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Bladder and bowel dysfunction in children : an investigation of cardiac autonomic nervous system activity… Fazeli, Mir Sohail 2017

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BLADDER AND BOWEL DYSFUNCTION IN CHILDREN: AN INVESTIGATION OF CARDIAC AUTONOMIC NERVOUS SYSTEM ACTIVITY AND RELATED THERAPIES   by Mir Sohail Fazeli  M.D., The University of Najafabad, 2006  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  October 2017  © Mir Sohail Fazeli, 2017  ii  Abstract Bladder and bowel dysfunction (BBD) consists of a prevalent group of conditions presenting mainly with lower urinary tract symptoms. The autonomic nervous system (ANS), comprising mainly of the sympathetic (SNS) and parasympathetic nervous systems (PNS), regulates lower urinary tract function. ANS role in the pathogenesis of BBD in not known. BBD management initiates with education and life-style modification (standard urotherapy or SU). Additionally, adjunctive treatments may be administered. Current treatments are often partially effective, costly, and sometimes associated with side-effects.  The overall goal of this dissertation was to describe cardiac ANS activity in children with BBD before receiving any treatments and subsequently after current and proposed adjunctive therapies.  Study 1: Forty children with BBD were compared with nineteen healthy controls for the activity of ANS measured via spectral analysis of heart rate variability (HRV). Results demonstrated a significantly lower overall ANS and PNS activity at baseline in BBD group. Study 2: The efficacy of pelvic floor biofeedback in children with BBD was assessed via a systematic literature review and meta-analysis of randomized controlled trials (RCT) which showed no improvement in resolution of urinary incontinence after 6 months of biofeedback compared to other adjunctive treatments. iii  Study 3: Ten children from Study 1 with overactive bladder (OAB) were followed receiving antimuscarinics for approximately 3 months. Results showed a significant reduction in the PNS activity after treatment accompanied by a failure to achieve complete symptom resolution. Study 4: Twenty-three children with BBD were randomized to SU plus diaphragmatic breathing (DB) or SU only in a pilot trial. Results showed a low overall feasibility for proposing DB in controlling symptoms mainly due to low adherence during an approximately 3 months of follow-up. Positive correlations were found between number of DB practices and change from baseline in overall ANS and PNS activity.  In summary, BBD was associated with a significantly low overall ANS and PNS activity. There is lack of evidence supporting relative treatment efficacy of biofeedback. Use of antimuscarinics in OAB was associated with further reduction in cardiac PNS activity and the pilot study showed low overall feasibility for proposing DB for three months.   iv  Lay Summary The autonomic nervous system (ANS), assures a smooth functioning of the lower urinary tract. The main goal of this dissertation was to understand the ANS role and activity in children with Bladder and Bowel Dysfunction (BBD) before and after receiving standard care and different types of interventions. Findings demonstrate that children with BBD have significantly weaker overall ANS activity compared to otherwise healthy children. A systematic review of published evidence did not confirm the efficacy of biofeedback for children with BBD. Use of oxybutynin, a recommended drug for overactive bladder, resulted in further reduction of the parasympathetic activity and generated side effects. Finally, the feasibility of a trial to assess diaphragmatic breathing (DB) to control symptoms of BBD was limited due to low adherence to DB practice for approximately 3 months. Overall, the projects comprising this dissertation demonstrate the importance of the ANS in the management of BBD in children.  v  Preface This certifies that the work presented in this dissertation is conceived, conducted, and written by Mir Sohail Fazeli. All studies were conducted after receiving approval from the University of British Columbia Research Ethics Board (Certificate No.: H12-03269 and H14-03205). Chapters 2 and 3 of the thesis are each composed of manuscripts, which have been published in peer-reviewed journals. I was responsible for developing the study proposal, conceptual framework, and analytic approaches for all analyses. For this, I received assistance from my thesis supervisor, Dr. Jean-Paul Collet, and my thesis committee members, Dr. Kourosh Afshar (co-supervisor), Dr. Rollin Brant, and Dr. Timothy Oberlander. I conducted all statistical analyses and wrote the first draft of all manuscripts. My supervisor, and thesis committee members made contributions to the study design, analysis and interpretation of data and revised each article for intellectual content. My contribution was 100% for each manuscript of this dissertation. CHAPTER 2. A version of this chapter has been published in a peer-reviewed journal. Fazeli MS, Collet JP, MacNeily AE, Afshar K. Cardiac Autonomic Nervous System Activity in Children with Bladder and Bowel Dysfunction. J Urol. 2016 Apr;195(4 Pt 2):1245-9. I developed the study idea, designed the experiments, collected all the data, performed all the statistical analyses for this study, and wrote the manuscript. Dr. Kourosh Afshar helped develop the study idea, helped with patient recruitment and data collection, provided clinical insight and support, helped with interpretation of the results, and revised the manuscript. Dr. Jean-Paul Collet helped develop the idea, supervised the data analyses, helped design the experiments, provided technical and methodological support, helped interpret the study results, and revised the vi  manuscript. Dr. Andrew E. MacNeily provided intellectual input to the study, helped with data collection, and revised the manuscript. Masoud Pourrahmat helped with data entry.  CHAPTER 3. A version of this chapter has been published in a peer-reviewed journal. Fazeli MS, Lin Y, Nikoo N, Jaggumantri S, Collet JP, Afshar K. Biofeedback for Nonneuropathic Daytime Voiding Disorders in Children: A Systematic Review and Meta-Analysis of Randomized Controlled Trials. J Urol. 2015 Jan;193(1):274-9. I developed the study idea, designed the systematic literature review protocol, performed the review, performed all the statistical analyses for this study, and wrote the manuscript. Dr. Kourosh Afshar helped develop the study idea, provided clinical insight and support, and revised the manuscript. Dr. Jean-Paul Collet helped develop the study idea, provided methodological support, and revised the manuscript. Dr. Nooshin Nikoo, Dr. Yiqun Lin, and Sravan Jaggumantri performed the review with me and revised the manuscript.  CHAPTER 4. This chapter is based on a work conducted at Urology department of BC Children’s Hospital. I developed the study idea, designed the experiments, collected all the data, and performed all the statistical analyses for this study. Dr. Jean-Paul Collet helped develop the idea, supervised the data analyses, and provided technical and methodological support. Dr. Kourosh Afshar helped develop the study idea and data collection, provided clinical insight and support, and helped with interpretation of the results. Masoud Pourrahmat helped with data entry and analysis.  CHAPTER 5. This chapter is based on a work conducted in Urology department of BC Children’s Hospital. I developed the study idea, designed the experiments, collected all the data, and performed all the statistical analyses for this study. Dr. Jean-Paul Collet helped develop the vii  idea, supervised the data analyses, provided technical and methodological support, and helped interpret the results. Dr. Kourosh Afshar helped develop the study idea and data collection, and provided clinical insight and support. Masoud Pourrahmat helped with data entry and analysis. Dr. Mojdeh Sharaf and Manoocher M. Civi helped partly with data collection.  viii  Table of Contents Abstract .......................................................................................................................................... ii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ....................................................................................................................... viii List of Tables ................................................................................................................................ xi List of Figures ............................................................................................................................. xiii List of Abbreviations ................................................................................................................. xiv Glossary ...................................................................................................................................... xvi Acknowledgements ................................................................................................................... xvii Dedication ................................................................................................................................... xix Chapter 1: Introduction ................................................................................................................1 1.1 Bladder and Bowel Dysfunction (BBD) in Children ...................................................... 1 1.2 Autonomic Nervous System in Normal and Abnormal Bladder Functions ................... 7 1.2.1 Measurement of the Cardiac ANS Activity by Means of Spectral Analysis of Heart Rate Variability (HRV) and Impedance Cardiography (ICG) .................................... 12 1.3 Study Rationale ............................................................................................................. 