interleukin-4, interleukin-5, and interleukin-10) (Subramanian et al., 2003). Oral

ethinylestradiol treatment decreased expression of chemokine receptors (CCR2, CCR3,

CCR4, CCR5, and CCR7) and matrix metalloproteinase-9 (Subramanian et al., 2003).

Matrix metalloproteinases are enzymes that have been found to be elevated in the plasma and cerebrospinal fluid of MS patients, and are implicated in the degeneration and regeneration of the central nervous system (Yong, 2005). Histopathological analysis of the spinal cord of the EAE mice found less infiltrating T cells after ethinylestradiol treatment

(Subramanian et al., 2003). The T cells drained from the control and treated mice were still capable of proliferating in response to myelin peptides, suggesting that the ethinylestradiol- mediated suppression of inflammation and demyelination in EAE is centered on preventing pathogenic T cell migration into the central nervous system (Subramanian et al., 2003).

There are three known estrogen receptors—two intracellular receptors, estrogen receptor-alpha and estrogen receptor-beta, and one membrane receptor, G-protein coupled estrogen receptor (GPER, also known as GPR30). Ethinylestradiol is thought to exert its protective effects chiefly through GPER (Yates et al., 2010b). Immune cells (Kvingedal and Smeland, 1997; Blasko et al., 2009), neurons (Brailoiu et al., 2007), and glia (Hirahara et al., 2013) widely express GPER. When white matter becomes demyelinated, as in MS, oligodendrocyte progenitor cells are the cells largely responsible for remyelinating the axons. In the toxin-induced animal model of demyelination whereby mice are feed the copper chelator cuprizone, GPER signalling has been found to enhance remyelination

(Hirahara et al., 2013). Additionally, vitamin D mediated prevention and protection from

EAE is believed to involve GPER (Subramanian et al., 2012), which may explain the

preferential effect of vitamin D in female EAE (Spach and Hayes, 2005) and its synergistic effects with estrogens (Nashold et al., 2009).

A study by Bouillon et al. compared vitamin D and vitamin D binding protein levels amongst 137 women, which included 29 women using OCs and 50 women who were pregnant (Bouillon et al., 1981). 25-hydroxyvitamin D3 is the circulating form of vitamin D (commonly measured as an indicator of vitamin D status), 1,25- dihydroxyvitamin D3 is the biologically active metabolite of vitamin D, and vitamin D binding protein is primarily responsible for transporting all forms of vitamin D throughout the body (Disanto et al., 2011). In the study, serum vitamin D binding protein and 1,25- dihydroxyvitamin D3 levels were increased during pregnancy and were increased in OC users to levels between that of control and pregnant women (Bouillon et al., 1981). Figure

3 shows the positive correlation found between the concentrations of vitamin D binding protein and 1,25-dihydroxyvitamin D3 in the control women and the women using OCs.

The 25-hydroxyvitamin D3 levels (circulating vitamin D) showed a seasonal variation but was uninfluenced by pregnancy or OC use (Bouillon et al., 1981). The mechanism by which oral contraception may affect vitamin D metabolism is not well understood, but increasing vitamin D binding protein may be a secondary, anabolic effect on protein synthesis and increasing the biologically active form may be due to stimulation of 1-alpha hydroxylase (the enzyme that converts 25-hydroxyvitamin D3 into 1,25-dihydroxyvitamin

D3) (Aarskog et al., 1983; Sowers et al., 1986). Given the protective effects of vitamin D in

MS (Ascherio et al., 2010), increasing the levels of vitamin D binding protein or the

metabolically active form of vitamin D may be one of the mechanisms by which OC use is found to be beneficial.

Few studies have directly compared the actions of different forms of estrogens.

Notably, unlike the endogenous form (estradiol), ethinylestradiol is also effective at reducing EAE severity when administered after disease induction (Subramanian et al.,

2003). In toxicology literature, vitellogenin (an egg yolk precursor protein) induction in male fish is commonly used to measure estrogenic potency of chemicals that may culminate in the aquatic environment (Van den Belt et al., 2004). According to this assay, ethinylestradiol has a potency 30 times that of estradiol (Van den Belt et al., 2004). Slight structural variations between physiological and synthetic estrogens may cause diverse binding affinities and receptor complex conformations, producing profound differences between natural and synthetic varieties.

Progestin

Progesterone is believed to have a role in oligodendrocyte precursor cell proliferation and differentiation (Gago et al., 2001; Schumacher et al., 2004; Ghoumari et al., 2005). The observed protective effects of progesterone in demyelinating and spinal cord injury models are supportive of this role (Labombarda et al., 2006; 2009; Garay et al.,

2011; Ye et al., 2013). Pregnancy levels of progesterone are also capable of reducing EAE severity by decreasing pro-inflammatory cytokines, increasing anti-inflammatory cytokines, and preserving axonal density (Garay et al., 2007; 2008; 2009; Yates et al.,

2010a; Garay et al., 2012; Giatti et al., 2012) although results are mixed (Kim et al., 1999;

Hoffman et al., 2001). There have also been reports of a synergistic effect of progesterone and estrogen (Garay et al., 2008; Acs et al., 2009).

Analogous to estradiol and ethinylestradiol, there are differences between progesterone and progestin. The distinction is potentially more important in the latter as progestins may have an opposing effect on neuroprotection. Early combined OCs, those containing the progestins medroxyprogesterone or norethynodrel, were found to be protective against EAE, but the OCs with the higher ethinylestradiol to progestin ratio remained effective longer (Arnason and Richman, 1969). Medroxyprogesterone acetate alone, a common progestin-only contraceptive, exacerbated the disease (Arnason and

Richman, 1969). Another progestin, melengestrol acetate, successfully reduced EAE without an ethinylestradiol complement (Greig et al., 1970) but melengestrol acetate is used to control ovulation in cattle, not humans. Many progestins have little or no affinity for the membrane progesterone receptors, leaving them unable to simulate the biological effects (Schumacher et al., 2008). Virtually all of the neuroprotection research relies on natural progesterone rather than a synthetic version or its combination with estrogen, offering a comforting prospect to the discontinuation of the phase III trial of high-dose progestin.

Part II: Summary

Much of what we know about MS and MS therapies comes from animal models.

According to animal models, ethinylestradiol alone, and in combination with a progestin, has therapeutic potential. While the exact mechanism is unknown, ethinylestradiol may

have both anti-inflammatory and neuroprotective roles. Given the encouraging evidence but also the shortcomings of observational studies shared in Part I, there is a need to investigate the effects of OCs in demyelinating diseases with an alternative, prospective approach.

Part III: Another approach to study oral contraceptives in multiple sclerosis

A model of multiple sclerosis

Multiple sclerosis is a complex disease of which inflammation and axonal degeneration are important aspects. Until the cause of MS is known, both of these features studied in animal models, while extremely valuable, fall short of replicating all of the processes that occur clinically. In the same way, clinical trials are neither warranted nor appropriate until convincing evidence accumulates. The high MS prevalence and the widespread use of OCs make observational studies favourable to perform, but the heterogeneity in central nervous system degeneration makes them difficult to interpret. In order to add simplicity to the central nervous system pathology, many have elected to focus on the optic nerve. The optic nerve is a common site of injury in MS. Moreover, the afferent visual pathway represents a functionally eloquent region of the central nervous system that can be interrogated to provide a structural-functional paradigm through which the acute and chronic effects of MS-related disease activity may be better understood. In essence, optic neuritis represents an acute inflammatory relapse model of MS that provides a unique opportunity to study the anti-inflammatory and neuroprotective effects of OCs.

Optic neuritis

Optic neuritis is a common harbinger of MS, and represents the initial clinical event in 20% of cases (Miller et al., 2005). Patients with optic neuritis typically report sub-acute onset vision loss, with associated pain (Balcer, 2006). Much of what we have come to understand about the clinical epidemiology of optic neuritis was gained from the experience of the Optic Neuritis Treatment Trial. This randomized, multicenter study was initially designed to compare the benefits of treatment with either oral prednisone (1.0 mg/kg/day for 14 days), intravenous methylprednisolone (250 mg every six hours for three days) followed by oral prednisone (1.0 mg/kg/day for 11 days), or oral placebo (for 14 days) in 457 patients with acute optic neuritis (Beck et al., 1992). From this trial, we learned that the majority of optic neuritis patients are young (mean age of 32 years),

Caucasian (85%), women (77%) (Beck et al., 1992). The long-term follow-up from the

Optic Neuritis Treatment Trial demonstrated that 72% of optic neuritis patients with evidence of brain MRI lesions at presentation developed clinically-definite MS over a 15- year period (Optic Neuritis Study Group, 2008). In patients with no white matter lesions evident on baseline MR imaging, the risk of MS was 25% (Optic Neuritis Study Group,

2008). Thus, the initial MRI scan is our most potent predictor of future MS risk in patients presenting with optic neuritis. While approximately 20% of MS patients experience optic neuritis as their first demyelinating event, optic neuritis affects the majority of MS patients during the course of their disease (Ikuta and Zimmerman, 1976).

Evaluating optic neuritis

The acute and chronic consequences of optic neuritis can be readily quantified with standardized and validated tests of visual function, and highly sensitive methods of measuring changes in retinal architecture. The battery of assessments useful in visualizing structure and measuring function of the anterior visual pathway, common for an optic neuritis diagnosis in a clinical or research setting, includes: high and low contrast acuity, optical coherence tomography (OCT), visual evoked potentials (VEPs), and MRI.

High and low contrast acuity

High contrast visual acuity tends to improve within two to three weeks of optic neuritis onset, regardless of steroid treatment, which has been shown only to hasten, not add to recovery (Beck et al., 1994). For most patients, visual acuity measured by a standard chart with black letters on a white background (e.g. Snellen, Early Treatment for Diabetic

Retinopathy Study; ETDRS) is completely restored by six months (Beck et al., 1994).

Contrast sensitivity, the identification of grey letters on a white background, is a more sensitive measure of visual dysfunction after optic neuritis. Abnormalities in contrast testing often persist after visual function appears otherwise normal (Trobe et al., 1996).

Reports dating back to the late 1970s sensed this discrepancy between contrast sensitivity and visual acuity testing in MS patients without any overt optic neuritis history (Regan et al., 1977). Because of its sensitivity, simplicity, and excellent inter-rater agreement (Balcer et al., 2000), low-contrast Sloan letter chart testing is increasingly being used as an outcome measure in longitudinal monitoring studies and MS clinical trials.

Optical coherence tomography

Optical coherence tomography is an ocular imaging technique that quantifies the thickness of various retinal layers within microns of resolution. This technology is analogous to ultrasonography but instead of sound waves, it employs infrared light. As the light beam passes through the layers of eye, retinal microstructures reflect light. By comparing these backscattered waves to those from a reference mirror, a high resolution (<

10 µm), cross-sectional image is generated in vivo (Figure 4) (Huang et al., 1991). Since

1991, the device has developed immensely in terms of resolution and speed. There is a high degree of correlation amongst the technological generations but they are not interchangeable (Kiernan et al., 2009; Grover et al., 2010; Chen et al., 2011; Lee et al.,

2011; Watson et al., 2011; Lange et al., 2012).

Retinal ganglion cells axons coalesce and exit the eye in the form of the optic nerve

(Figure 5). With a hand-held ophthalmoscope, Frisén and Hoyt described atrophy of retinal ganglion cell axons in MS patients and acknowledged RNFL defects to be a consequence of retrograde degeneration from visual pathway demyelination (Frisén and

Hoyt, 1974). More than 20 years later, this subsequent loss of RNFL thickness was captured with OCT testing of MS patients with (Parisi et al., 1999) and without an optic neuritis history (Fisher et al., 2006) .

On average, RNFL thickness is approximately 97 µm in healthy young adults and decreases 0.2 µm per year (Bendschneider et al., 2010; Alasil et al., 2012). A meta-analysis of MS patients reported that RNFL thickness was reduced by approximately 20 µm in optic

neuritis-affected eyes and seven microns in unaffected eyes compared to healthy controls

(Petzold et al., 2010). A reduction in RNFL thickness occurs in 74% of patients three to six months after an optic neuritis event (Costello et al., 2006). In a study of RNFL thinning outside the acute optic neuritis phase, MS eyes lost an average of 2.0 µm per year (Talman et al., 2010), suggesting the RNFL thins 10 times faster in MS patients. The swelling associated with optic neuritis may initially cause an increase in RNFL values, but the degree of swelling seen acutely is not predictive of later RNFL loss (Henderson et al.,

2010). Moreover, Costello et al. found a threshold thickness of 75 µm, below which persistent visual defects may exist (Costello et al., 2006).

Approximately five millimeters temporally of the optic nerve head is the fovea centralis, the center of the macula (Williams and Wilkinson, 1992) (Figure 5C). Optical coherence tomography-derived macular volumes are calculated from the entire thickness of the retina, which includes a greater proportion of retinal ganglion cell bodies than retinal ganglion axons (Ishikawa et al., 2005), especially towards the fovea (Burkholder et al.,

2009). Therefore, macular volume is often examined longitudinally in optic neuritis patients as a measure of neuronal degeneration to supplement RNFL thickness (axonal in nature) (Figure 6). Studies of patients with clinically isolated optic neuritis (i.e. presenting with optic neuritis as their first and only demyelinating event) have reported both normal

(Kallenbach et al., 2010) and reduced macular volumes (Outteryck et al., 2009) in the affected eyes, although patients were tested sooner after their event in the former. A decrease in macular volume is more visible in optic neuritis patients with a longer MS

duration (Pulicken et al., 2007; Henderson et al., 2008) or with incomplete visual recovery

(Trip et al., 2005).

Visual evoked potentials

Visual signals relayed by the retina travel through the optic nerve, enter the lateral geniculate nucleus, and continue on to the visual cortex. Typically while viewing a black and white pattern, the signal’s conduction velocity and wave amplitude can be measured through carefully placed electrodes on the scalp (Figure 7A). In 1972, Halliday et al. characterized this visual response from a cohort of 19 patients with unilateral optic neuritis.

The affected eyes had increased peak latency (milliseconds, msec) and smaller amplitudes

(microvolts, µV) compared to the unaffected eyes as well as eyes from healthy individuals

(Halliday et al., 1972) (Figure 7B and 7C). Delayed latency is reflective of demyelination

(You et al., 2011). Signal amplitude correlates well with visual acuity and is therefore thought to represent the number of functional axons in the pathway, which may temporarily be blocked by active inflammation or permanently reduced with axonal degeneration

(Jones and Brusa, 2003; Klistorner et al., 2008).

Studies using repeat testing found 77 to 97% of optic neuritis-affected eyes will have an abnormal VEP pattern (Frederiksen and Petrera, 1999; Fraser et al., 2006). During follow-up, measurements may improve or worsen, and a smaller percentage will completely normalize. In clinically isolated optic neuritis patients, normalization occurred in 11 to 19% after six months to three years (Hely et al., 1986; Hidajat and Goode, 2003).

In a mixed population consisting of mostly MS and neuromyelitis optica (NMO; a

demyelinating condition of the optic nerve and spinal cord) patients, more unaffected eyes had an abnormal VEP patterns compared to having an abnormal average RNFL thickness, suggesting VEP may be superior to OCT for detecting subclinical attacks (Naismith et al.,

2009b).

Magnetic resonance imaging

Currently, MRI is routinely performed in patients after a first demyelinating event and is the gold standard for predicting MS risk (Miller et al., 2012). As previously mentioned, the risk of developing MS in the 15 years following optic neuritis is 72% or

25%, depending on the presence of demyelinating lesions. Brain MRIs of acute optic neuritis patients frequently show contrast enhancement of the optic nerve (Kupersmith et al., 2002). Magnetic resonance imaging that targets the optic nerve, while not routinely ordered, is potentially very useful in understanding the natural history of lesions. The clinical use of advanced MRI techniques is also promising, especially as a means to study recovery. Diffusion tensor imaging, for instance, maps water diffusion. Increased radial diffusivity indicates myelin loss (Song et al., 2005) and decreased axial diffusivity correlates with axonal loss (Budde et al., 2009). When applied to the optic nerve and supplemented with VEP testing, diffusion tensor imaging may be used to study factors that contribute to remyelination after optic neuritis. Similarly, texture analysis is a novel post- processing approach that categorizes tissue as “fine” or “coarse” based on interpixel relationships (Zhang, 2012). In a prospective study of acute optic neuritis patients, we found the texture of the injured optic nerve useful in predicting visual acuity recovery

(Zhang et al., unpublished).

Evidence that sex steroids may influence optic neuritis characteristics

For optic neuritis to be a useful model to study the effects of OCs, the characteristics of optic neuritis should be sensitive to hormonal changes. While this has not been directly examined, some evidence already exists that indicates sex steroids may influence retinal measures, including RNFL thickness.

Sex differences in retinal nerve fiber layer thickness after optic neuritis

In a cohort of 144 acute optic neuritis patients, Costello et al. initially found comparable average RNFL values in affected eyes between men and women. Six months after their optic neuritis event, however, average RNFL values were lower in men (74 µm) than women (91 µm) (Costello et al., 2012). Sex-specific RNFL thinning has also been noticed in our current cohort of 27 acute optic neuritis patients who have completed month six testing (Burton et al., 2013). Extended follow-up studies are needed to establish if the difference persists or if the difference is in the rate of thinning. In a separate heterogeneous cohort, women were more likely to have three or more optic neuritis events so accumulated

RNFL loss will also need to be addressed (unpublished data).

Exogenous estradiol protects retinal ganglion cells in models of optic atrophy

Many scientists focus on quantifying retinal ganglion cell bodies rather than the

RNFL. This is more challenging clinically, as the number of retinal ganglion cell axons requires calculating the RNFL area from the OCT scan height at each pixel and the axon density from an age-dependent variable (Harwerth and Wheat, 2008). It is encouraging to know that estrogen effects have been detected beyond the axon, at the cell body. Evidence

suggests estradiol can directly protect retinal ganglion cells in varying models of ocular diseases, including glaucoma induced by optic nerve axotomy (Nakazawa et al., 2006), ischemia induced by transient middle cerebral artery occlusion (Kaja et al., 2003), neuropathy by N-methyl-D-aspartate-induced retinal neurotoxicity (Hayashi et al., 2007), and degeneration induced by hydrogen peroxide (Yu et al., 2004) and tumor necrosis factor

(Kitaoka et al., 2011). Furthermore, estradiol significantly reduced cellular pathology in

Leber’s hereditary optic neuropathy, a disease caused by retinal ganglion cell degeneration

(Giordano et al., 2011).

States of hormonal fluctuation influence retinal measures

Clinically, various studies have examined retinal changes during times of hormonal fluctuations. For instance, Demir et al. measured RNFL thickness of 40 healthy pregnant and 37 healthy non-pregnant women. The eyes of pregnant women had increased RNFL values in upper, temporal, and inferior quadrants of parafoveal areas (Demir et al., 2011)

(quadrants listed clockwise for the right eye are: superior or upper, nasal, inferior, and temporal). Akar et al. compared characteristics of the optic nerve head on 38 normally menstruating women during the follicular phase, ovulatory phase, and in the late luteal phase. Neuroretinal rim area decreased during the luteal phase (low estrogen, high progesterone), while linear cup to disc ratio, cup to disc area ratio and cup area were significantly higher (Akar et al., 2004). Another study of 15 healthy women found increased VEP latencies when tested during menstruation compared to the mid-follicular phase (Azarmina et al., 2011). Deschênes et al. compared retinal and optic nerve head measurements of 35 women who continuously used hormone replacement therapy since

menopause onset with 29 women who never used of the therapy. The hormone replacement therapy users had an increased average inferotemporal retinal artery diameter and blood flow, and an increased inferotemporal RNFL thickness (Deschênes et al., 2010).

Pregnancy, menstrual cycle phases, and hormone therapy may influence the retinal characteristics in healthy women; the same has yet to be explored in pathological situations.

Part III: Summary

Given its ability to non-invasively “biopsy” the retina, OCT testing is becoming widespread in clinical care for many ophthalmic conditions. As a component of the central nervous system, imaging the retina also provides an opportunity to monitor the inflammatory and neurodegenerative effects in many neurological disorders, including MS.

An inflammatory injury to the central nervous system can be affirmed in the form of optic neuritis. Multiple measures (e.g. OCT, VEP, MRI) exist to evaluate nerve damage and recovery for optic neuritis patients. There is laboratory evidence that estrogen protects retinal ganglion cells in pathological situations and clinical evidence that the healthy retina changes during times of hormonal fluctuations. Therefore, measures of retinal integrity in the optic neuritis model of MS may be useful in detecting the therapeutic potential of OCs.

Chapter summary

Epidemiological data, along with pregnancy observations, supports a role for sex hormones in MS. Oral contraceptives are a source of the exogenous hormones, typically ethinylestradiol (estrogen derivative) and a type of progestin (progesterone derivative).

Ethinylestradiol alone, or in combination with a progestin, but not progestin alone, is known to reduce disease severity in animal models. Clinically, how OC agents influence

MS is unresolved. Experimental trials to study the effect of hormonal interventions in patients are inherently complicated and controversial without an appropriate model and understanding the mechanism of action. On account of an ocular imaging technique through which we can infer axonal and neuronal loss, optic neuritis has emerged as a model through which we can better understand factors that influence brain injury and repair in

MS. Therefore, optic neuritis is a simpler, yet still clinically applicable, approach to study the neuroprotective properties of OCs.

A. Estradiol

B. Progesterone

C. Prolactin

Figure 1 Changes in maternal serum concentrations of sex hormones during pregnancy Increasing levels of (A) estradiol, (B) progesterone, and (C) prolactin during pregnancy. Blood samples were collected from 60 women during weeks 6 to 14, 16 to 28, and 27 to 39 of pregnancy. The solid lines correspond to the mean and 95% confidence intervals. Non- pregnant (NP) and luteal phase (LP) reference intervals are displayed at the bottom left of the graphs. Modified from (O'Leary et al., 1991). 38

A. Estradiol

B. Progesterone

Figure 2 Changes in serum concentrations of sex hormones during the natural and oral contraceptive-treated menstrual cycle Serum levels of (A) estradiol and (B) progesterone during normal cycles (n = 34) compared to cycles treated with Ovostat 1375 (a combined oral contraceptive consisting of 0.0375 mg of ethinylestradiol and 1 mg of lynestrenol) (n = 9). The Ovostat 1375 tablets were taken daily between day -27 and day -6. For the normal cycles, means and 95% confidence limits are given and for the treated cycles, means and total range. Modified from (Johansson, 1975).

Figure 3 Relationship between the serum concentrations of 1,25-dihydroxyvitamin D3 and vitamin D binding protein in oral contraceptive users and non-users

1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and vitamin D binding protein (DBP) in 29 women using oral contraceptives (red) and 56 healthy women not using oral contraceptives (black). Modified from (Bouillon et al., 1981) .

Figure 4 High-resolution images of the internal retinal structure taken with optical coherence tomography, demonstrating the processes involved in using this technology (A) Low-coherence infrared light is transmitted into the eye through use of an interoferometer. (B) The infrared light is transmitted through the pupil and then penetrates through the transparent nine layers of the retina. Subsequently, the light backscatters and returns through the pupil, where detectors can analyze the interference of light returning from the layers of the retina compared with light traveling a reference path (mirror #2). An algorithm mathematically uses this information to construct a gray-scale or false-color image representing the anatomy of the retina. (C) A fundus image from the optical coherence tomography device, showing the optic disc appropriately centered and surrounded by the target image circumference marker for analysis of the retinal nerve fiber layer. Modified from (Frohman et al., 2008).

Figure 5 The human optic nerve (A) The optic nerve exits the eye posteriorly. Inset (red) is detailed in (B). (B) The cell bodies of the retinal ganglion cells (RGCs; approximately 1 million in each eye) are in the innermost cellular layer of retina. The axons extending from the RGCs form the retinal nerve fiber layer. At the optic nerve head, the axons combine to form the optic nerve and exit the eye. (C) An example of a retinal fundus image of a healthy left eye, showing the optic nerve head and the fovea. Modified from (Huizing et al., 2005) and (Zhang et al., 2010).

A. Retinal nerve fiber layer B. Macular volume

Figure 6 Optical coherence tomography measured peripapillary retinal nerve fiber layer and macular volume reports A retinal fundus image indicating the scan location of retinal nerve fiber layer (RNFL) and macular reports generated by an optical coherence tomography (OCT) device (Carl Zeiss Meditec, Dublin, California) in a relapsing-remitting multiple sclerosis (MS) patient. (A) The average RNFL thicknesses in the right (OD) and left (OS) eyes (as well as multiple quadrants and clock-hours) are represented in red indicating reduced RNFL thicknesses bilaterally (red denotes values below 1% of what would be expected when compared to an age-matched reference population; yellow denotes values between 1% and 5% of what would be expected compared to an age-matched reference population; green indicates values within normal range). The RNFL values for this patient suggest loss of ganglion cell axons. (B) The macular scan of the right eye in the same patient. The macular thickness and volume (in red) is inferred as neuronal loss. Modified from (Saidha et al., 2011) and (Monteiro et al., 2012).

B. C.

Figure 7 Visual evoked potential setup and recordings (A) The typical recording arrangement for visual evoked potentials (VEPs). The patient is seated at an appropriate distance from the visual stimulus wearing the appropriate refraction. The appropriate recording electrode is attached to the posterior scalp and connected to the positive input of the differential amplifier. A similar reference electrode is attached to a visually neutral site on the head and connected to the negative input of the differential amplifier. A ground (GND) electrode is attached to the forehead or arm. A stimulus generator is used to select the desired stimulus type. (B) An example of a normal VEP pattern and (C) VEP recordings from a patient with left (L) eye optic neuritis. Note the reduced amplitude (µV) and increased latency (mS) of the P100 wave in the affected eye. Modified from and (Trick and Skarf, 2006) and (Holder, 2004)

CHAPTER THREE: THE ROLE OF REPRODUCTIVE HORMONES AND ORAL

CONTRACEPTIVES IN OPTIC NEURITIS STUDY- DESIGN AND OPERATION

Chapter overview

The potential benefit of OCs in MS is unresolved. The OC pill is an established medication and if it proves beneficial in optic neuritis, it would be a relatively easy treatment option for women with optic neuritis or MS, and possibly even other neurological diseases. We designed a study to help test our hypothesis that oral contraception during an acute inflammatory event will be associated with beneficial outcomes for optic nerve health. Evidence to this effect may ultimately support the use of sex hormone therapies in demyelinating diseases. In this chapter, I explain my role in the development and implementation of the Role of Reproductive Hormones and Oral Contraception in Optic

Neuritis (ROC-ON) study.