16 1.4 Dissertation Objectives ................................................................................................. 16 1.5 Dissertation Structure.................................................................................................... 17 Chapter 2: Cardiac Autonomic Nervous System Activity in Children with Bladder and Bowel Dysfunction .......................................................................................................................19 2.1 Synopsis ........................................................................................................................ 19 2.2 Background and Objectives .......................................................................................... 22 ix  2.3 Methods......................................................................................................................... 24 2.4 Results ........................................................................................................................... 28 2.5 Discussion ..................................................................................................................... 31 Chapter 3: Biofeedback for Bladder and Bowel Dysfunction in Children: A Systematic Review and Meta-analysis of Randomized Controlled Trials .................................................39 3.1 Synopsis ........................................................................................................................ 39 3.2 Background and Objectives .......................................................................................... 41 3.3 Methods......................................................................................................................... 43 3.4 Results ........................................................................................................................... 48 3.5 Discussion ..................................................................................................................... 61 Chapter 4: The Effects of Anti-muscarinic Agents on the Activity of the Cardiac Autonomic Nervous System in Children with Functional Overactive Bladder (A Subtype of BBD) ..............................................................................................................................................66 4.1 Synopsis ........................................................................................................................ 66 4.2 Background and Objectives .......................................................................................... 69 4.3 Methods......................................................................................................................... 74 4.4 Results ........................................................................................................................... 77 4.5 Discussion ..................................................................................................................... 81 Chapter 5: Diaphragmatic Breathing as an Adjunctive Therapy in the Management of Children with Bladder and Bowel Disorders: A Pilot Randomized Clinical Trial................85 5.1 Synopsis ........................................................................................................................ 85 5.2 Background and Objectives .......................................................................................... 88 5.3 Methods......................................................................................................................... 99 x  5.4 Results ......................................................................................................................... 107 5.5 Discussion ................................................................................................................... 127 Chapter 6: Conclusions .............................................................................................................142 6.1 Summary of Findings and Contributions to the Scientific Knowledge ...................... 142 6.2 Strengths and Limitations ........................................................................................... 144 6.3 Significance and Future Research Directions ............................................................. 147 6.4 Concluding Remarks ................................................................................................... 149 References ...................................................................................................................................151 Appendices ..................................................................................................................................166  Dysfunctional Voiding Questionnaire ................................................................ 166  Pediatric Incontinence Quality of Life Questionnaire (PinQ) ............................ 168  Protocol for Recording Heart Rate Variability ................................................... 170  Search Strategies for the Systematic Review ..................................................... 176  Diaphragmatic Breathing Figures ....................................................................... 182  Treatment Acceptability Questionnaire (TAQ) .................................................. 184 xi List of Tables Table 2.1 Table of baseline characteristics. .................................................................................. 29 Table 2.2 Autonomic and uroflowmetric profile at baseline. ....................................................... 30 Table 2.3 Relative change in autonomic profile from baseline to voiding ................................... 31  Table 3.1 Study selection criteria for systematic literature review (PICO) .................................. 44 Table 3.2 Characteristics of included studies. .............................................................................. 51 Table 3.3 Summary of findings. ................................................................................................... 58  Table 4.1 Table of baseline characteristics ................................................................................... 77 Table 4.2 Symptoms reported by BBD questionnaire at baseline and post-treatment. ................ 78 Table 4.3 Autonomic and uroflowmetric profile at baseline and post-treatment. ........................ 80 Table 4.4 Relative change in autonomic profile from filling to voiding phase during baseline and after receiving full treatment of oxybutynin. ................................................................................ 81  Table 5.1 Table of baseline characteristics. ................................................................................ 109 Table 5.2 Table of study treatment adherence and follow-up .................................................... 113 Table 5.3 Intention-to-treat analyses........................................................................................... 119 Table 5.4 Per-protocol analyses. ................................................................................................. 123 Table 5.5 Correlation between the total number of DB practice and the relative change (in % except otherwise specified) from Visit 1 to Visit 2 in the other outcome measures. ................. 126  Table D.1 MEDLINE® search strategy for the systematic review and results. ......................... 176 xii  Table D.2 Embase search strategy for the systematic review and results. ................................. 178 Table D. 3 Cochrane Central Register of Controlled Trials (CENTRAL) search strategy for the systematic review and results...................................................................................................... 180 Table D.4 Excluded studies and reasons for exclusion............................................................... 181  xiii  List of Figures Figure 1.1 Schematic depictions of the innervation and neural circuits in LUT .......................... 10 Figure 1.2 Schematic depictions of the innervation and neural pathways in LUT during storage (a) and voiding (b) phases of the bladder function ....................................................................... 11 Figure 3.1 Modified PRISMA flow chart of search results and publication selection. ................ 49 Figure 3.2 Review authors' judgments about each risk of bias item presented as percentages across all included studies. ............................................................................................................ 55 Figure 3.3 Review authors' judgements about each risk of bias item for each included study. Green = low risk, yellow = unclear risk, red = high risk. ............................................................. 56 Figure 3.4 Forest plot of comparison between biofeedback and standard treatment, outcome resolved incontinence (OR). M-H, Mantel-Haenszel test. ............................................................ 59 Figure 3.5 Forest plot of comparison between biofeedback and standard treatment, outcome resolved incontinence (RD). M-H, Mantel-Haenszel test. ............................................................ 59 Figure 3.6 Forest plot of comparison between biofeedback and standard treatment, outcome reduction in incidence of UTI. M-H, Mantel-Haenszel test. ........................................................ 60 Figure 3.7 Forest plot of comparison between biofeedback and standard treatment, outcome Q-max. M-H, Mantel-Haenszel test. ................................................................................................. 61 Figure 5.1 Study flow diagram ................................................................................................... 111 Figure E.1 Diaphragmatic breathing. .......................................................................................... 182 Figure E.2 Diaphragmatic breathing – continued. ...................................................................... 183    xiv  List of Abbreviations ADHD Attention Deficit Hyperactivity Disorder ANS Autonomic Nervous System BBD Bladder and Bowel Dysfunction CENTRAL Cochrane Central Register of Controlled Trials CI Confidence Interval CNS Central Nervous System CONSORT Consolidated Standards of Reporting Trials DB Diaphragmatic Breathing DBP Diastolic Blood Pressure EMG Electromyography FDA Food and Drug Administration HF High Frequency HRV Heart Rate Variability ICCS International Children’s Continence Society ICG Impedance Cardiography IHD Ischemic Heart Disease ITT Intention-to-treat LF Low Frequency LUT Lower Urinary Tract MRI Magnetic Resonance Imaging NVD Nonneuropathic Voiding Disorders xv  OAB Overactive Bladder OR Odds Ratio PEP Pre-ejection Period PFM Pelvic Floor Muscle Retraining PNS Parasympathetic Nervous System PP Per-protocol PSD Power Spectral Density analysis PVR Post-void residual volume RCT Randomized Controlled Trials RD Risk Difference REDCap Research Electronic Data Capture SBP Systolic Blood Pressure SD Standard Deviation SNS Sympathetic Nervous System T2 National Center for Telehealth & Technology TP Total Power UTI Urinary Tract Infection VLF Very Low Frequency VUR Vesicoureteral Reflux WHR Waist-hip Ratio  xvi  Glossary Decreased voiding frequency in children – Voiding ≤ 3 times per day. Dysuria – Sensation of burning or discomfort during voiding. Encopresis – When soiling of the underwear with stool happens in children who are past the age of toilet training. Enuresis – When intermittent incontinence happens during sleeping periods. Hesitancy – Difficulty to initiate voiding when the child feels ready to void. Incontinence (Urinary Incontinence) – Involuntary leakage of urine. Increased voiding frequency in children – Voiding ≥ 8 times per day. Intermittency – Discontinuous micturition representing by several distinct stops and start spurts. Nocturia – Waking at night to void. Post-micturition dribble – Involuntary leakage of urine immediately after voiding has finished. Q-Average – Average urinary flow rate. Q-Max – Maximum urinary flow rate. Straining – Making an intense effort by increasing intra-abdominal pressure in order to start and maintain voiding. Stress urinary incontinence (Stress incontinence) – Involuntary leakage of small amounts of urine with physical movement or activity such as coughing, or sneezing. Urgency – Sudden and unexpected experience of an immediate and compelling need to void. Urgency incontinence – Complaint of involuntary loss of urine associated with urgency.    xvii  Acknowledgements Firstly, I would like to acknowledge my supervisor, Dr. Jean-Paul Collet, for his exceptional guidance, knowledge, patience and support throughout my time as his student. Dr. Collet has set an example of excellence as a researcher, mentor and instructor. Special thanks are extended to Dr. Kourosh Afshar, my co-supervisor, for his outstanding mentorship, knowledge, expertise, support, and willingness to offer suggestions on this work. I would also like to express my gratitude to my supervisory committee members, Dr. Rollin Brant and Dr. Timothy Oberlander, for their feedback, direction and assistance when needed. Special thanks to my fellow colleague, collaborator, and dear friend, Masoud Pourrahmat, for always being ready to lend a hand and for having the courage to comment, question and improve this work.  To the patients and families from the urology department at BC Children’s Hospital who participated in the studies we conducted, thank you for your patience, time and readiness to participate in our studies. Also, I would like to extend a warm thank you to the staff and colleagues at the Urology Clinic, for their support during the study process and all the faculty and staff in the Department of Experimental Medicine at the University of British Columbia for giving me the privilege and opportunity to complete my doctoral degree. To my wife, Anousheh, I owe you endless gratitude for the love, patience and encouragement you have offered me specifically over the past 7 years – I could not have done it without you.  To my parents, Nina and Ali, thank you for your unconditional love, for being the wonderful examples you are and for always pushing me to do more with my studies, career, and life. To my oldest best friend and brother, Mirfarhang, thank you for always being there for me, standing by xviii  me, loving me, and supporting me throughout my life. To my lovely sister, Afsoon, thank you for your kind love and support.  Finally, this work was supported by the iACT Clinical Investigator Trainee Seed grant, Canadian Urological Association together with Astellas Pharma Canada Inc. grant, and The Sullivan Urology Research, Education and Development Foundation grant. I therefore would like to use this opportunity to thank them for supporting my ideas and work.  xix  Dedication This dissertation is dedicated to my beautiful, loving and understanding wife, Anousheh, and to my always encouraging, and supportive parents Nina and Ali, my extremely supportive brother Mirfarhang, and lovely sister Afsoon.  This work is also dedicated to all children and patients with BBD; thank you for participating in these studies, being patient with me and letting me be a part of your lives during the process of my studies. 1  Chapter 1: Introduction  1.1 Bladder and Bowel Dysfunction (BBD) in Children Disorders of the lower urinary tract (LUT) and bowel in pediatric population can be classified as either a) Organic or Neuropathic with an underlying disease process which can be neurogenic or structural in nature; or b) Functional or Nonneuropathic with no anatomic and/or neurologic lesions identified.(1, 2)  Functional daytime disorders of the lower urinary tract and bowel comprise a heterogeneous group of syndromes(3) collectively known as Bladder and Bowel Dysfunction (BBD).(4) The term “BBD” does not explain the pathogenesis but is rather a comprehensive descriptive term working as an “umbrella” to cover the concomitant dysfunction of the bladder and bowel due to their close functional relationship which is widely established. The International Children’s Continence Society (ICCS) has recently published a classification with a standardized set of terminology recommending the use of the term “BBD” as well as subcategorizing BBD into lower urinary tract dysfunction (LUT dysfunction) and bowel dysfunction in the absence of a combined bladder and bowel disturbance.(5) My work is more focused on the urinary expression of BBD symptoms which are the chief complaints that lead to frequent referrals of children with BBD to the pediatric urology department. Having said that, my work does not exclude reporting of the co-existing bowel symptoms of BBD patients as many of these symptoms are caused in conjunction with (or as a result of) the LUT dysfunction.   Children with BBD present mainly with LUT symptoms which are variable based on their relation to the storage and/or voiding phase of bladder function. These symptoms include 2  increased or decreased voiding frequency, incontinence, enuresis, nocturia, hesitancy, straining, intermittency, dysuria, feeling of incomplete emptying, and post-micturition dribbling (please see glossary of terms for the definition of these symptoms).(5) These symptoms should not be rigidly viewed as separate and distinct symptoms because one type of bladder dysfunction may often progress with time and unperceivably evolve into another without a sharp distinction between the different stages.(2) While LUT symptoms are the main symptoms seen in children with BBD, some of the patients may also experience co-existing bowel symptoms such as constipation or fecal soiling which are mainly caused because of the inappropriate compensatory postponement of defecation to avoid initial LUT symptoms like incontinence.(6, 7) These bowel symptoms and their relationship with overactive bladder (a subtype of BBD) will be more described in Chapter 4 of this dissertation.    Often, BBD is also associated with recurrent urinary tract infections (UTIs) as well as vesicoureteral reflux (VUR).(8, 9) The prevalence of UTI and VUR in children with voiding dysfunction has been shown through urodynamic studies with UTIs identified in 34% of girls and 5% of boys with urinary incontinence(10) and in 16% of children with previous and 20% with current daytime wetting.(11) VUR was also seen in 18.2% of children with urge syndrome (a subtype of BBD).(10)  Epidemiology: the epidemiological studies mostly investigate the prevalence of urinary incontinence as the main presenting symptom of BBD and rarely focus on fecal symptoms. According to ICD-10 and DSM-V criteria and definitions, for the symptoms of incontinence to be termed a condition the child has to have at least 5 years of age and frequency of incontinence needs to occur at least once per month for a minimum duration of 3 months.(5) From studies 3  investigating the prevalence of BBD, a large longitudinal population based study consisting of a cohort of nearly 11,000 children in the United Kingdom investigated gender specific rates of daytime wetting and soiling (reported by parents) in children between the ages of 4.5 to 9.5 years.(12) The authors of this study reported the rate of daytime wetting to be 10.5% in boys and 11% in girls at the age of 4.5 year, and 2.2% in boys and 3.2% in girls at the age of 9.5 years. Consecutively, daytime fecal soiling was reported to be 6% in boys and 3.9% in girls at the age of 4.5 years, and 3.6% in boys and 2.1% in girls at the age of 9.5 years.