Study development

The Role of Vitamin D Status on Optic Neuritis Recovery study as a prototype

In May 2011, the Role of Vitamin D Status on Optic Neuritis Recovery (VitaDON) study started recruiting patients. Optic neuritis patients were followed for six months to determine if vitamin D status as the time of an optic neuritis event impacts optic nerve recovery. The optic neuritis event can be isolated or part of a known demyelinating disease phenotype (MS or NMO). The target sample size is 50 adult patients without a previous optic neuritis in the same eye or any other underlying ophthalmological disorder. Patients with optic neuritis routinely undergo neurological and visual testing, including acuity and

OCT testing near the time of symptom onset, month three, and month six. These results, as well as vitamin D levels, were collected for study participants. In addition, participants were asked to complete a demographic/lifestyle questionnaire at baseline and month six.

The primary outcome is the relative risk of having RNFL thickness greater than 75 to 80

µm (threshold below which persistent visual defects may exist) at month six in those with vitamin D (25-hydroxyvitamin D3) levels greater than or equal to 75 nmol/L (sufficient circulating vitamin D levels). As the principle investigator of the study, Dr. Jodie Burton welcomed my participation. Given the overlapping elements of hormone testing and optic neuritis, the VitaDON study was a useful template for the development of the ROC-ON study.

Helping define the population

The initial proposal of the ROC-ON study was to enrol female optic neuritis patients of reproductive age and currently still menstruating to eliminate the potential effects of puberty and menopause. Women were to be excluded for current pregnancy, amenorrhea, oophorectomy, or hysterectomy, as these conditions may also cause drastic alterations in sex hormones. We also collectively decided to investigate OC use in the form of the combined OC pill because: 1) There would be too much variability if all forms of hormonal contraception were studied at this stage; and 2) studies have shown that in North

America, the overwhelming majority of hormonal contraception users choose an oral ethinylestradiol and progestin form (Black et al., 2009; Mosher and Jones, 2010). However, there were still a number of questions concerning the hormonal details that needed to be resolved to further define the study population.

How long must participants be oral contraceptive users or non-users?

Previous studies suggest the serum hormone profile requires five to 21 days to stabilize after starting OC treatment (Dibbelt et al., 1991; Kuhnz et al., 1993b; Heuner et al., 1995) or to resume after ending OC treatment (Klein and Mishell, 1977). Therefore, we conservatively decided that women must be either OC users or non-users for at least four weeks prior to the optic neuritis episode. With this criterion, we focus on the short-term benefits of OC use. Indeed, efforts to investigate the short-term effects are more defensible.

Alonso et al. found no clear trend of MS risk reduction with OC duration but had results that suggested the highest risk reduction occurred with OC use in the year preceding the first symptoms (Alonso et al., 2005). Most epidemiology studies that failed to find a relationship between MS risk and OC use can not exclude the possibility that OC use reduces MS risk or symptoms with current ingestion and that the protection is merely lost after discontinuation of the pill (Villard-Mackintosh and Vessey, 1993; Hernán et al.,

2000). Finally, despite the convincing temporary benefit of pregnancy, there remains uncertainty surrounding any lasting protective effects (Thompson et al., 1986; Weinshenker et al., 1989; Stenager et al., 1994; Verdru et al., 1994; Runmarker and Andersen, 1995;

Hernán et al., 2000; D'hooghe et al., 2010). Therefore, in order to uncover any potential therapeutic effect of OCs, it is more suitable to focus on the immediate effects.

Should all participants have their blood tested for hormones?

Given the current questions surrounding neuroendocrine axis and synergistic effects in MS, we are interested in testing serum levels of: estradiol, progesterone, prolactin, testosterone, and vitamin D. Studies, however, found differences in the levels of these

hormones during the natural early follicular phase compared to the pill free interval (van der Spuy et al., 1990; Aden et al., 1998; van den Berg et al., 2010), making the hormone profile inherently different between OC users and non-users throughout the month. Oral contraceptive users also have predictable levels of estradiol (Mishell et al., 1972) and levels of ethinylestradiol that typically correlate with OC dosage (Kaufman et al., 1981). Based on this and the expert opinion of Dr. Bernard Corenblum, blood testing of sex hormones for the study is only appropriate for non-OC users.

When should blood testing occur?

Deciding on a window for blood testing was difficult because most of the hormones of interest fluctuate monthly or even daily. The unique opportunity to further explore hormonal concentrations in a relatively homogenous cohort, serially after an inflammatory injury, made our efforts worthwhile. To reduce variability amongst the non-OC users, we ultimately agreed on the mid-follicular phase of the menstrual cycle for testing (days five to

10; the menstrual diary located in Appendix C is provided to participants). Estradiol levels around this time seem to correlate to total estradiol exposure during the menstrual cycle

(Ahmad et al., 2002).

Should recruitment be limited to a specific demyelinating disease subtype?

Demyelinating optic neuritis may occur either in the context of CIS, MS, or NMO.

Based on evidence of NMO and progressive forms of MS having a greater degree of subclinical RNFL thinning (Pulicken et al., 2007; Naismith et al., 2009a; Costello et al.,

2010; Monteiro et al., 2012), patients diagnosed with these subtypes were to be excluded

from study participation. This decision was strengthened by hormonal knowledge. The potential benefits of OCs have been noticed primary in relapsing-remitting MS patients with a relatively short disease duration (Sena et al., 2012). It has been hypothesized that a

“healthy cell bias” exists in which estrogen exerts a robust protection of healthy neurons if present prior to the insult (Brinton, 2005). Estriol pilot trial data (Sicotte et al., 2002) and other work in MS (D'hooghe et al., 2012; Sena et al., 2012) supports this theory. Thus, if

OCs exhibit neuroprotective properties, a benefit is most likely to be seen in the population we are studying.

Logistics of the outcome variables

The primary study aim is to examine whether OC use at the time of optic neuritis onset impacts optic nerve injury or recovery. Secondary aims include: to explore select reproductive hormones and correlations with RNFL thickness and to determine if OC use is associated with improved MRI outcomes in the subset of patients with clinically isolated optic neuritis (i.e. those with a baseline MRI). For each of these aims and the related outcome variables, I helped establish various logistical processes, many of which were an extension from my involvement with the VitaDON study.

Communication between multiple sites

Physically separating the neurological exam and consent process at the MS Clinic

(Foothills Medical Center) from ophthalmic testing at the Eye Clinic (Rockyview General

Hospital) necessitates clarity and consistency among study personnel and participants.

These procedures, however, are far from independent. Baseline testing, in particular,

requires coordination between sites to prevent patients from exceeding the enrolment window. My familiarity with the scheduling and filing system at both sites enabled me to verify visits are scheduled and attended. Should any issues arise, my familiarity with the management and staff enables me to troubleshoot directly with the appropriate person. As with any collaboration, communication is critical for the success of the VitaDON and

ROC-ON studies.

Access to results

The majority of study data is acquired from electronic databases. For access, I completed the corresponding applications and training upon their approval. Synergy® is the newly implemented database used by the Eye Clinic; it stores most of the ophthalmological results. Other visual outcomes, such as the worst recorded visual acuity, are collected directly from paper charts at the Eye Clinic. Netcare® is a province-wide health record.

For study purposes, this database is used to assemble vitamin D levels, and for verification of hormonal contraceptive status and if applicable, the brand and dose. Laboratory requisitions not included in standard care (e.g. estradiol, progesterone, testosterone, prolactin) are mailed directly to Dr. Burton at the MS Clinic. Information from the neurological exam, including MS onset dates, can be collected from the Sunrise Clinical

Manager® database at the Calgary MS Clinic. Case report forms are standardized data sheets used to ensure complete data collection during study visits. For the ROC-ON study,

I constructed forms to assemble participant’s optic neuritis, MS, and hormonal history, as well as a checklist for inclusion and exclusion criteria, and visit tasks (Appendix D).

Results from the case report forms are directly added to a study database.

Database development

Designing a database to effectively capture the data for the ROC-ON study required several meetings with statisticians Dr. Misha Eliasziw and Dr. Gordon Fick. After finalizing the variables of interest, I learned proper coding and organization to aid the translation of data into statistical software. For example, we collect the participant’s current medications and vitamins, however most drugs are not of particular interest for this study.

A useful approach is to list common medication categories in the database (disease modifying therapies, anti-depressants, vitamins/minerals, etc.) and respond with ‘yes’ or

‘no’ for each. Whenever appropriate, drop-down lists are used to minimize error and maintain consistency. Also, a complex Excel sheet with all outcomes for each time point is difficult to directly input into statistical software. Our database contains separate pages for patient history, visual outcomes, blood testing, and MRI outcomes. Patient IDs span horizontally and variables are listed vertically to facilitate data entry. With one glance, there are a greater number of rows than columns; displaying more of a complete individual dataset is conducive for quick referencing to the original document.

Tailoring the questionnaire

Questionnaires are often used in observational studies to collect additional information necessary for proper interpretation. If the questionnaire is too long, the participant is less likely to complete it, but if it is too short, important data may be missed.

Using the VitaDON questionnaire as a template, the ROC-ON study questionnaire requests information on ethnicity, comorbidities, vitamins and medications, smoking history, physical activity, pregnancy, and birth control history (Appendix E). The questions were

selected based on variables that may influence MS risk or progression, or OC use. With the questionnaire information, we can compare relevant demographical and lifestyle characteristics between the OC users and non-users to assess the similarity of the two groups. Ideally, we would like OC status to be the only difference between the two groups, so we can more accurately compare optic neuritis recovery with OC use.

Funding

In 2011, we applied for a seed grant from the University of Calgary. For the application, I drafted the proposal, including a section on student training and a summary for the public. I also contributed to the budget justification. Later in the year, we were awarded the University Research Grants Committee Seed Grant for $16, 091. Since then, I have assisted Dr. Burton in her efforts to obtain external funding through operating grant competitions. A graduate student support grant was awarded to Dr. Burton and the project from the Alberta endMS Regional Research and Training Centre.

Process of submitting to the Conjoint Health Research Ethics Board

Application overview

The ROC-ON study required Conjoint Health Research Ethics Board (CHREB) review. The submission was comprised of the study protocol and an application form, in which we disclosed all potential risks and benefits, as well as our plans to safeguard participant’s privacy and confidentiality. This prospective cohort study will be tested largely within routine care, with minimal study-specific procedures. Study-specific procedures include a lifestyle questionnaire and serum testing for various hormones. Blood

testing has minimal risks, but includes bruising and soreness. More serious risks, such as infection, can be eliminated with sterile technique. All patients are made aware of these risks and gain a thorough understanding of the study through the consenting process.

Informed consent forms

For the CHREB application, I also assisted with the construction of the informed consent form. Informed consent forms should contain the following sections: information on the rationale for the study, its design and methodology, the purpose, a detailed description of what the study entails for the participant, the risks and benefits, whether participation will require any costs or reimbursements, who has access to information collected during the study, and a compensation clause should a research injury arise.

Sections should be concise and located under easily observable titles. Informed consent forms are ideally written for a fourth to sixth grade reading level (Paasche-Orlow et al.,

2003).

Approval and modifications

The CHREB approval took approximately four months, during which time minor modifications were made to the protocol (e.g. changed the maximum allowable age from

50 to 45) and consent form. These modifications were approved in a second correspondence from CHREB, approximately eight months after initial submission. The updated protocol is located in Appendix F. An annual progress report will be submitted to

CHREB every August until study closure.

Study implementation

Recruitment

Urgent ophthalmology clinics and urgent neurology clinics are skilled in referring optic neuritis patients to Dr. Fiona Costello. One way I could assist and encourage recruitment was by presenting the ROC-ON study at MS Rounds shortly after enrolment began. Furthermore, my presentation gave clinic staff and other colleagues an opportunity to ask questions or provide us with suggestions.

Ongoing involvement

Upon notice of potential study candidates, I prepare new study charts for their visit at the MS Clinic. This includes gathering the consent form, case report forms and the

EDSS workbook, questionnaire, blood requisition, and menstrual diary. On occasion, I attend the clinic visit and witness the neurological exam performed by Dr. Burton. These are tremendous learning opportunities where I am exposed to the delicate patient-doctor dialogue. After patients are enrolled, I communicate with the Eye Clinic to verify future tests are arranged and attended. I also make myself available to participants should they need any reminders or clarifications.

On a regular basis, I visit the MS Clinic and Eye Clinic for data collection. To ensure confidentiality, I remove identifying information from test results obtained from the paper and electronic charts, and patient questionnaires. Then, I re-label results and enter data into the study database according to code numbers, and store hard copies of the results

in study binders. The database management is an integral part of keeping the team members informed, professional and attentive.

Chapter summary

The ROC-ON study will help recognize if oral contraception has a potential beneficial effect in MS. Through my extensive involvement in the groundwork and implementation of the study, I have gained valuable experience in experimental design and protocol development, ethical considerations, patient recruitment and communication, outcome measurement, data collection, and statistical analysis as well as presentation skills.

As of early March, we enrolled four patients and the study is ongoing.

CHAPTER FOUR: PRELIMINARY DATA

Chapter overview

In this chapter I present preliminary results pooled from two ongoing OCT observational studies. Due to the small sample size of 26 participants, the focus is to describe the study population, and include the mean and confidence intervals (CI) of test results. I use the interim analysis as an opportunity to identify outliers and address any foreseeable issues, such as potential confounding variables and our screening strategy for eliminating optic neuritis mimics. The discussion also emphasizes study limitations.