(12) Another large cross-sectional study has reported the prevalence of daytime incontinence at the age of 7 years to be 6% among females and 3.8% among males.(13) Also, a 6-year report (2002 to 2007 merged) of the nationally representative number of outpatient hospital or physician office appointments in the Medicare/Medicaid system revealed a staggering number of over 2.6 million visits made for pediatric urinary incontinence listed as any diagnosis.(14)   It has also been shown through a number of cohort and case-control studies that children with BBD have a good chance of becoming adults who continue to have problems both with their LUT function as well as their sexual performance.(15-17) Furthermore, studies have shown the cost of urinary incontinence management for adults in the United States to have increased from $3.94 billion in 1984 to $19.5 billion per year in 2004.(18)   Diagnosis: The hallmark of diagnostic tools for evaluation of a child with BBD is a thorough history and physical examination.(5) During this evaluation, the physicians are also advised to closely observe the child for any suspicious holding maneuvers, signs of urgency, as well as any behavioral issues that may exist. In addition, the physician may ask for documentation of the bladder and bowel function in order to record number of voiding episodes per day, incontinence 4  episodes, as well as approximate urine volume measurements through bladder and bowel diaries.(19) Other non-invasive tools used in clinics are validated and reliable questionnaires to quantify severity of LUTS and bowel symptoms,(20, 21) and the emotional impact of BBD on the child’s quality of life.(22) Uroflow study is another useful tool which facilitates the diagnosis and screening of children with BBD by measuring the flow rate, volume voided, voiding time, pattern of voiding during urination, and post-void residual (PVR).(19) Uroflow studies are considered very useful for revealing LUT dysfunction in children due to the least invasiveness nature of the method.(23, 24)   Etiology: The etiology of BBD has mostly been discussed in the context of overactive bladder (OAB), which is one of the most prevalent subtypes of BBD. OAB is not well understood and several different theories have previously been proposed for its etiopathogenesis. They vary from associating dysfunctional elimination syndrome (generic old term used for LUT dysfunction of BBD patients) to a congenital urethral stricture that obstructs urine flow, completely ignoring the possible bowel dysfunction that sometimes accompanies BBD,(25) to non-physiologic contraction of the pelvic floor during elimination, thought to be a continuation of the repeated habit of children to delay or inhibit micturition/defecation,(26) however these theories still remain a matter of debate.(27) The predominant theory behind the cause of OAB is focused on the LUT itself, and thought to be due to uninhibited detrusor contractions.(15) Maturational delays in adjusting bladder sphincter coordination during voiding leads to detrusor overactivity, which meets voluntary external urethral sphincter contractions and leads to increased intravesical pressure. Symptoms of increased intravesical pressure include urgency and urge incontinence. 5  Continuous uninhibited detrusor contractions meeting a tightened sphincter causes bladder muscle hypertrophy leading to loss of bladder functionality and worsening of symptoms.(15)  Recently, a more neurocentric approach has emerged from epidemiological and brain imaging studies linking cerebral defects with functional urinary problems.(28) Patients with disorders such as anxiety, depression, and schizophrenia have been found to display cerebral defects around the anterior cingulate gyrus (ACG) and the prefrontal cortex (PFC), which is the same area where defects are seen in patients with urgency and urge incontinence. It has been suggested that the ACG and the PFC play major roles in processing information from the bladder, and that inability to process these signals could be the cause of OAB.(28) Management: The basic conservative treatment strategy for children with BBD starts with urotherapy. This nonpharmacological treatment is based on the concept of educating the patients and their families on the normal and abnormal LUT and bowel functions and training them on resolving BBD by adopting proper voiding and bowel habits as well as modifying their lifestyle by changing their diet and fluid intake.(29) During this intervention, the caregiver will also provide the patients with bladder and bowel diaries and follow-up with them regularly.   Other treatment modalities may also be used in addition to standard urotherapy as follows:(29) - Alarm treatment: This therapy can be used during the day but is mostly used during night with a minimal treatment duration of 3 months and is based on the use of an electronic device that senses moisture on the child’s underwear or a pad and produces a strong acoustic signal to create a response in the child to wake-up and go to the bathroom.   6  - Pelvic floor muscle retraining (Biofeedback): This method has been used in various settings to treat acquired behavioral dysfunctions through visualizing the physiological processes, increasing awareness, and developing a better voluntary control. In case of children with BBD, biofeedback helps the patients learn about the physiological activity occurring during the voiding and filling phases through the use of audio and visual aids and obtain voluntary control over pelvic floor muscles during voiding.     - Pharmacological therapy: If the above interventions fail in achieving continence, pharmacological therapies are introduced. The main drugs that are used for this purpose are anti-muscarinic (anticholinergic) agents including oybutynin, propiverine, tolterodin, and solifenacin of which oxybutynin is the most widely used and is approved by the U.S. Food and Drug Administration (FDA) for use in children with overactive bladder (OAB – a subtype of BBD). As of January 10, 2017, propiverine has also been approved by Health Canada for use in both adult and pediatric patients with OAB.  - Neuromodulation: This form of therapy claims to reduce LUT symptoms or restores LUT function based on the concept of activating neural structures of the central nervous system through central and/or peripheral electrical stimulation which in turn may modulate the innervation of the bladder.   - Botulin Toxin A (BTA):  BTA is neurotoxic protein that is produced by the bacterium Clostridium botulinum and is introduced into the bladder detrusor muscle in form of multiple injections (20-50) during cystoscopy procedure. BTA is used as the third-line 7  treatment option in children with OAB when all previous procedures fail to achieve continence. BTA injection can cause bladder smooth muscle relaxation through inhibition of acetylcholine release at the presynaptic cholinergic junction. When evaluating children with BBD the treating physician should also look for any signs of co-morbid behavioral or psychiatric disorder. In case of diagnosing a psychiatric/psychologic co-morbidity the care provider should follow with full psychiatric/psychologic assessment and if necessary initiate the treatment of the co-morbidity in addition to any specific treatment of BBD since behavioral problems may affect both compliance and motivation of BBD therapy.(30) A cohort study analyzing large databases in Germany assessing the percentage of patients receiving different treatment modalities for 3,188 outpatient children with functional urinary incontinence has recently published its results.(31) According to this study, an approximately 78% of the population under investigation were not prescribed any specific treatments for their urinary incontinence. The results of this study show that the current treatment modalities only partially comply with the current practice for treating children with BBD. This implies that the efficacy of current treatment modalities for urinary incontinence in childcare is still under question. 1.2 Autonomic Nervous System in Normal and Abnormal Bladder Functions Understanding the anatomy and physiology of normal micturition may provide us with some clues in investigating the underlying causes of urinary incontinence as the most prominent symptom of BBD.  8  LUT is made up of the bladder and the urethra that together form a functional unit for the urine storage and voiding process. The detrusor is a smooth muscle that forms the bulk of the bladder wall and by contraction generates pressure and forces the urine out through the urethra. Detrusor is innervated by fibers from both the sympathetic and parasympathetic branches of the autonomic nervous system.(32)  The Autonomic Nervous System (ANS) is an ancient part of our nervous system that constantly regulates and controls all unconscious bodily functions, including heart rate, blood pressure, temperature regulation, bladder function, gastrointestinal secretion, immune, metabolic and endocrine systems based on signals it receives from our central nervous system (CNS). The ANS is composed of two primary components; 1) the sympathetic nervous system (SNS): also referred to as the “catabolic nervous system” which is in charge of “fight or flight” response. SNS activity  results in an increased utilization of nutrients and hormones coincident with a general increased tissue destruction, and 2) the parasympathetic nervous system (PNS): also referred to as the “anabolic nervous system” is in charge of digestion, elimination and regulation of the body repair and immune system mostly during rest.(33)  The SNS and PNS have constant and complex interactions in order to keep the body functioning in a balanced (homeostatic) state. When homeostasis is challenged by internal or external stressors, the balance between SNS and PNS branches can be disrupted. This state of threatened homeostasis is called ‘stress’.(34) In situations of persistent and uncontrolled stress, this state of imbalance can become prolonged and lead to a disease condition.(35) Micturition control in healthy children and adults is organized as a switch-like activity which involves complex interactions between the autonomic and somatic nervous systems that control 9  the muscles of the LUT. Urination is indeed a voluntary act that is affected by learned behavior and physiology. This is strikingly contrary to the tonic neural control characteristic of most visceral organs. The degree of bladder fullness and sensations is matched with an individual’s social situations and emotional state, allowing for micturition to be delayed, or even accelerated.(36)  The elimination of urine from the LUT is performed at timely intervals following these two phases:(32, 37) 1) Storage phase: which is controlled centrally by a storage center within the rostral pons (also called L-center). During the storage phase, SNS outflow in the hypogastric nerve is dominant and acts via β3 receptors on detrusor muscle to inhibit its contraction while it simultaneously stimulates α-adrenergic receptors at bladder neck that causes an increase in outlet resistance. Also, an excitatory pudendal (somatic) outflow to the external urethral sphincter and a direct inhibition of detrusor motor neurons by sacral spinal cord happens during this phase (Figure 1.1 and Figure 1.2 ). 2) Voiding phase: which is centrally controlled by the micturition center in the rostral pons (also called M-center). During this phase, PNS outflow in the pelvic nerve is dominant and acts via M3 receptors on detrusor and urethral smooth muscles, SNS and pudendal nerves are inhibited and as a result the detrusor is contracted, internal and external sphincters are relaxed and voiding takes place (Figure 1.2 ).   10  Figure 1.1 Schematic depictions of the innervation and neural circuits in LUT        Source: Fowler, C. J., Griffiths D., and de Groat W.C. (2008). "The neural control of micturition." Nature reviews. Neuroscience 9(6): 453-466.          11  Figure 1.2 Schematic depictions of the innervation and neural pathways in LUT during storage (a) and voiding (b) phases of the bladder function                                     Source: Fowler, C. J., Griffiths D., and de Groat W.C. (2008). "The neural control of micturition." Nature reviews. Neuroscience 9(6): 453-466.  While the ANS plays a crucial role in normal urinary function, its possible role in the pathogenesis of BBD in children has not been studied in the past. However; a few studies investigating children with monosymptomatic nocturnal enuresis (urinary incontinence happening only during night time with a different pathophysiology than daytime urinary incontinence) via conventional or non-conventional methods of evaluating the ANS function have shown an imbalanced ANS system with an interpretation of marked hypo-(38-40) or hyper-(41-43) activity in the PNS branch of these patients. 12  It is worth noting that the normal physiology of micturition is comprised of complex mechanisms involving more than just the central and peripheral ANS and somatic nervous system. It is shown that factors like stretch, inflammatory insult, acidity, and change in temperature in urothelium and underlying lamina propria can cause increased bladder excitability, hypersensitivity, and sensation of urgency via local release of a variety of neuropeptides (e.g. nerve growth factor, ATP, Substance P, and neurokinin A). Additionally, a long list of biochemical mediators including but not limited to neurotrophins, purines, proteases, cytokines, nitric oxide, serotonin, glycine, and gamma-aminobutyric acid are essential in the normal functioning of the neural pathways.(44) An unusual inhibition or increase in these mediators has shown to cause disruption in normal bladder function.(44) It is not clear whether ANS role in LUT function is also mediated by these factors, however, this dissertation is focused only on the cardiac ANS activity in children with BBD.         1.2.1 Measurement of the Cardiac ANS Activity by Means of Spectral Analysis of Heart Rate Variability (HRV) and Impedance Cardiography (ICG) The ANS function can be evaluated using different invasive or non-invasive techniques. The two main methods for invasive measurement of the ANS function are: 1) Neurophysiological approach of microneurography which directly records the efferent post-ganglionic sympathetic nerve activity by insertion of a tungsten microelectrode in a nerve fascile(45) and 2) Haemodynamic measurements through direct evaluation of the haemodynamic responses to stressful stimuli(46) or pharmacological blockade of the adrenergic receptors located at the level of the heart and peripheral vessels.(47) These methods suffer from important limitations, which include not only the invasiveness of the method but also (1) measurement of only the SNS 13  activity (2) lack of a standard method of comparing the amplitudes between subjects in case of microneuropathy (3) restriction of these measurements to the laboratory environment (4) limitation within subject reproducibility of the haemodynamic responses evaluated and (5) lack of correlation between haemodynamic responses to different stimuli.(48)  The non-invasive methods of measuring the ANS activity work by using changes in an organ’s response as a proxy for changes in the overall ANS activity. These techniques vary from pupillary light reflex,(49) Valsalva maneuver,(50) and test of sudomotor (sweating) function,(51) to the analysis of heart rate variability (HRV) which has recently been introduced as a diagnostic tool and is the most popular and practical method of measuring the activity of the ANS.(52, 53) In this dissertation, I will be using the analysis of HRV method for assessing the functionality of the ANS.    It has been known since 18th century that our heart rate, even at rest, fluctuates on a beat-to-beat basis.(54) Using pharmacological agents, it has been demonstrated that these fluctuations are mainly the results of interplay between the sympathetic and vagal modulations of the heart’s sinus node pacemaker activity.(55, 56) Therefore, the overall state of the cardiac ANS as well as the interaction between the SNS and PNS branches can be explored by assessing the HRV with appropriate methodologies and in particular the novel method of spectral analysis of the physiological beat-to-beat cardiac signal variations which is considered the most precise non-invasive procedure to investigate the status of the ANS.(53) Clinical significance of HRV has been demonstrated through a strong body of literature(53, 57) and a decreased HRV has been shown to be a predictor of mortality in post-myocardial infarction patients.(58, 59) The pattern of reduced HRV has also been described as a marker for chronic stress associated with increased 14  inflammation in the body,(34, 60) as well as in major chronic diseases such as diabetes and in patients with higher cardiovascular disease risk factors.(61-64)  A search in the Ovid web-based platform for scientific literature has revealed that over the last 3 decades (from 1981 to 2010), the number of publications on HRV has increased exponentially reaching approximately 8000 papers by year 2010.(57) The most widely used methods of HRV analysis are time-domain and frequency-domain methods which can be converted into one another using mathematical method of Fourier transform.(65)  The time-domain is the simplest method of HRV analysis in which different variables (such as mean, standard deviation (SD), or the difference between the longest and shortest intervals), mostly representing the overall HRV, are calculated based on the sequence of intervals between successive fiducial points of R peaks (also known as NN intervals) of QRS complexes in the ECG. The main limitations of the time-domain method are its lack of discrimination between the PNS and SNS effects of the ANS, and the fact that this method is not the preferred method for the short-term and stationary measurements of the HRV.(57) Conversely, with the frequency domain analysis, using a more advanced method of power spectral density analysis (PSD), the heart rate signal is decomposed into its frequency components providing us with a spectrum of relative and absolute values of the signal intensity (so called “power”). In short, PSD provides information on how the power of HRV is distributed as a function of frequency. The main advantages of the frequency-domain method is its reliability in measuring the overall HRV and more specific estimates of each individual ANS components (mostly PNS) as well as its preference in the short-term and stationary assessment of HRV.(57) In this dissertation I will focus on PSD frequency-domain method of HRV analysis. 15  Spectral analysis of HRV provides us with three frequency bands as follows: (1) very low frequency (VLF, < 0.04 Hz), which does not have a well-defined physiological explanation, but is often related to the vasomotor tone of thermoregulation or hormonal regulation, (2) low frequency (LF, 0.04 – 0.15 Hz), which is the marker of primarily SNS cardiac modulation with some influence from the PNS and (3) high frequency (HF, 0.15 – 0.4 Hz), which is a marker of pure cardiac PNS modulation. By summing all three abovementioned bands (HF + LF + VLF), we are able to calculate a new important variable which provides us with an estimate of the overall ANS’ regulatory ability in coping with physiological or non-physiological stress. This variable is called “total power (TP, approximately ≤0.4 Hz)”.(53) Low TP and HF have been documented in several conditions associated with chronic inflammation/stress including, but not limited to, diabetes and cardiovascular diseases.