Sources of data

The Role of Reproductive Hormones and Oral Contraceptives in Optic Neuritis study

Discussed in Chapter Three, the ROC-ON study is a prospective study of premenopausal women with acute optic neuritis (refer to Appendix F for study details). The primary objective is to examine whether OC use at the time of optic neuritis onset impacts optic nerve injury or recovery. Enrolment commenced in late October 2012, and as of

March 2013, we have enrolled four patients. The study is funded by the University of

Calgary and by an Alberta endMS Graduate Student Support Grant.

The Role of Vitamin D Status on Optic Neuritis Recovery study

Also introduced in Chapter Three, the other source of data used in this chapter is the

Role of Vitamin D Status on Optic Neuritis Recovery study (VitaDON; Primary

Investigator: Dr. Burton). From May 2011 to March 2013, the VitaDON study accrued 46 participants (out of a projected 50). Data presented in this chapter is limited to the women

younger than 45 years of age with unilateral demyelinating optic neuritis (26 of the current

46 subjects). There is considerable overlap between the ROC-ON study and VitaDON study; all four ROC-ON patients are also in VitaDON.

Preliminary data

Baseline characteristics

The pooled data includes 26 women, aged 20 to 45 years. Four of the 26 (15%) were OC users. At the time of enrolment, 10 (38%) were diagnosed with MS and 16 (62%) experienced a clinically isolated optic neuritis. The baseline characteristics for the participants are summarized in Table 1 (categorical variables) and Table 2 (continuous variables).

Neurological outcomes

Median EDSS scores were 1.5 at baseline (interquartile range; IQR 1 – 3) and 1.0

(IQR 0 – 1.75) at month six. At time of enrolment, 16 out of 26 (62%) carried a CIS diagnosis. By the sixth month of follow-up, five out of 11 (45%) CIS patients converted to

MS.

Recovery of visual acuity after optic neuritis

Worst recorded visual acuity in the affected eye ranged from 20/20 to hand motions. By month six, visual acuity recovered to 20/25 or better in 19 out of 20 (95%) patients; and 20/30 or better in all patients.

Optical coherence tomography outcomes

The average RNFL thickness for the affected eyes at baseline and month six was

125 µm (95% CI 98 – 152) and 82 µm (95% CI 75 – 89), respectively. The fellow

(contralateral) eyes had consistent average RNFL thickness of 92 µm at baseline (95% CI

80 – 99) and month six (95% CI 84 – 99). Similarly, the mean macular volume decreased from 10.1 mm3 (95% CI 9.8 – 10.3) to 9.6 mm3 (95% CI 9.3 –9.9) in the affected eyes and remained 9.8 mm3 (95% CI 9.6 – 10) in the fellow eyes.

Visual evoked potential outcomes

The mean VEP latencies for the affected eyes at baseline and month six were 133 msec (95% CI 126 – 141) and 129 msec (95% CI 120 – 137), respectively. The fellow eyes had mean VEP latencies of 118 msec (95% CI 110 – 126) and 117 msec (95% CI 110

– 125). At both time points, VEP amplitudes were approximately 8 µV (baseline 95% CI 5

– 10; month six 95% CI 6 – 10) for the affected eyes and 10 µV (baseline 95% CI 8 – 13; month six 95% CI 8 – 14) for the fellow eyes.

Discussion

At this time, there is limited data for interpretation, especially when comparing OC users to non-users. Instead, I sought to use the preliminary data to address outliers, potential confounders, validate or strengthen our screening process, and discuss study limitations.

Discussion topic 1: normality of data and outliers

Previous studies have found RNFL thickness, macular volume, and VEP amplitudes and latencies to follow a normal distribution (Khanifar et al., 2010; Garcia-Martin et al.,

2011; Warner et al., 2011). The skewness and kurtosis of these variables from the cohort of

26 female optic neuritis patients are listed in Table 3. Focusing on the RNFL thickness of the affected eyes at baseline, the non-normality of the data may be due to outliers. Based on

IQR calculations, there were two mild outliers: 215 µm and 240 µm (these values exceeded the upper inner fence; third quartile + 1.5(IQR) = 131 + 1.5(44) = 197). There were also two extreme outliers: 293 µm and 315 µm (these values exceeded the upper outer fence; third quartile + 3(IQR) = 131 + 3(44) = 263). I confirmed these values with the original results. In fact, the RNFL thickness remained increased at follow-up testing (data not shown), suggesting these outliers did not occur in error and may be examples of excessive swelling. At this point in time, there was no reason to exclude the four data points of baseline RNFL thickness in the affected eyes and for the remainder of the chapter, this variable was analyzed according to a non-normal distribution. Month six RNFL thickness of the affected eye, however, was analysed according to a normal distribution.

Discussion topic 2: potential confounders

There is always the possibility of confounding variables in observational studies. In our investigation, a confounding variable would need to be independently associated with

OC status and optic nerve health. Oral contraceptive use, for instance, decreases with age

(Mosher and Jones, 2010). In our population, OC users have a mean age of 25 years (95%

CI 19.6 – 30.6) versus 33 years (95% CI 30.5 – 36.9) for non-users. Also, many DMTs

have an unsafe or unknown safety profile during pregnancy (Lu et al., 2012), and thus women using DMTs are encouraged to use adequate birth control. As a result, OC use may be more common amongst MS patients compared to CIS patients. In our baseline cohort, two out of 10 (20%) MS patients were OC users and two out of 16 (13%) CIS patients were

OC users. Finally, some studies have found higher circulating vitamin D levels in exogenous estrogen users compared to non-users (Sowers et al., 1986; Harris and Dawson-

Hughes, 1998; Gagnon et al., 2010) but not all (Bouillon et al., 1981). The authors explained that OCs might increase the levels of vitamin D binding protein, consequently increasing the amount of circulating vitamin D (Sowers et al., 1986). At baseline, the mean vitamin D level was 93 nmol/L (95% CI 31 – 156) for the four OC users and 71 nmol/L

(95% CI 57 – 84) for the 22 non-users. In this section, I explored these variables—age, diagnosis, and vitamin D status—with RNFL thickness to appreciate their potential to influence our final results. Depending on whether a variable amplifies or dilutes the protective effect of OC use, it would be considered to have a positive or negative confounding effect.

Age as a confounding variable

Retinal nerve fiber layer thickness is lost at a rate of 0.2 to 2.0 µm per year in healthy controls and MS patients, respectively (Bendschneider et al., 2010; Talman et al.,

2010). Using the current age difference of eight years (mean age 25 years for OC users and

33 years for non-users), OC users could have a maximum gain of 16 µm compared to non- users due to age alone, thereby potentiating a beneficial relationship between OC use and

RNFL preservation. A correlation coefficient was computed to assess the relationship

between average RNFL thickness of the affected eye and age. There was a moderate negative correlation between the two variables at baseline (Spearman r = -0.43, 95% CI

-0.71 – -0.036, n = 26) and a similar trend for month six (Pearson r = -0.41, 95% CI

-0.72 – 0.039, n = 20), suggesting age will have a positive confounding effect (Figure 8). In other words, age may overestimate any protective effects of OC use. Of note, the magnitude of the relationship between age and RNFL thickness may be larger at baseline in presence of other factors, such as inflammation.

Disease subtype as a confounding variable

Limiting enrolment to CIS and relapsing-remitting MS was an effort to reduce disease subtype (and disease duration) as a potential confounder of RNFL thinning. While progressive MS subtypes and NMO patients have a greater degree subclinical RNFL thinning compared to patients with CIS or relapsing-remitting MS (Pulicken et al., 2007;

Naismith et al., 2009a; Costello et al., 2010; Monteiro et al., 2012), Costello et al. found differences between the latter two diagnoses (Costello et al., 2010). Based on diagnosis at enrolment, average RNFL thickness for the affected eyes was 140 µm (95% CI 98 – 183) for CIS and 100 µm (95% CI 86 – 115) for MS patients at baseline. After six months,

RNFL thickness was 81 µm (95% CI 71 – 92) for CIS patients and 83 µm (95% CI 72 –

95) for MS patients (Figure 9). Of note, eyes displaying sizeable baseline edema (that is, potential outliers) belong to CIS patients. The exclusion of outliers would narrow the gap between CIS and MS patients.

Vitamin D status as a confounding variable

Using a standard vitamin D level of 75 to 80 nmol/L as the upper limit for deficiency, a review of Canadian research found 70 to 97% of Canadians to be vitamin D deficient (Schwalfenberg et al., 2010). There is growing evidence that higher vitamin D exposure (van der Mei et al., 2003) and sufficient levels (Salzer et al., 2012) are associated with a reduced risk of MS. It has been hypothesized that vitamin D may be a more potent anti-inflammatory agent in women because of its synergistic effect with estradiol (Correale et al., 2010). Therefore, vitamin D status may be a confounding variable in our cohort of women. Figure 10 shows the range and standard deviation of RNFL thickness for affected eyes between two vitamin D categories: sufficient and low. The category was assigned according to initial levels (as Dr. Burton encouraged all patients during their first visit to take vitamin D supplements) using the more generous definition of vitamin D sufficiency

(≥ 75 nmol/L). At baseline, the mean RNFL thickness was 110 µm (95% CI 68 – 152) for the sufficient group (≥ 75 nmol/L) and 137 µm (95% CI 99 – 176) for the low group (< 75 nmol/L). At month six, the mean RNFL thickness was 78 µm (95% CI 65 – 91) for the sufficient group and 85 µm (95% CI 76 – 94) for the low group. The removal of the two extreme outliers at baseline changes the mean and 95% confidence intervals to 92 µm

(95% CI 82 – 101) for the sufficient group and 125 µm (95% CI 95 – 156) for the low group. Therefore, whether vitamin D status is a confounding variable may depend on the treatment of outliers.

Discussion topic 3: addressing the screening process for optic neuritis mimics

Recruiting patients promptly after initial optic neuritis symptoms makes us vulnerable to enrolling patients with other diseases that mimic demyelinating optic neuritis.

Differential diagnosis of demyelinating optic neuritis includes: ischemic optic neuropathy, compressive optic neuropathy, Leber’s hereditary optic neuropathy, traumatic optic neuropathy, radiation-induced optic neuropathy, retinal diseases, sarcoidosis, lupus, syphilis, and Lyme disease (Bianchi Marzoli and Martinelli, 2001; Balcer, 2006). A retrospective study at a large MS Center found neurosarcoidosis to be the most common inflammatory neurological disease misdiagnosed as MS, albeit a rare occurrence (Scott et al., 2010). Thus far, we have experienced two optic neuritis mimics—neurosarcoidosis and retinopathy. In the Optic Neuritis Treatment Trial, 92% of patients suffered from pain

(Optic Neuritis Study Group, 1991). Indeed, both of the mimics lacked a clear worsening of pain with extraocular movements. It may be beneficial to document and categorize clinical presentation in a study database, not to preclude enrolment but to further characterize the study population. Ultimately, it was the rigorous evaluation these patients underwent that helped detect their mimics and being involved in a research study would have only assisted the process.

Discussion topic 4: study limitations

Oral contraceptive behaviour bias

As discussed previously, confounding variables are characteristic of observational studies. All study participants are women of reproductive age, experiencing a similar life stressor. There are, however, a number of other possible inherent differences between OC

users and non-users. We have attempted to identify many of them a priori, including smoking and pregnancy history through the study’s lifestyle questionnaire (Appendix E), however, it is possible that a confounding variable may be missed.

Variability in oral contraceptive regimes

Another study limitation is the diversity of OC formulas. While we are only studying OC use in the form of the combined OC pill, there is variation in the possible ethinylestradiol dose, and progestin type and dose. There is conflicting data on the extent with which progestin influences in the pharmacokinetics of ethinylestradiol (Jung-Hoffman and Kuhl, 1990; Hammerstein et al., 1993). Therefore, if a certain threshold or regime is beneficial, it may remain undetected. Our sample size may not be large enough to make subgroup analysis.

Variability in serum hormone levels

While we are limiting blood testing to a five-day window during the menstrual cycle, there is still a great deal of variability. For instance, Calgary Laboratory Services® uses a reference interval of 90 to 700 pmol/L for estradiol in the follicular phase (days one to 13) (The reference intervals for all the hormones that may be tested for study purposes are listed in Appendix G). Nonetheless, investigations have succeeded in finding altered sex hormone profiles in both EAE (Foster et al., 2003) and MS patients (Wei and

Lightman, 1997; Tomassini et al., 2005; Safarinejad, 2008; Zakrzewska-Pniewska et al.,

2011; Trenova et al., 2013). Even in a study where estradiol and progesterone levels in corresponding phases on the menstrual cycle were similar between MS women and

controls, a high progesterone to estradiol ratio corresponded to increased MRI activity

(Pozzilli et al., 1999). Therefore, exploring the ratio of the hormones may be more valuable than absolute levels.

Sample size calculation

The two pieces of information that are needed for the ROC-ON study’s sample size calculation are: the ratio of OC users to non-users and the outcome that can be equated to a benefit. Between 25 to 40% of women of reproductive age use hormonal contraception, with the vast majority using oral contraception (Black et al., 2009; Mosher and Jones,

2010). As existing data regarding the prevalence of OC use in an optic neuritis cohort and the average RNFL thickness of OC users and non-users in not available, we were are not in a position to make an estimate of effect size and possible differences between groups.