(34, 60-64) Although the frequency-domain is a preferred method of HRV analysis, it has its own limitations with the most important one being its lack of estimating the pure activity of the SNS due to the questionable physiological explanation of VLF and LF components.(53, 66-68) For this reason, pre-ejection period (PEP) is obtained from analysis of Impedance Cardiography (ICG), a non-invasive technology that was introduced in 1940s and later developed by NASA in 1960s for detecting total electrical conductivity and changes in thorax,(69) as an important hemodynamic parameter representing the pure activity of the SNS.(70) PEP is the time interval from the beginning of electrical stimulation of the ventricles to the opening of the aortic valve (electrical systole) and has been shown in several studies using pharmacological blockades alone or in combination with psychological stressors or exercise to be inversely correlated with the pure SNS influence upon the heart.(70-76)  16  1.3 Study Rationale As discussed in earlier sections of this chapter, BBD and in specific the urinary incontinence as the main presenting symptom of BBD comprise a significant number of outpatient referrals made to pediatricians. Although ANS innervates most of the LUT and is highly important in the normal bladder function, the ANS activity and its potential role in the pathophysiologic mechanism of BBD in children has been poorly investigated. In fact, this gap in the published scientific literature does not stop at the etiopathogenesis level but as mentioned earlier in the background section of this chapter, the efficacy of current treatment modalities for BBD in childcare is also still under question and in many cases the patients are left without being prescribed any treatments. The above issues highlight the importance of BBD and its complications, its management, and the need for conducting detailed and methodologically rigorous studies describing the ANS activity in children with BBD as well as assessing the efficacy of current widely used and proposed treatments for managing this condition.   1.4 Dissertation Objectives To address the abovementioned deficiencies in the literature, in this dissertation I chose the following objectives which are described in more detail in each relevant chapter: The overall primary objective of this dissertation was to describe the activity of the cardiac ANS in children with BBD. Given the notable role of the ANS in normal bladder function, we hypothesized finding a significantly lower cardiac ANS activity in children with BBD compared to otherwise healthy controls.  17  The secondary objectives of this dissertation were to: 1) Systematically review the current scientific literature on the relative efficacy of biofeedback (perceived as working through up-regulation of the ANS activity) as one of the noninvasive and widely used adjunctive treatment modalities in resolution of urinary incontinence symptom of children with BBD.   2) Investigate the effects of anti-muscarinic agents, used in management of children with OAB (a subtype of BBD), on the cardiac ANS activity.  3) Assess the feasibility of conducting a large trial to investigate the effect of a proposed intervention of diaphragmatic breathing technique as an adjunctive therapy in the management of children with BBD.    1.5 Dissertation Structure Chapter 2 of this thesis explores the association between BBD and ANS dysfunction via a cross-sectional study in which the cardiac autonomic activity is measured and described in children with BBD and compared with otherwise healthy controls. Chapter 3 builds on the findings of chapter 2 and provides a systematic review and critical appraisal of the existing research literature on the relative efficacy of biofeedback as an adjunctive therapy and a potential treatment candidate in the management of children with BBD. Chapter 4 builds on the work in chapter 2 and investigates the effects of anti-muscarinic agents, used as part of the treatment of children with overactive bladder (a subtype of BBD), on the cardiac ANS activity through a prospective follow-up of a subset of recruited BBD patients with OAB whom received prescription for anti-muscarinic agents. Chapter 5 builds on the results of the work in all previous 18  chapters and assesses the feasibility of conducting a large trial to investigate the effect of a proposed intervention of diaphragmatic breathing technique as an adjunctive therapy in the management of children with BBD. Finally, chapter 6 (Conclusions chapter) synthesizes the findings of all previous chapters, highlights the significance of this thesis, and proposes new directions for future research. 19  Chapter 2: Cardiac Autonomic Nervous System Activity in Children with Bladder and Bowel Dysfunction1  2.1 Synopsis Background: Bladder and bowel dysfunction (BBD) is a functional daytime disorder of the lower urinary tract and bowel and a common reason for referral to pediatric urology. The autonomic nervous system (ANS) plays a key role in normal bladder function; however, its role in the etiopathogenesis of BBD has not been well investigated. I assessed cardiac ANS activity in children diagnosed with BBD and compared it to that in healthy controls hypothesizing that children with BBD would show a marked difference in their ANS profile compared to the controls as measured by parameters captured from spectral analysis of heart rate variability (HRV) and impedance cardiography (ICG).  Methods: A cross-sectional study was conducted at the department of Urology in the Children's & Women's Health Centre of British Columbia between October 2013 and April 2015 to recruit children clinically diagnosed with BBD using a validated scoring tool, as well as otherwise healthy children for the comparison. Cardiac autonomic activity was assessed via spectral analysis of HRV at baseline and during voiding. The primary outcome measures were the main parameters of HRV/ICG, including total power (TP), high frequency (HF), as well as pre-ejection period (PEP) and heart rate (HR). Uroflow study outcomes including volume voided, average and peak urinary flow rate, pattern of voiding during urination, and post-void residual                                                  1 A version of this chapter has been published as Fazeli MS, Collet JP, MacNeily AE, Afshar K. Cardiac Autonomic Nervous System Activity in Children with Bladder and Bowel Dysfunction. J Urol. 2016 Apr;195(4 Pt 2):1245-9. 20  (PVR) were also measured during voiding. Questionnaires were also used to assess the severity of LUT symptoms (BBD questionnaire) and quality of life (PinQ questionnaire) in children with BBD. Results: Forty children clinically diagnosed with BBD, including 25 girls, with a median age of 10 years (range 5 to 18) and 19 healthy controls, including 9 girls, with a median age of 8 years (range 5 to 16) were recruited into this study. There was no significant difference between BBD cases and healthy controls with respect to their baseline characteristics (age, gender, and BMI). The median BBD score was significantly higher among BBD cases compared to healthy controls (p < 0.0001) with high scores corresponding to irritative LUTS. Children with BBD also had a median PinQ score of 30 (range 4 to 75) indicative of a moderately severe impact on the quality of life. Comparison of outcomes at baseline showed significantly lower TP and HF component of HRV in BBD cases compared to healthy controls (p = 0.001 and 0.002, respectively) when adjusted for age, gender, and BMI. Children with BBD also had a higher heart rate compared to healthy controls (p = 0.151). During voiding, PEP did not change in BBD cases while significantly decreasing in healthy controls (p = 0.05). Children with BBD also showed a significantly higher percentage of abnormal uroflow curve pattern, lower average urinary flow rate and voided volume (p = 0.009, p = 0.013, p = 0.016, respectively), and a trend of higher PVR (27 ml in BBD vs. 14 ml in controls, p = 0.282) compared to the controls. Conclusion: The results of this study demonstrate that children with BBD compared to healthy controls have a different cardiac ANS profile with a significantly lower overall HRV, lower cardiac PNS activity, and a higher heart rate at rest as well as an almost un-responsive cardiac SNS activity during voiding. This phenomenon can be explained by a possible positive feedback 21  loop creating a reflex cardiovascular response to overactivity of the bladder by increasing heart rate and thus decreasing HRV and cardiac PNS activity as well as a central attempt to regulate this unresponsive overactive detrusor by lowering the entire PNS output. This difference in the ANS profile of these children may be used in future studies to further clarify the pathophysiology of BBD, and introduce novel treatment targets to manage the condition.22  2.2 Background and Objectives Bladder and bowel dysfunction (BBD) in children is a common functional disorder of the lower urinary tract and bowel comprising a heterogeneous group of syndromes.(2, 4) Children with BBD mainly present with inconstant LUT or bowel symptoms based on their relation to the storage and/or voiding phase of bladder function. These symptoms vary from increased voiding frequency, incontinence, nocturia, and enuresis to decreased voiding frequency, hesitancy, straining, intermittency, dysuria, feeling of incomplete emptying, constipation, and post-micturition dribbling(5) (please see glossary of specialized terms for the definition of these symptoms). There is a significant social and cultural variation in toilet-training but most children are toilet-trained by the age of three and by the age of five it is assumed that the child is able to void at will and be able to postpone voiding in a socially acceptable manner. By this age, daytime or night-time wetting becomes a social problem and a cause for therapeutic intervention.