The present protocol is a prospective pilot cohort study to explore the potential relationship OC use has in optic neuritis. Recruitment estimates based on similar research at our centre have shown that within one month, three female patients can be recruited, for a total of roughly 50 to 60 female patients in 18 to 20 months. The goal is to complete this preliminary study in 18 to 24 months so that trends to support a larger undertaking can be detected in a reasonable time frame and planning for a larger-scale multicentre study can begin if indicated. We feel it is neither feasible nor prudent to undertake such a large multicentre study of this research question without obtaining supportive data first.

Clinical significance of retinal nerve fiber layer thickening

According to an expert panel consensus on OCT use in MS, one question that needs answering to further clarify the clinical significance of changes in RNFL thickness is: “how early after an optic neuritis episode should OCT be used as a baseline to measure RNFL thinning?” (Sergott et al., 2007). Optic disc swelling is present in one third of optic neuritis patients (Balcer, 2006). Baseline OCT testing reveals RNFL swelling (RNFL thicker than the 95th percentile of the fellow eye) in 82 or 33% of affected eyes when assessing quadrants or global averages, respectively (Kupersmith et al., 2011). It has been suggested that RNFL thickening at baseline does not predict later RNFL thinning (Henderson et al.,

2010) and is not related to the proximity of the optic nerve lesion visible on MRI

(Kupersmith et al., 2011). Therefore, its relevance remains unknown and is a study limitation. Inflammation resolution will exaggerate RNFL thinning and complicate the interpretation of axonal loss. Gaining better understanding of RNFL thickening remains an important goal, as it would help define outliers and determine if there is an optimal time after an optic neuritis episode for OCT testing. Baseline testing in the ROC-ON study spans a maximum of 45 days as the first detectable signs of atrophy occur temporally and are not generally evident until 60 to 90 days after optic neuritis (Costello et al., 2006;

Costello et al., 2008a; Henderson et al., 2010). This variability in baseline testing, however, does not seem to determine whether inflammation is observed (Spearman r = 0.0015, 95%

CI -0.40 – 0.40, n = 26) (Figure 11). Of note, the largest RNFL values were most noticeable around the second week after optic neuritis onset.

Retinal nerve fiber layer is not completely axonal in nature

Optical coherence tomography-derived RNFL thickness has been found to correlate with global measures of central nervous system pathology in MS (Saidha et al., 2013).

With the added knowledge that the retina lacks myelin, researchers often use axonal loss interchangeably with RNFL thinning. It is important to remember that RNFL is not completely axonal in nature. There are also glia cells (Ogden, 1983) and blood vessels

(Hood et al., 2008), for example. Harweth and Wheat investigated the discordance between age-related loss of retinal ganglion cells from histologic counts (0.6% per year) and age- related thinning of RNFL (0.2% per year) (Harwerth and Wheat, 2008). The authors eloquently demonstrated that as the neuronal component of the RNFL decreases with age, the non-neuronal component increases, dampening the overall observed RNFL thinning

(Figure 12) (Harwerth and Wheat, 2008). Segmentation techniques capable of quantifying other retinal layers, such as the ganglion cell layer and inner nuclear layer, may help reduce the impact of the non-neuronal RNFL component.

Chapter summary

This chapter characterized a study population that will address if OC use is beneficial in the context of optic neuritis. We anticipant age to cause a positive confounding effect, although the magnitude of the relationship may be altered between baseline and month six assessments. Thus far, a diagnosis of CIS or relapsing-remitting MS does not appear to affect RNFL thickness, however our population is comprised of MS patients with relatively short disease durations. Vitamin D status as a potential confounding variable depends on the treatment of outliers. Future discussions are needed regarding the

operational definition of outliers. The infrequency of enrolling optic neuritis mimics and the observational nature of our study does not support changing our recruitment strategy, although documenting clinical presentation may be useful. The ROC-ON study is not without limitations including the confounding variables, the variability of hormone levels, the basis for sample size calculations, and that there is still much to understand about

RNFL thickening and thinning in the context of MS and optic neuritis.

Table 1 Categorical variables at baseline Characteristic Number of patients (%) Disease subtype CIS 16 (62) RRMS 10 (38) Acute optic neuritis-affected eye* OD 11 (42) OS 15 (58) Steroid use at optic neuritis onset Yes 8 (31) No 18 (69) DMT use at optic neuritis onset Yes 3 (12) No 23 (88) OC use at optic neuritis onset Yes 4 (15) No 22 (85) Previous optic neuritis† Yes 4 (15) No 22 (85) CIS = clinically isolated syndrome; DMT = disease modifying therapy; OC = oral contraceptive; OD = right eye; OS = left eye; RRMS = relapsing-remitting multiple sclerosis *Optic neuritis in last 45 days †Previous optic neuritis occurred in the fellow eye (previous optic neuritis in affected eye was an exclusion criterion)

Table 2 Continuous variables at baseline Percentiles Mean SD Range 25% 50% 75% Age (N = 26) 32.3 7.4 20.3 – 44.8 27.5 30.7 38.3 RRMS duration* (N = 10) 4.0 2.6 0 – 8.4 1.8 4.9 5.3 N = number; RRMS = relapsing-remitting multiple sclerosis; SD = standard deviation *In cases where the optic neuritis episode established the MS diagnosis, a disease duration of zero years was used. Clinically isolated syndrome patients were not included.

Table 3 Normality of optical coherence tomography and visual evoked potential outcome variables at baseline and month six Time point Outcome Eye† Skewness‡ Kurtosis§ Baseline* RNFL thickness Affected (n = 26) 1.8 5.2 Fellow (n = 26) 0.69 3.4 Macular volume Affected (n = 23) 0.090 2.3 Fellow (n = 24) 0.12 2.1 VEP latency Affected (n = 21) -0.20 2.2 Fellow (n = 21) 1.2 3.6 VEP amplitude Affected (n = 21) 1.2 3.8 Fellow (n = 21) 0.14 2.0 Month 6 RNFL thickness Affected (n = 20) 0.75 3.7 Fellow (n = 20) -0.21 2.4 Macular volume Affected (n = 20) 0.15 2.4 Fellow (n = 20) 0.35 2.1 VEP latency Affected (n = 16) 0.18 2.1 Fellow (n = 16) 1.3 4.2 VEP amplitude Affected (n = 16) 1.3 5.1 Fellow (n = 16) 0.27 2.1 RNFL = retinal nerve fiber layer; VEP = visual evoked potential *Unilateral optic neuritis within 45 days of baseline testing †Optic neuritis-affected eye is the affected eye and the contralateral eye is the fellow eye ‡For a normally distributed variable, skewness is near 0 §For a normally distributed variable, kurtosis is near 3

A. Baseline B. Month 6

Figure 8 Scattor plot showing the relationship between retinal nerve fiber layer thickness and age Average retinal nerve fiber layer (RNFL) thickness for the affected eyes at (A) baseline and (B) six months post optic neuritis.

Figure 9 Retinal nerve fiber layer thickness in clinically isolated optic neuritis and multiple sclerosis patients The average retinal nerve fiber layer (RNFL) thickness for affected eyes of patients diagnosed at enrolment with clinically isolated syndrome (CIS) or multiple sclerosis (MS). The horizontal lines represent mean and standard deviation.

Figure 10 Retinal nerve fiber layer thickness in patients with sufficient and low vitamin D status The average retinal nerve fiber layer (RNFL) thickness in patients with vitamin D status classified at enrolment as sufficient (≥ 75 nmol/L) or low (< 75 nmol/L) for those eyes affected by optic neuritis. The horizontal lines represent mean and standard deviation.

Figure 11 Scattor plot showing the relationship between retinal nerve fiber layer thickness and the delay in testing from optic neuritis onset Patients are enrolled during a window of 45 days after optic neuritis onset and consequently have baseline testing on variable days. The scatter plot shows the average retinal nerve fiber layer (RNFL) thickness for the affected eyes relative to the number of days since optic neuritis onset.

Figure 12 Total retinal nerve fiber layer has a neuronal and non-neuronal component The retinal nerve fiber layer (RNFL) thickness at each age (solid line) represents the sum of two components of the total thickness: the age-dependent loss of neuronal tissue (dashed line) and a compensating increase of non-neuronal tissue (dot-dash line). Proposed by (Harwerth and Wheat, 2008) and modified from (Harwerth et al., 2008).

CHAPTER FIVE: LESSONS LEARNED

Chapter overview

Optical coherence tomography is a widely used tool for ophthalmic conditions such as glaucoma and macular degeneration. In the field of MS, OCT has become recognized as a promising way to further our understanding of central nervous system injury and to complement current techniques for patient management. The application of OCT has recently infiltrated other areas in neurology, including Parkinson disease and Alzheimer disease (Jindahra et al., 2010). As technology continues to improve and our knowledge of

OCT-measured values increases, so will the applications. In my final chapter I share lessons learned from my involvement with OCT studies. I also share lessons learned from studying reproductive hormones and being involved with human research. To conclude, I review my three thesis objectives introduced in the first chapter and discuss future directions.

Lessons learned from optical coherence tomography research

Lesson 1: optical coherence tomography compliments current techniques

Magnetic resonance imaging remains the most powerful technique in MS. Its foundation began in 1946, and in 1981, Young et al. introduced us to the possibilities of

MRI in MS (Young et al., 1981). Twenty years later, MRI was integrated into the diagnosis of MS (McDonald et al., 2001). A similarly slow path has followed for incorporating OCT into the MS research and clinic world. Only recently has OCT been used as an outcome

measure (primary and secondary) in clinical trials. Researchers with a full understanding of its benefits and pitfalls remain cautious about over-relying on this technology.

Benefits

In MS, OCT measurements correlate with visual outcomes (Balcer and Frohman,

2010; Talman et al., 2010), brain atrophy (Gordon-Lipkin et al., 2007; Grazioli et al., 2008;

Siger et al., 2008), and disease activity (Saidha et al., 2012; Ratchford et al., 2013). For example, in a study of 164 MS patients without optic neuritis and 59 healthy controls, an accelerated rate of annual ganglion cell/inner plexiform layer thinning was observed in patients with new T2-weighted lesions and new gadolinium-enhancing lesions (Ratchford et al., 2013). Optical coherence tomography has augmented our understanding of MS with quantifiable evidence of axonal loss in the optic nerves of MS eyes even without optic neuritis (Fisher et al., 2006; Sepulcre et al., 2007; Pueyo et al., 2008; Talman et al., 2010).

Optic neuritis is a clinically overt symptom from a relatively isolated location, and often precedes an MS diagnosis. By providing a transparent and early instance to study the rapid and gradual axonal loss in optic neuritis-affected and unaffected eyes, respectively, the disease (optic neuritis) and the technology (OCT) have shaped an opportunity to investigate factors that contribute to neuroprotection. Equally responsible to the success of OCT are its logistical advantages. Optical coherence tomography testing is relatively inexpensive, safe, concise, and easily tolerated by patients.

Pitfalls

Despite the benefits, there are a number of reasons physicians and researchers cannot solely rely on OCT testing. Currently, RNFL measurements have little prognostic ability; RNFL thickness of CIS patients (with or without optic neuritis) does not predict conversion to MS (Costello et al., 2008b; Outteryck et al., 2009). Single OCT measurements have limited impact on therapy decisions and long-term OCT data from large patient cohorts is lacking. The estimated yearly RNFL thinning of MS patients is less than the resolution of any given OCT device. There remains uncertainty on how to optimally analyze results. There is considerable variability among and within patients, questioning the clinical relevance of minor changes. As explained in Chapter Three as a study limitation, RNFL thinning and thickening is not axonal specific. Arguably the greatest caveat to the wide applicability of OCT is the indefinite number of clinical characteristics capable of altering its dynamics.

Lesson 2: retinal nerve fiber layer interpretation is complicated by various factors

Clinical characteristics influence RNFL values, making interpretation challenging and producing many inconsistencies in the literature. For instance, the RNFL difference between eyes of MS patients without optic neuritis and the unaffected fellow eyes of MS patients with unilateral optic neuritis is conflicting. Some studies have found lower RNFL values in eyes of MS patients without optic neuritis (Fisher et al., 2006; Costello et al.,

2010) and others reported lower RNFL thickness in unaffected eyes of MS patients with unilateral optic neuritis (Pulicken et al., 2007; Siger et al., 2008). These differences are likely due to other clinical features, such as MS disease duration, between the

heterogeneous study populations. Researchers should consider the following parameters to help explain or mitigate RNFL thickness discrepancy between OCT studies:

Optic neuritis history

Time since optic neuritis. Longitudinal studies have established that RNFL values often initially increase at optic neuritis presentation, then demonstrate accelerated thinning over the next six months, and finally stabilize thereafter (Costello et al., 2008a; Henderson et al., 2010; Kupersmith et al., 2011). Consequently, optic neuritis studies typically focus on either the acute phase, which encompasses swelling and subsequent axonal loss, or the chronic phase, which is characterized by RNFL thinning at a relatively slower rate. A serial

OCT study by Henderson et al. tested 23 patients regularly after a first episode of unilateral optic neuritis. The speed of RNFL thinning within the first three months predicted RNFL thickness at one year (Henderson et al., 2010). Talman et al. studied one thousand MS patients outside the acute phase. Almost 300 patients returned for follow-up testing at various time points, ranging from six months to five years. After accounting for age, the length of follow-up (defined by time intervals or a continuous variable) influenced the gradual RNFL thinning of the cohort (Talman et al., 2010).