(77)  The studies investigating the prevalence of BBD report sparse and at times un-interpretable results. Most of these studies focus on the prevalence of urinary incontinence as the main diagnostic and therapeutic target outcome of BBD. The prevalence of BBD symptoms has already been briefly described in previous chapter. In addition to what has been described, the prevalence of daytime wetting has been reported to be 1% in children between the ages of 7 and 12 in Netherlands(78) while in another large study investigating the micturition habits of 7 year-olds in a population of 3556 school entrants in Sweden, daytime wetting was reported in 6% of the girls and 3.8% of the boys.(13) Another study in Finland reported daytime wetting of 1.8% in a random sample of 7-year-old children. They also reported 50.7% of children with daytime or 23  mixed wetting to suffer from urgency with 79.1% wetting themselves at least once in 10 days.(79) These symptoms of urgency seem to increase between the ages of 6 to 9 years and decrease towards puberty with an estimated spontaneous cure rate of 14% per year in daytime wetting.(77)  In a 6-year period from 2002 to 2007 almost 2.6 million hospital or physician visits with diagnosis of pediatric urinary incontinence have been documented only in Medicare and Medicaid system in the U.S.(14) Observational studies have also established an association between childhood and adulthood urinary and sexual dysfunction(16, 17) and it has been shown that some children with overactive bladder (a subtype of BBD) can carry this problem into their adulthood.(15) As already described in Chapter 1, the etiopathogenesis of BBD is still unclear and several debatable theories have been proposed. The autonomic nervous system (ANS) function has been described during normal storage and voiding phases of the bladder(32) however little is known about ANS role in pathogenesis of BBD. This could partly be due to lack of reliable and non-invasive methods to assess the ANS activity in the past.  Changes in cardiac ANS activity, which is a proxy for changes in the overall ANS activity, can be explored by assessing the cardiac rhythm with appropriate non-invasive methods and in particular the novel method of spectral analysis of the beat-to-beat cardiac signal variations (HRV/ICG) which is considered the most precise non-invasive procedure to investigate the status of the ANS.(53) Additional details regarding the ANS activity during normal bladder function as well as the measurement of the cardiac ANS activity by means of spectral analysis of heart rate 24  variability (HRV) and impedance cardiography (ICG) has been described in Chapter 1 under sections 1.2 and 1.2.1. I conducted a cross-sectional study to address the question - Is there a difference between the ANS activity of children with BBD compared to otherwise healthy population? The primary objective of this study was to describe the activity of the cardiac ANS in children with BBD in comparison with the otherwise healthy children. I hypothesized to find a significantly lower cardiac ANS activity in children with BBD compared to the otherwise healthy group measured by parameters of HRV/ICG.   2.3 Methods After obtaining institutional research ethics approval from the University of British Columbia’s Children and Women’s Research Ethics Board, I conducted a cross-sectional study at the department of Urology in the Children's & Women's Health Centre of British Columbia between October 2013 and April 2015 to compare the ANS activity in children with BBD to an otherwise healthy group of controls.  The study involved recruitment of 40 children with BBD and 19 otherwise healthy controls. Inclusion criteria for cases were children between the ages of 5 to 18 and diagnosed with BBD. Diagnosis was made by our attending pediatric urologists (Drs. KA and AEM) via a combination of thorough clinical history, physical examination, validated questionnaire,(21) uroflowmetric evaluation, as well as voiding and stool diary. Exclusion criteria consisted of any anatomical or neurological lower urinary tract abnormalities such as posterior urethral valve, urethral obstruction or stricture, ectopic ureters, and congenital abnormalities of the spinal cord, as well as the current use of medication or treatments which were known to affect bladder function or 25  the activity of the ANS. Children with psychological/behavioral abnormalities known to affect the activity of the ANS (such as ADHD or severe anxiety disorder) or prevent the cooperation of the child with the study coordinator or the urologist were also excluded from participating. Healthy controls between the ages of 5 to 18 with no evidence of BBD or ANS abnormality were also recruited as control group for this comparison.  After a full discussion of the study procedure and obtaining participant/parents’ informed consent/assent, each participant’s anthropometric data was measured. This measurement included weight, using a digital scale and height using a wall-mounted stadiometer. Subsequently, each participant was asked to fill out the Dysfunctional Voiding Questionnaire, which has undergone rigorous assessment of its psychometric properties (Appendix A).(21) This questionnaire contains 13 items assessing the severity of the following symptoms: urgency, constipation, incontinence, nocturia, voiding frequency, interrupted urinary pattern, enuresis, encopresis, and dysuria. The items are scored on a 5-point Likert scale from 0 to 4, with higher scores indicating more severe symptoms. The results of this questionnaire are reported as “BBD score” in this dissertation. Participants with BBD were also asked to fill out the Pediatric Incontinence Quality of Life Questionnaire (PinQ), which is a validated quality of life questionnaire specific to children with voiding disorders (Appendix B).(22) This questionnaire consists of 20 items scored on a 5-point Likert scale from 0 to 4, with higher scores indicating a lower quality of life.  Measurement of the cardiac ANS activity was then ensued in a controlled lab environment and by following a protocol for capturing and analyzing HRV and ICG (Appendix C). Data were collected by Biopac MP150 data acquisition system in a controlled environment by placing 6 26  electrodes on the skin surface of the chest, neck, and lower back. Signals were sampled at 500 Hz throughout all the testing. All participants were asked to sit comfortably on a chair for 5 minutes prior to HRV data recording. After the 5-minute adaptation period, baseline data recording was pursued for 5 minutes according to the standard recommended length of recording for a short-term spectral analysis of HRV.(53) Consequently, the participants were asked to sit on the toilet which measured their uroflow parameters while they urinated into the toilet. The main uroflow parameters measured were average and peak urinary flow rate, and voided volume. These parameters measure the volumetric flow rate of urine (how fast a person can empty their bladder) as well as the volume of urine expelled and were compared by our pediatric urologists against normal normograms of healthy boys and girls based on their body surface to help with diagnosis of LUT dysfunction.(80)  HRV/ICG data was recorded at baseline when participants were seated on a comfortable chair as well as during the voiding period. At the end of urination, the electrodes were detached from each participant’s body and they were asked to lie down on a bed so an attending urologist could measure their post-void residual volume (PVR) using an ultrasonic-bladder scanner device. PVR is the amount of urine that is left in the bladder after voluntary urination. PVR has shown to be influenced by bladder over distension, age, and possibly extra-hydration before assessment and is used as one of the screening tests for evaluating voiding dysfunction.(81, 82) PVR values greater than 20 ml (or more than 10% of bladder capacity) on repeated voiding tests without bladder over distention in kindergarteners have been defined as abnormal.(82)    After completion of data collection, I manually reviewed recorded HRV/ICG data for ectopic beats and analyzed them using AcqKnowledge® software version 4.2 manufactured by Biopac 27  Systems Inc. Fast Fourier Transformation (FFT) technique was used to perform frequency domain HRV analysis. Three frequency bands were obtained from our HRV analysis as follows: (1) Very Low Frequency (< 0.04 Hz), which has debatable and much less defined physiological explanation compared to other bands and its interpretation should be avoided in short-term recordings (e.g. ≤ 5 minutes) (2) Low Frequency (0.04 – 0.15 Hz), which is the marker of primarily sympathetic cardiac modulation with some influence from parasympathetic and (3) High Frequency (0.15 – 0.4 Hz), which is a marker of pure cardiac parasympathetic modulation. Total Power (TP) ( 0.4 Hz), which represents the variance of all cardiac beat-to-beat intervals, was also calculated by summing all frequency bands (HF + LF + VLF).(53) All frequency bands described above (except for VLF) as well as LF/HF ratio and normalized HF (𝐻𝐹𝑛𝑢 =HFTP−VLF) values are captured, calculated, and reported. However, due to questionable physiological explanations of the VLF and LF components(53, 66-68), and thus the inaccuracy of LF/HF ratio(83) or HFnu(84) in measuring the cardiac sympatho-vagal balance I only considered HF and TP for our analysis and interpretation of the results.  