Number of events. Naismith et al. performed a cross-sectional study of 65 patients and clearly illustrated a negative linear association between average RNFL thickness and the number of optic neuritis events (Naismith et al., 2009b). Similarly, Costello et al. demonstrated in 154 affected eyes among MS subtypes, RNFL values were lower in eyes

that were recurrently affected compared to singly affected and in singly affected eyes compared to unaffected eyes (Costello et al., 2010).

Multiple sclerosis history

Disease subtype. Defining MS disease course is challenging due to the complex pathology and lack of biomarkers. Interestingly, studies of RNFL thickness have found differences between the various MS subtypes. Pulicken et al. compared RNFL values between 135 relapsing-remitting patients, 16 secondary progressive patients and 12 primary progressive patients. Compared to controls, RNFL values were most reduced in the secondary progressive patients and least reduced in the relapsing-remitting patients

(Pulicken et al., 2007). In a smaller population that included CIS patients, Costello et al. observed decreased RNFL values in secondary progressive patients relative to relapsing- remitting and in relapsing-remitting patients compared to CIS (Costello et al., 2010).

Disease duration. Disease duration correlations with RNFL will remain less concrete until we can objectively determine when MS begins. An effort by Siger et al., in which the study population consisted of untreated relapsing-remitting MS patients with (n

= 20) and without (n = 31) a history of optic neuritis, found average RNFL thickness of unaffected eyes (both the fellow eyes of unilateral optic neuritis patients and eyes of non- optic neuritis patients), negatively correlated to disease duration (Siger et al., 2008). Other studies of predominately relapsing-remitting patients have reinforced this relationship between disease duration and RNFL loss (Fisher et al., 2006; Sepulcre et al., 2007; Spain et

al., 2009; Costello et al., 2010), while a study of progressive MS patients have found no significant correlation (Henderson et al., 2008).

Demographics

Average RNFL thickness is known to decrease by 0.15 to 0.2 µm per year

(Kanamori et al., 2003; Budenz et al., 2007; Parikh et al., 2007; Bendschneider et al., 2010;

Alasil et al., 2012; Knight et al., 2012). It has also been established that healthy men and women have similar RNFL values (Budenz et al., 2007; Girkin et al., 2011; Alasil et al.,

2012; Knight et al., 2012). Hispanic and Asian ethnicities tend to have thicker average

RNFL, while people of European descent tend to have the thinnest (Girkin et al., 2011;

Alasil et al., 2012; Knight et al., 2012). In strictly an MS population, however, the effects of aging, sex, and ethnicity are underexplored. In a study by Talman et al., MS eyes lost on average 2.0 µm of RNFL thickness per year (Talman et al., 2010), suggesting the RNFL of

MS eyes thins 10 times faster than that of non-MS patients. The authors noted the rate of

RNFL thinning is potentially overestimated because the group of participants that returned for follow-up testing had a greater proportion of optic neuritis-affected eyes than the initial cohort (Talman et al., 2010). This is in contrast to the effects of race and gender on RNFL thinning in MS; MS cohorts are predominately Caucasian females, therefore any differences in these areas may be overlooked. Only recently has a sex difference in RNFL thinning been observed, where the RNFL thickness of the affected eyes was more preserved in women compared to men six months after optic neuritis (Costello et al., 2012;

Burton et al., 2013).

Comorbidities

Finally, identifying coexisting ocular pathology and systemic disease with ocular manifestations is necessary for understanding data gathered via OCT. A comprehensive ophthalmological exam is required to diagnose conditions such as glaucoma or congenital abnormalities of the optic nerve (Sergott et al., 2007). These conditions may better explain an abnormal OCT result and such patients may need to be excluded from study participation. Corrective lens prescription also needs to be addressed. Specifically, as myopia increases, RNFL thickness decreases in the non-temporal quadrants (Leung et al.,

2006; Wang et al., 2011). A refractive error of greater than six dioptres is often used as an exclusion criterion for OCT studies. Furthermore, an ophthalmological exam is essential to exclude optic neuritis mimics including: neurosarcoidosis, Behcet’s disease, and lymphoma.

Lesson 3: optical coherence tomography technology and interpretation continues to evolve

The Stratus time domain (TD) OCT was the first OCT device used in a MS study.

With this device, retinal depth is determined by echo-delay information. The development of the spectral domain (SD; also called Fourier domain) OCT technology allows for the detection of all light echoes simultaneously, advancing two key operations: speed and resolution (Figure 13). Instead of producing 400 A-scans per second, SD-OCT acquires

18000 to 40000 A-scans per second, greatly reducing movement artefacts (Sakata et al.,

2009). Recent technology has an axial resolution of three to six microns compared to approximately 10 µm for prior models (Sakata et al., 2009). Retinal images now show impressive detail and allow for the analysis of additional layers (Wolf-Schnurrbusch et al.,

2009). Today, there are several SD-OCT options, each with additional abilities. The

Spectralis SD-OCT (by Heidelberg), for example, offers an attractive feature for longitudinal monitoring—the ability to pinpoint the location of retina and re-align to it at later exams.

These big gains in technology came at the cost of longitudinal and universal collection. Retinal nerve fiber layer measurements in the Stratus TD-OCT are significantly increased compared to the Cirrus SD-OCT (both by Carl Zeiss Meditec) (Knight et al.,

2009; Vizzeri et al., 2009). Studies have consistently demonstrated that even across SD-

OCT models, measurements are not interchangeable because of altered scan paths and segmentation methodologies (Wolf-Schnurrbusch et al., 2009; Giani et al., 2010; Seibold et al., 2010; Leite et al., 2011; Watson et al., 2011; Patel et al., 2012; Pierro et al., 2012).

Specifically, retinal thickness is calculated from the inner limiting membrane to the retinal pigment epithelium with the Cirrus SD-OCT whereas the Spectralis SD-OCT uses Bruch's membrane as the posterior reference line (Warner et al., 2011). Such variations have thwarted long-term, multicenter studies currently lacking in OCT research. Automated segmentation software that further defines retinal layers is an attractive solution to improve device compatibility and will ultimately enrich our understanding of pathology in MS

(Seigo et al., 2012). With segmentation, Saidha et al. discovered an increase in inner nuclear layer thickness among active MS patients, suggesting inflammation is compartmentalized (Saidha et al., 2013). Thus endeavours around the consequences of changing technology ironically pushes for further scientific advancements and superior interpretation.

Another example of how our interpretation has improved involves identifying the quality of the scan itself. Recently, Tewarie et al. put forth a set of criteria, identified with the acronym “OSCAR IB,” to assist with the quality control of OCT images. The criteria describes seven reasons to reject scans from study interpretation, including: (O) obvious failures (e.g. protocol violation); (S) signal strength less than 15 decibels; (C) de-centration of the scan; (A) algorithm failures (e.g. boundary line errors); (R) retinal pathology other than MS related; (I) poor illumination; and (B) de-centration of the laser beam (Tewarie et al., 2012). The development of the OSCAR IB criteria was an important step for the application of OCT measures in multicenter trials and will need to be revised in conjunction with evolving technology.

Lessons learned from reproductive hormone research

Lesson 4: reproductive hormone research is topical and data must be communicated with caution

In 2002, the Women’s Health Initiative (WHI) trial of hormone therapy in postmenopausal women was discontinued because of the greater than expected potential risk in the treatment arms. The lessons learned with 10 years of hindsight should forever remain in the minds of all researchers in this field. One of the more universal lessons pertains to the way scientific research is communicated.

Hormones are extremely relatable to both young and older women. The majority of women have used hormones for contraceptive or menopausal purposes at least once in their lives (Carney et al., 2006; Mosher and Jones, 2010). In the case of the WHI trial, media

headlines linking estrogen plus progestin use with increased breast cancer risk profoundly changed hormone replacement therapy discussions between patients and doctors. There has been considerable evolution of the WHI message as results from the estrogen-only treatment arm surfaced and variables, including age and years since menopause, were looked at with greater scrutiny. Specifically, women aged 50 to 59 years randomized to receive conjugated equine estrogens experienced cardiovascular benefits (Manson et al.,

2007) and had a reduced incidence of invasive breast cancer (Anderson et al., 2012).

Unfortunately, the first impression is longer lasting and the negative perspective of hormone replacement therapy continues to deter women from this treatment even in cases where the benefits outweigh the risks (Langer et al., 2012).

Lesson 5: hormone type, dose, and route of administration matter

Hormone type

The endless incorporation of new drug generations, modified regimes, and now bioidentical hormones that grew in popularity largely because of the WHI trial makes studying the effects of exogenous hormones a moving target. In animal models of MS, research has found clear distinctions between estradiol compared to ethinylestradiol, and progesterone compared to progestin, yet clinical trials continue to blur the lines. At a minimum, sex hormone research should be categorized as physiological or synthetic. Recently, the FDA approved the first OC preparation containing “natural” estrogen (Natazia, which contains estradiol valerate), marking the beginning of the next generation of combined OCs.

Dosage

Prospective studies of exogenous estradiol or ethinylestradiol will help determine if beneficial thresholds exist in MS. In a randomized trial of either transdermal estradiol (0.05 mg) and oral norethisterone (1.0 mg) or placebo as an adjunct therapy in 200 postmenopausal rheumatoid arthritis patients, women who achieved estradiol levels greater than 100 pmol/L had significant improvements with regards to pain and stiffness (Hall et al., 1994). Comparable to MS, the majority of rheumatoid arthritis patients report an improvement in disease during pregnancy and a worsening postpartum (Barrett et al.,

1999). Pregnancy data in MS suggests third trimester levels substantially lower relapse rate but first trimester changes offer some relapse protection (Confavreux et al., 1998). Assisted reproductive techniques achieve a complicated hormonal milieu that depends on ovarian desensitization and stimulation protocols, but imparts a relatively new and underexplored opportunity to study hormone manipulation (Voskuhl, 2012). In vitro fertilization (IVF) has been associated with a temporary increased relapse rate, particularly in patients for whom IVF failed or in IVF protocols where gonadotrophin releasing hormone agonists were used (Correale et al., 2010; Michel et al., 2012).

Route of administration

Van den Heuvel et al. compared the ethinylestradiol pharmacokinetics of three common hormonal contraceptive agents with different routes of administration—the contraceptive ring (NuvaRing; 0.15 mg ethinylestradiol and 0.120 mg etonogestrel), the transdermal patch (0.20 mg ethinylestradiol and 0.150 mg norelgestromin), and contraceptive pill (0.30 mg ethinylestradiol and 0.150 mg desogestrel) (van den Heuvel et

al., 2005). In this randomized trial, average ethinylestradiol concentrations were lowest for the contraceptive ring (21 pg/mL) and highest for the patch (71 pg/mL), differing from the order expected based on initial concentrations (Figure 14) (van den Heuvel et al., 2005).

The highest peak ethinylestradiol concentrations were seen in the OC group (van den

Heuvel et al., 2005).

Lessons learned from human research

Lesson 6: study design

Observational studies

Randomized controlled trials are the gold standard of study designs to assess clinical efficacy and safety of therapeutic strategies. Observational studies are often a valuable first step and can provide additional information, such as long-term effects, if done properly. The two key issues in the design of observational studies are: the impact of bias and confounding variables.

Bias. In order to prevent bias, careful attention should be given to choice of study population and sources of data (Mayrent, 1987). In the ROC-ON study, selection bias is less of a concern because of the prospective nature and the unlikely possibility that OC status alters the referral process for demyelinating optic neuritis. The effects of nonparticipation and losses to follow-up, on the other hand, will need to be addressed in the future. To prevent misclassification, OC status will be explicitly addressed during physician interviews, and specific OC ingredients will be obtained or verified with pre- existing records. The categorization of “user” or “non-user” will be based on OC use

during the month prior to optic neuritis onset, minimizing any recall bias. Finally visual outcomes and MRI results are objective.

Confounding. Recognizing and controlling confounding variables is essential to the interpretation of all observational studies. Through medical records and questionnaires, we will collect additional data to address this issue in the ROC-ON study. As displayed in

Chapter Four, we anticipate age as a possible confounding variable. Restriction (e.g. limiting inclusion criteria to a narrow age range) and matching (e.g. selecting patients to achieve an equal age distribution between OC users and non-users) are design methods frequently used to control confounding in an observational setting (Mayrent, 1987). More stringent recruitment was not appropriate for the ROC-ON study given the limited number study candidates. Instead, we have opted to control for age and other potential confounders in our analysis through stratified or multivariate techniques.

Recruitment and retention strategies

We employed a number of strategies for patient accrual in the VitaDON and ROC-

ON studies. First and foremost, testing heavily overlapped with standard of care to reduce patient burden. The possibility of an MS diagnosis was often discussed for the first time with the study patients. Too little or too many clinic visits can be anxiety provoking. We found that month three visits were irregular and that appointments between baseline and month six were dictated by the patient’s condition and concerns. For example, cases where vision had not recovered or those with persistent inflammation were followed more closely compared to those patients who had promptly returned to normal. Six-month intervals are

more practical study targets for long-term monitoring of a relatively healthy population.

Our baseline phase was extended from a 30-day period to a 45-day period based on early attempts to enrol within the first month. Lastly, our inclusion and exclusion criteria mirrors the most frequently optic neuritis-affected population—women of reproductive age.

Lesson 7: ethical framework

Institutions across Canada follow the guidelines of the Tri-Council Policy

Statement: Ethical Conduct for Research Involving Humans (TCPS-2). Research involving human participants almost always requires ethics review and approval by a Research Ethics

Board (REB). The process is time-consuming, much of which is dependent on outside factors. There are, however, steps that can be taken to facilitate the submission. For example, a general understanding of the REB’s resources before initiating the application will save time later in the process. A well-planned recruitment strategy is also important.

The REB will look closely at the target population and how participants will be contacted to assess justice and respect for persons, two of the three core principles of the TCPS-2

(Cummings, 2012). Moreover, recruitment is a common hurdle in research studies and a carefully considered approach will facilitate recruitment goals. Finally, the TCPS-2 is subject to diverse interpretations. Specific guidelines are dynamic within the institution and vary between institutions. It is not uncommon for reviewers to ask for clarification or for the application to slightly change between back-to-back submissions.

Discussion of thesis objectives

The first objective of my thesis was to review the literature on the effects of hormonal variations in women with MS, explore the rational for sex hormones as anti- inflammatory and/or neuroprotective therapies in women, and analyze the current approaches to studying certain hormones in MS. Ultimately, this work was the foundation for the ROC-ON study and was a necessary, time-consuming undertaking. The literature review revealed important gaps and inconsistencies in our understanding of oral contraception in MS. Epidemiology studies suggest OC use likely does not influence a women’s ultimate risk of developing MS, but how OC use can alter disease progression is very much unknown. Oral contraceptive use has been associated with more of a benign disease course in early relapsing-remitting MS patients (Sena et al., 2012) and an increased risk of reaching EDSS of 6.0 in progressive patients (D'hooghe et al., 2012). The literature review fortified our efforts towards studying CIS and relapsing-remitting MS patients, and the short-term OC effects. Frank Westheimer, an American chemist, is credited for saying,

“A couple of days in the laboratory can frequently save a couple of months in the library.”

Contrary to his view, I believe a couple months in the library can save a couple of years in the laboratory. This notion is even more pertinent when the laboratory is the clinic.

The second objective was to assist in designing an observational research study that offers new insight into the use of sex hormone therapy in demyelinating conditions.

Through implementing and managing this observational study, as well as analyzing preliminary data, I focused on any foreseeable issues. Study implementation commanded more time than anticipated. This was largely due to delays from CHREB restructuring and

relocating. As of early March 2013, four patients were enrolled into the ROC-ON study.

This is somewhat unexpected and concerning, although no eligible patients declined study participation thus far. In the near future, the research team should meet to discuss our recruitment efforts.

Heterogeneity in MS disease course and disability is arguably the largest limitation of previous work investigating the effects of oral contraception in MS. By focusing on young optic neuritis patients, we reduced the diversity considerably. Our efforts will help overcome current sample size calculation challenges, provide a superior opportunity to explore the effect of age at optic neuritis onset, and contribute to understanding RNFL thickness variability in a homogenous cohort. These are all of growing importance because of the difference found in RNFL thickness between men and women after optic neuritis, and the drive to use RNFL thickness as an outcome for neuroprotection trials.

Having two complementary studies targeting a similar population has been beneficial rather than competitive. The VitaDON study started enrolling patients approximately a year and a half earlier than the ROC-ON study. This staggered start helped establish processes and improve the protocol as we recognized the need to forgo the collection of month three data and increase the baseline window from 30 to 45 days.

Furthermore, as a way to enhance the validity of the ROC-ON study with no extra patient burden, we decided to include information from baseline MRIs ordered for CIS patients.

With similar background information, procedures and risks, both studies could simultaneously be explained to an interested patient.

The final objective was to share general lessons learned. Over the years, persistent themes have emerged from my involvement with research. With these lessons come the following recommendations for future students or researchers: 1) Complement OCT studies with an additional objective outcome measure and adequate patient information, including optic neuritis history (affected eyes, time since last event, and number of events), MS disease history (disease subtype and duration), demographics (age, sex, ethnicity), and comorbidities (including myopia) identified though an ophthalmological exam. 2) When assessing the effects of therapies that are already available, especially widespread hormonal treatments, be cautious in the interpretation to prevent widespread frenzies. 3) Use computerized patient records methodically to assist retrospective queries and to prepare researchers for the next generation of hormones (bioidentical therapies). 4) Resist generalizing estradiol outcomes for all estrogens and equating progesterone to progestin. 5)

Long before beginning an ethics application, become familiar with the guidelines, forms, and templates made available by the local REB. 6) Have a well-planned recruitment strategy. 7) Approach every application with some degree of clean slate and be accessible following the submission for ethics review.

Conclusions

Objective 1: the story behind the study

In earlier work, we found that six months after an optic neuritis event, women had superior RNFL preservation compared to men (Costello et al., 2012). This added to the accumulating evidence of sex differences in MS. As we better understand sex differences in MS, we better appreciate the therapeutic potential of sex hormones in demyelinating

disease. In women, there are several opportunities to observe the effects of sex hormones in

MS. Oral contraceptive use is of particular interest because research on hormonal therapy in MS is lacking and inconsistent. Furthermore, many women are diagnosed with MS in their childbearing years and contraception has immediate relevance. Using optic neuritis as a manifestation of acute demyelination and having already detected sex differences in optic neuritis recovery, we were in a position to unite the question of oral contraception with optic neuritis outcomes. We created a novel project in a disease and patient group that are relatively common.

Objective 2: initial experiences

Conducting a research study requires a great deal of planning. Over a year was devoted to developing a sound proposal and securing funding. Additionally, ethics approval took longer than anticipated. Collaborating between sites grew straightforward and more streamlined over time, as long as proper communication and enthusiasm were maintained. Data management and troubleshooting remain as ongoing activities.

At this time, the limited sample precludes addressing the hypothesis that OC use in our population will be associated with beneficial outcomes. Interim analysis drew attention to outliers in the data, prepared us for age and vitamin D status as confounding variables, and reinforced the screening process.

The study has several limitations including: its observational nature, the variability of hormones, information available for sample size calculations, and the unknown degree

by which the optic nerve reflects overall central nervous system health. By reducing disease heterogeneity and complimenting OCT testing with other outcomes (e.g. VEP and in many cases MRI), we hope to have more confidence in our interpretation and pave the path for future work.

Objective 3: lessons learned

The ease and speed at which quantifiable data is collected from OCT testing outrivals many other imaging techniques. Optical coherence tomography testing is useful for defining damage to the anterior visual pathway and confirming suspicions. Its ability to detect changes over a relatively short amount of time makes OCT a promising tool for disease monitoring. However, as a relatively new and evolving technique for neurological diseases, the importance of these changes needs to be better understood, such as the clinical relevance of temporary RNFL thickness increases during optic neuritis and minor RNFL thickness decreases between follow-up visits. As this field expands and develops, OCT in

MS has the potential to be implicated in clinical diagnosis and prognosis. Until then, OCT testing must be paired with other diagnostic tools and knowledge, particularly demographics and comorbidities.

Hormonal research avenues in MS are attractive but delicate. One of the advantages to studies involving hormones is that many treatments, such as the OC pill, already exist.

Therefore, researchers have an opportunity to explore relationships prior to clinical trials. It also provides a certain level of comfort with drug administration. Hormonal studies are relatable; patients may be more inclined to enroll into and remain in a study that peaks their

interest. As a consequence, research concerning hormones is prone to public misinformation. Trials must be carefully interpreted and communicated. In the wake of controversial research in MS, improving communications between researchers and participants is more imperative than ever. Finally, not all estrogens are equal, nor are natural and synthetic progesterones equivalent, and the differences need to be better defined.

The usefulness of an observational study is dictated by the prevention of selection bias, misclassification, or recall bias, and the use of objective outcomes. Confounding variables are inherent and so a coping strategy (e.g. restriction, matching, multivariate analysis) must be developed into the design. Careful consideration should be given to the population of interest and recruitment strategy. In a disease as diverse and prevalent as MS, future observational studies will continue to play an important role in understanding disease progression, recognizing risk factors, and monitoring therapies.

Future directions

The ROC-ON study will be ongoing for years. In the meantime, more research is needed to expand our understanding of OCT-derived measurements. As suggested by

Sergott et al. and explained in this thesis as a study limitation, clinically relevant variability must be determined for sample size calculations (Sergott et al., 2007). While RNFL thinning is considered a measure of axonal loss, research has yet to determine the degree of

RNFL preservation that can be considered neuroprotective. Similarly, studies suggest that

RNFL loss correlates with global measures of disability (Gordon-Lipkin et al., 2007;

Grazioli et al., 2008; Siger et al., 2008), but it remains uncertain if other areas of the central nervous system are spared when the RNFL thickness is protected.

Another area that needs to be further developed is the possibility of hormonal thresholds or beneficial regimes. Animal experiments of neuroprotection primarily treat with estradiol, although a few have used ethinylestradiol and estrogen receptor ligands. In the literature review, there was only one research study that administered true combined

OCs in EAE. According to these experiments (Arnason and Richman, 1969) and others

(Rosario et al., 2006), the progestin medroxyprogesterone acetate alone may have deleterious affects on neurons. In the same way progesterone and progestin are not equivalent, the negative effects of medroxyprogesterone acetate cannot be generalized to the entire class of progestins (Schumacher et al., 2008). More research is needed to compare the effects of OC agents, with varying ethinylestradiol doses and progestin types, in multiple animal models of MS to help identify potentially harmful or particularly helpful formulas.

Finally, I think a way to improve communication between researchers and participants, and the participant’s overall experience, is to keep patients updated and engaged on their contribution. Newsletters that report research progress are greatly appreciated by participants and double as an excellent retention strategy for long-term studies. Specifically for the ROC-ON study, I would like to provide participants with a letter upon study completion. The message would thank patients and reiterate the goal of the study. Ideally, this could also serve as an opportunity for participants to be added to a

mailing list where published results can be distributed. Showing an extra effort will improve researcher-patient relationships, promote research, and ultimately help give participants the recognition they deserve.

Figure 13 Time domain versus spectral domain optic coherence tomography The comparison of time domain (Stratus, Carl Zeiss Meditec) (bottom) and spectral domain (Spectralis, Heidelberg Engineering) (top) optical coherence tomography (OCT) scans of retinal nerve fiber layer (RNFL) (right) and macular thickness (left) in subjects with multiple sclerosis. In all four panels, OCT scans are accompanied by the corresponding fundus images of the optic nerve head and macula to indicate the scan locations. Modified from (Khanifar et al., 2010).

Figure 14 Mean serum ethinylestradiol concentrations for subjects treated with contraceptive agents through various routes of administration Subjects were treated with the contraceptive ring (n = 8), the transdermal patch (n = 6), and the combined oral contraceptive (COC) pill (n = 8). Modified from (van den Heuvel et al., 2005).

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APPENDIX B: LIST OF COMBINED ORAL CONTRACEPTIVES

Combined oral Progestin Ethinylestradiol Monophasic Day contraceptive brand component dose (µg) (M) vs. supply multiphasic (Ph) dosing Ortho Tri-Cyclen Lo® Norgestimate 25 Ph 21/7 Yasmin® Drospirenone 30 M 21/7 Yaz® Drospirenone 20 M 24/4 Ortho Tri-Cyclen® Norgestimate 35 Ph 21/7 Tri-Sprintec® Norgestimate 35 Ph 21/7 Loestrin FE 1/20® Norethindrone 20 M 21/7 acetate Trinessa® Norgestimate 35 Ph 21/7 Ortho-Cyclen® Norgestimate 35 M 21/7 Microgestin 1.5/30® Norethindrone 30 M 21/7 acetate Aviane® Levonorgestrel 20 M 21/7 LuteraTM Levonorgestrel 20 M 21/7 Seasonale® Levonorgestrel 30 M 84/7 Apri® Desogestrel 30 M 21/7 Sprintec® Norgestimate 35 M 21/7 Lo/Ovral® Norgestrel 30 M 21/7 Kariva® Desogestrel 20 M 23/5 Estrostep FE® Norethindrone 20-35 Ph 21/7 acetate Necon 1/35® Norethindrone 35 M 21/7 Levora® Levonorgestrel 30 M 21/7 Trivora® Levonorgestrel 30-40 Ph 21/7 Ortho-Novum 7/7/7® Norethindrone 35 Ph 21/7 Ortho-Novum 1/35® Norethindrone 35 M 21/7 Alesse® Levonorgestrel 20 M 21/7 Desogen® Desogestrel 30 M 21/7 Ortho-Cept® Desogestrel 30 M 21/7 Mononesessa® Norgestimate 35 M 21/7 Seasonique® Levonorgestrel 30 M 84/7 Zovia 1/50® Ethynodiol 50 M 21/7 diacetate Junel FE 20TM Norethindrone 20 M 21/7 Loestrin 21 1.5/30® Norethindrone 30 M 21/7 acetate Mircette® Desogestrel 20 M 21/2/5 Cryselle® Norgestrel 30 M 21/7 Ovcon 35® Norethindrone 35 M 21/7 160

Combined oral Progestin Ethinylestradiol Monophasic Day contraceptive brand component dose (µg) (M) vs. supply multiphasic (Ph) dosing Levlen® Levonorgestrel 30 M 21/7 Nortrel 28® Norethindrone 35 M 21/7 Oegstrel® Norgestrel 50 M 21/7 Triphasil® Levonorgestrel 30-40 Ph 21/7 Tri-Levlen® Levonorgestrel 30-40 Ph 21/7 Necon 7/7/7® Norethindrone 35 Ph 21/7 Low-Ogestel® Norgestrel 30 M 21/7 Cyclessa® Desogestrel 25 Ph 21/7 Enpresse® Levonorgestrel 30-40 Ph 21/7 Portia® Levonorgestrel 30 M 21/7 Nortrel 7/7/7® Norethindrone 35 Ph 21/7 Femcon FETM Norethindrone 35 M 21/7 Ortho-Novum 1/50® Norethindrone 50* M 21/7 Zovia 1/35E® Ethynodiol 35 M 21/7 diacetate Nordette® Levonorgestrel 30 M 21/7 Norethin 1/35E Norethindrone 35 M 21/7 Loestrin 24FE® Norethindrone 20 M 24/4 acetate Jenest® Norethindrone 35 Ph 21/7 OcellaTM Drospirenone 30 M 21/7 LevliteTM Levonorgestrel 20 M 21/7 Nortrel 0.5/0.035® Norethindrone 35 M 21/7 Ovcon 50® Norethindrone 50 M 21/7 ReclipsenTM Desogestrel 30 M 21/7 Necon 1/50® Norethindrone 50* M 21/7 Demulen 1/35® Ethynodiol 35 M 21/7 diacetate Modicon® Norethindrone 35 M 21/7 Ortho-Novum®† Norethindrone NS M 21/7 Ortho-Novum 10/11® Norethindrone 35 Ph 21/7 KelnorTM Ethynodiol 35 M 21/7 diacetate VelivetTM Desogestrel 25 Ph 21/7 JolessaTM Levonorgestrel 30 M 84/7 Demulen1/50® Ethynodiol 50 M 21/7 diacetate JunelTM Norethindrone 30 M 21/7 Tri-PrevifemTM Norgestimate 35 Ph 21/7 Junel FE 30TM Norethindrone 30 M 21/7

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Combined oral Progestin Ethinylestradiol Monophasic Day contraceptive brand component dose (µg) (M) vs. supply multiphasic (Ph) dosing Norinyl 1+35® Norethindrone 35 M 21/7 Ovral® Norgestrel 50 M 21/7 Nortrel® Norethindrone 35 M 21 Lessina® Levonorgestrel 20 M 21/7 Zenchent® Norethindrone 35 M 21/7 Zovia®† Ethynodiol NS M 21/7 diacetate Microgestin FE 1/20® Norethindrone 20 M 21/7 acetate PrevifemTM Norgestimate 35 M 21/7 Necon 10/11® Norethindrone 35 Ph 21/7 Norinyl 1/50® Norethindrone 50* M 21/7 Junel 20TM Norethindrone 20 M 21/7 BalzivaTM Norethindrone 35 M 21 or 21/7 AzuretteTM Desogestrel 20 M 21/7 Mercilon® Desogestrel 20 M 23/5 Miranova‡ Levonorgestrel 20 M 21/7 Nueva Perla(minulet) ‡ Gestodene 30 M 21/7 Microgynone‡ Levonorgestrel 30 M 21/7 Valette‡ Dienogest 30 M 21/7 Nocicline‡ Norethindrone 30 M 21/7 Gynera‡ Gestodene 30 M 21/7 Modified from (Hall and Trussell, 2012)

*Estrogen component is mestranol

†Specific pill/dosage not specified (NS)

‡Products not available in the United States

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APPENDIX D: CASE REPORT FORMS

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APPENDIX F: THE ROLE OF REPRODUCTIVE HORMONES AND ORAL

CONTRACEPTIVES IN OPTIC NEURITIS (ROC-ON) STUDY PROTOCOL

Study overview

This study will examine the recovery features of optic neuritis in women based on oral contraceptive (OC) use. Reproductive hormones figure heavily into theories of why women develop multiple sclerosis (MS) more often than men, and why women may have milder disease. These theories are supported by evidence from studies of pregnancy, the menstrual cycle and oral contraceptive use in MS, in which certain reproductive hormone states appear to be beneficial in the disease. MS is a complex disease, but the optic nerve, commonly affected in MS, is easily assessed with the advent of optical coherence tomography (OCT) and specifically assessment of the retinal nerve fiber layer (RNFL). We will look at the optic nerve in the acute and chronic phases of optic neuritis to determine if

OC use is associated with less optic nerve injury (i.e. a reduced degree of inflammation and thinning).

Inclusion and exclusion criteria

Inclusion criteria

1. Female gender

2. Aged 18 to 45 years

3. Currently still menstruating

4. Confirmed optic neuritis event within 45 days of onset

5. Either a combined hormonal OC user or a non-user for ≥ 4 weeks.

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Exclusion criteria

1. Non-oral hormonal contraception

2. Pregnancy

3. Any cause of menopause

4. Amenorrhea ≥ 6 months

5. Oophorectomy

6. Hysterectomy

7. Prior optic neuritis in the affected eye

8. Patients with neuromyelitis optica or progressive forms of MS

9. Ophthalmological disorders affecting vision or OCT measures

10. Inability to undergo OCT testing

11. Inability to provide informed consent

Study procedures

Optical coherence tomography

Optical coherence tomography testing will be performed with the Cirrus HD-OCT.

The protocols to be used are macular cube 200 X 200 and optic disc cube 200 X 200. The outcome measures are macular volume, and average and quadrant (superior, inferior, nasal and temporal) RNFL thickness. A trained ophthalmic technologist or trained research assistant/student will perform the testing. Pupils will only be dilated if imaging is impaired by a small pupil size (2 mm). Only OCT scans with signal strength greater than or equal to

7 (maximum 10) will be included in the study.

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Visual evoked potentials

Visual evoked potential (VEP) testing will be performed with the Espion E2

(Diagnosys LLC, Lowell, Massachusetts) system. Pattern VEPs will be obtained with checkerboard stimulation (60, 30 and 15 minutes of arc), subtending 15° visual angle and presented at 2 reversals per second. VEPs will be recorded between 0.1 to 100 Hz bandwidth and 100 sweeps of 250-Msec duration will be averaged. A minimum of 2 recordings for each VEP condition will be acquired. Pattern VEPs will be recorded from the occiput (Oz) referred to linked ears. Subjects will be asked to wear glasses or contact lenses when necessary. Eyes will be individually tested and the unstimulated eye will be covered with an opaque patch. The N70 and P100 components will be identified through visual inspection. A trained electro-diagnostic expert will interpret the results. Amplitudes and latencies (at 30 minutes of arc and the midline active electrode) will be collected.

Visual acuity

High contrast visual acuity will be tested using Early Treatment Diabetic

Retinopathy Study (ETDRS) charts at 4 m (Lighthouse Low Vision Products, Long Island

City, NY). Low contrast visual acuity 2.5% and 1.25% will be tested using Sloan letter charts (Precision Vision, Laselle, IL). Best-corrected visual acuity will be used in all cases.

Expanded Disability Status Scale

The Expanded Disability Status Scale (EDSS) is the standard measure of neurologic impairment in MS (Kurtzke, 1983). This scale measures 7 functional systems: optic, brain stem, pyramidal, cerebellar, sensory, bowel/bladder, cerebral function, and ambulation,

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from which EDSS score is derived. The EDSS scores patients from 0 (no signs) to 10

(death due to MS). Patients will undergo EDSS testing by an accredited physician.

Magnetic resonance imaging

Patients normally undergo magnetic resonance imaging (MRI) brain studies with gadolinium and after a first demyelinating event as part of the diagnostic workup (provided there are no contra-indications). In Southern Alberta, routine MRI studies use a 1.5 Tesla coil, and the “MS protocol” requested typically includes brain axial and sagittal FLAIR,

Axial T2, and Axial T1 pre and post-gadolinium sequences.

Serum hormone testing

All study participants that do not use OCs will have the following serum testing between 5 and 10 days after the start of their menstrual cycle at baseline, month 6, and month 12: estradiol, progesterone, prolactin, total testosterone (TT), and sex hormone binding globulin (SHBG). Total testosterone and SHBG are used to calculate the free androgen index (FAI; TT/SHBG x 100 = FAI), which is a method to compare bioavailable testosterone in our study patients. All patients (OC and non-OC users) will have serum testing for vitamin D (25-hydroxyvitamin D3) levels. If necessary, beta-human chorionic gonadotropin will also be tested at baseline to rule out pregnancy. Testing will be done by

Calgary Laboratory Services®.

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Menstrual/parity history

Reproductive history (and other lifestyle/demographic factors) will be documented at baseline, month 6, and month 12 with a patient questionnaire and chart review. Monthly menstrual diaries will be made available to patients in an effort to more accurately record period frequency between study visits and plan serum testing. Last menstrual period and period frequency during the prior 3 months will also be collected to aid in hormone level interpretation.

Schedule of study activities and evaluations

Baseline Month 6 Month 12 (within 45 days of (+/- 30 days) (+/- 30 days) onset) Informed consent • Review of eligibility • History and chart review • • • Routine medical • • • (e.g. EDSS, baseline MRI) Routine • • • ophthalmological testing (e.g. OCT, VEP, and visual acuity)

Serum hormone testing • • • Demographic/lifestyle • • • questionnaire Menstrual diary • • •

Outcome variables

Primary outcomes

1. The primary outcome is difference in RNFL thickness between OC and non-OC

users in the affected eye at month 6 and month 12 post optic neuritis.

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Secondary/exploratory outcomes

2. Mean RNFL values at baseline between OC and non-OC users

3. Proportion of patients with month 6 and month 12 mean RNFL thickness < 75 µm

between OC and non-OC users

4. High and low contrast visual acuity scores and visual functional status scores at

baseline, months 6, and month 12 between OC and non-OC users

5. Outcomes 1 and 2 for temporal RNFL and macular volume

6. Visual evoked potentials at baseline, months 6, and month 12 between OC and non-

OC users

7. Correlation between mean RNFL values and VEP latency and amplitude at

baseline, months 6, and month 12

8. Presence vs. absence of demyelinating lesions on baseline scan in CIS patients

between OC and non-OC users

9. Presence and number of gadolinium-enhancing lesions on baseline scan in CIS

patients between OC and non-OC users

10. Exploratory assessment of mean RNFL values in non-OC users based on levels of

estradiol, progesterone, prolactin, and testosterone.

Planned analyses

Primary analysis

Patient demographics and clinical characteristics at baseline will be determined for

OC and non-OC users. As existing data regarding the primary outcome (RNFL values 6 to

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12 months after optic neuritis based on OC use at onset) is not available, we are not in a position to make an estimate of effect size and possible differences between groups. We will evaluate the mean value of RNFL at both 6 and 12 months in both groups with 95% confidence intervals with half-widths of about 20%, speaking to the preliminary nature of this research. If the data is normally distributed, we may explore these results using the student t-test, if not, the Mann-Whitney U test.

Secondary/exploratory analyses

We will examine the proportion of patients in each group with 6 and 12 month

RNFL thickness < 75 µm using Chi-square or Fisher exact testing. Descriptive statistics describing the other secondary outcomes of interest in the study including means and proportions (with 95% confidence intervals) will be generated.

Ethical considerations

Ethical conduct of the study

The study will be conducted in accordance with the ethical principles of the

Declaration of Helsinki and in the agreement with ICH-GCP (International Conference on

Harmonisation-Good Clinical Practice) guidelines. All study personnel have ICH-GCP training.

Patient information and consent

The investigator and/or delegate will explain the nature of the study, its purpose, associated procedures, the expected duration, and the potential benefits and risks of

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participation to each patient prior to her entry into the study before any examinations and procedures are performed for the purpose of selection for the study. Each patient will have ample opportunity to ask questions. The patient will be informed of their right to withdraw from the study at any time without any disadvantage or explanation required. All patients will be informed throughout the study if relevant changes medical practice occur.

Patient confidentiality

Information obtained in this study that is part of the routine patient health record is already confidential, and for review only by the patient’s caregivers and study personnel.

All study documents will be kept in a locked file cabinet in a locked research office at our site only for review by the study research team. All non-routine bloodwork obtained will be assigned an anonymized, non-identifiable patient code that will be known only to study personnel. The use of these samples will occur only after informed consent from patients is obtained. Digital databases and files are encrypted and password protected.

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APPENDIX G: HORMONE REFERENCE INTERVALS

Hormone Reference interval for premenopausal women* Beta human chorionic gonadotropin (BHCG) Non pregnant: 0 - 5 IU/L Estradiol (E2) Follicular: 90 - 700 pmol/L Luteal: 150 - 950 pmol/L Mid-cycle: 250 - 1500 pmol/L Progesterone (P4) Follicular: 0.0 - 5.0 nmol/L Luteal: 13.0 - 108.0 nmol/L Prolactin (PRO) 0 - 25 mg/L Sex hormone binding globulin (SHBG) 20 - 100 nmol/L Total testosterone (TT) 0.5 - 3.0 nmol/L 25-hydroxyvitamin D3 (VD) Severe deficiency: <25 nmol/L Moderate-mild deficiency: 25 - 80 nmol/L Optimum levels: 80 - 200 nmol/L Toxicity possible: >250 nmol/L *According to Calgary Laboratory Services®

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