The analysis of HF and TP provided us with the necessary tools to estimate the activity of the cardiac PNS (HF) as well as the overall regulatory ability of the ANS in coping with physiological or non-physiological stress (TP). In addition, PEP was also obtained and reported from ICG analysis and measured the time interval from the onset of the electromechanical systole to the onset of the opening of the aortic valve. The analysis of PEP provided us with an estimate of the activity of the SNS influence upon the heart (there is an established inverse correlation between PEP and pure SNS influence upon the heart).(70, 74, 76) Finally, beat-to-28  beat heart rate (HR), which is under the influence of both SNS and PNS was also obtained and reported from ICG data analysis. Descriptive statistics on baseline characteristics were used to report the results of this study. Relative percentage change from baseline to the voiding period was also calculated in order to control for the baseline differences between participants, as well as to test if the two groups differed in their HRV/ICG parameters with regards to a change from the bladder filling phase to the voiding phase. For a between-groups comparison of the ANS and uroflowmetric profile, analysis of covariance test was used adjusting for age, gender and BMI as co-variates. Mann–Whitney U test was used as a non-parametric approach to test the between-groups difference in BBD score, BMI, and age. Pearson’s Chi-squared test was also used for categorical variables (gender, and the uroflow curve pattern).   For all statistical tests, a P value of < 0.05 was considered as being statistically significant. Statistical analyses were performed using SPSS® v.22.0. 2.4 Results A total of 40 children with BBD (15 boys and 25 girls with median age of 10 years, range 5 to 18) and 19 healthy controls (10 boys and 9 girls with median age of 8 years, range: 5 to 16) were recruited into this study. No significant difference was found between the cases and the healthy controls with respect to their baseline characteristics (age, gender, and BMI). As expected, the median BBD score was found to be significantly higher among cases compared to healthy controls:18 (range 8 to 41) in cases vs. 5 (range 2 to 11) in controls. (Table 2.1 ). The majority of BBD patients were clinically diagnosed as having symptoms of OAB (a subtype of BBD) with high scores corresponding to irritative LUTS as defined by ICCS.(5) The emotional impact of 29  BBD on the quality of life was also measured only in BBD group using PinQ questionnaire with a median PinQ score of 30 (range 4 to 75) indicative of a moderately severe impact on the quality of life as suggested by Thibodeau et al. 2013.(85) The main uroflow parameters (average urinary flow rate, voided volume, and pattern of uroflow curve) differed significantly between the two groups when adjusted for age, gender, and BMI with BBD group having significantly higher percentage of abnormal uroflow curve pattern (68.6% vs. 31.6%, p = 0.009), lower average urinary flow rate (6.47 ml/sec in BBD vs. 9.11 ml/sec in controls, p = 0.013) and voided volume (116 ml in BBD vs. 193 ml in controls, p = 0.016) and a higher PVR (27 ml in BBD vs. 14 in controls, p = 0.282) compared to the controls (Table 2.1  and Table 2.2). Table 2.1 Table of baseline characteristics. Variables BBD (n = 40) Control (n = 19) P-Value Gender (% female) 62.5 47.4 0.272 Median Age year (range) 10 (5-18) 8 (5-16) 0.529 Median BMI kg/m2 (range) 16.5 (12-28) 17.2 (14.0-24.0) 0.942 Uroflow curve (% abnormal) 68.6 31.6 0.009* Median BBD score (range) 18 (8-41) 5 (2-11) < 0.0001* * P values are reported using Mann–Whitney U, or Pearson’s Chi-squared tests.   30  Table 2.2 Autonomic and uroflowmetric profile at baseline.  Autonomic Variables BBD (n = 39) Control (n = 19) p value TP (ms2/Hz) 944.03 (133.97) 1756.22 (193.13) 0.001* HF (ms2/Hz) 490.64 (99.20) 1061.68 (143.00) 0.002* LF (ms2/Hz) 307.19 (44.25) 500.12 (63.79) 0.017* LF/HF ratio 0.90 (0.10) 0.73 (0.15) 0.351 HFnu (%) 58.44 (2.55) 61.77 (3.68) 0.464 PEP (msec) 111.58 (1.02) 111.10 (1.46) 0.790 HR (beats/min) 82.47 (1.50) 78.62 (2.16) 0.151 Uroflowmetric Variables BBD  Control  p value Peak urinary flow rate (mL/sec)  15.54 (2.53) 18.89 (3.55)  0.451 Average urinary flow rate (ml/sec) 6.47 (0.59) 9.11 (0.83) 0.013* Voided volume (ml)  115.54 (17.92) 193.49 (25.17) 0.016* PVR (ml) 26.77 (6.74) 14.02 (9.46) 0.282 * Univariate analysis using Analysis of Covariance adjusted for age, sex, and BMI. Values are adjusted mean (SEM). TP, Total Power; HF, High Frequency; PEP, Pre-ejection Period; HR, heart rate; PVR, Post-void residual volume   Comparison of the ANS profiles at baseline and during voiding: Of a total of 40 children with BBD, the recorded HRV data on 1 child contained significant number of artifact and noise due to the child not cooperating with the investigator at the time of the assessment and as a result the data were uninterpretable and erroneous and therefore excluded from the analysis.  At baseline, when adjusted for age, sex, and BMI, children with BBD had a significantly lower TP, and HF compared to the healthy controls (mean TP at rest = 944.03 ms2/Hz in BBD vs. 1756.22 ms2/Hz in controls, p = 0.001) (mean HF at rest = 490.64 ms2/Hz in BBD vs. 1061.68 ms2/Hz in controls, p = 0.002). Children with BBD also had a higher heart rate at rest compared 31  to the controls (mean HR at rest = 82.47 bpm in BBD vs. 78.62 bpm in controls, p = 0.151). However, PEP was similar in the 2 groups at baseline (Table 2.2).   Comparison of the between-groups difference for the relative change from baseline to the voiding period was only performed for variables extracted from ICG data analysis (PEP and HR) because in order to assess the HF and TP component of the HRV a recording of a minimum of 1 and 2 minutes is required, respectively(53) which is clearly not feasible for the voiding period. Voiding was associated with almost no change in PEP values for BBD cases which was significantly different than an observed decrease in PEP in healthy controls (relative PEP change (%) from baseline to voiding = -0.80 % in BBD vs. -3.36 % in controls, p = 0.05). We also observed a larger change in heart rate in BBD group during voiding however this between-groups difference was minimal and non-significant (relative HR change (%) from baseline to voiding = 3.39 % in BBD vs. 2.24 % in controls, p = 0.678) (Table 2.3 ).  Table 2.3 Relative change in autonomic profile from baseline to voiding Autonomic Variables Relative change (baseline rest to voiding) (in %) p Value BBD (n = 39) Control (n = 19) PEP (msec)  - 0.80 (0.73) - 3.36 (1.03) 0.05* HR (beats/min) + 3.39 (1.57) + 2.24 (2.24) 0.678 *Univariate analysis using Analysis of Covariance adjusted for age, sex, and BMI. Values are adjusted mean (SEM). PEP, Pre-ejection Period; HR, heart rate   2.5 Discussion While the ANS plays a key role in the regulation of normal urinary function, the possibility of ANS involvement in the pathogenesis of daytime BBD has not been investigated before.  32  To my knowledge, this is the first study that objectively measures the activity of the ANS in children with daytime BBD and compares it with healthy children using spectral analysis of cardiovascular signal variability methodology. Through my preliminary literature search, I found a few studies that had previously investigated the activity of the ANS, via conventional and non-conventional methods of measuring ANS activity, in children diagnosed with monosymptomatic nocturnal enuresis (urinary incontinence occurring only during night time with a different pathophysiology than daytime BBD). The results of these studies show an imbalance in the harmonious activity of the ANS components with significant discrepancies between authors’ interpretation of this imbalance reporting hypo-(38-40) or hyper-(41-43) activity in the PNS component of these patients. The main limitations of these studies were that all of them were structurally different than the current study with regards to the indication under investigation (monosymptomatic nocturnal enuresis) with dissimilar pathophysiology to daytime BBD. They also did not investigate patients during voiding phase. In addition, some of these studies used unconventional methods of assessing ANS activity including; Valsalva ratio(38) which is known to measure vagal afferent and efferent limb function and is prone to bias due to improper performance of Valsalva manoeuver by the patients. Pupil diameter measurement(40) using infrared detector (Pupilometric assessment) which measures the ocular PNS activity for a short period of time providing an indirect and not fully accurate method to evaluate ANS imbalance.(86) 24-hour ambulatory electrocardiographic recordings(39, 42, 43) and time-domain method of HRV analysis.(39)    The results of the current study provide valuable information suggesting that children with BBD, during storage phase of bladder function, have a significantly lower overall regulatory ability of 33  the ANS (mean TP in children with BBD is almost half its value in healthy controls) with lower PNS modulation upon the heart (mean HF in children with BBD is less than half its value in healthy controls). Since SNS and PNS directly influence the heart rate, the above finding (lower PNS in BBD group) is also reinforced with the observation of BBD children having a higher heart rate at rest compared to the healthy controls (mean HR at rest = 82.47 bpm in BBD vs. 78.62 bpm in controls), although not reaching statistical significance at alpha = 0.05. Due to the normal length of voiding being less than 1 minute, we were not able to directly measure some of the ANS components (i.e. HF, and TP) during voiding (also described in the methods section), however, measurement of the relative change in PEP and HR from baseline to during voiding enabled us to indirectly assess cardiac ANS activity during the voiding phase. Our findings comparing the relative change in ICG parameters from baseline to during voiding in the two groups showed that children with BBD had an almost un-responsive cardiac SNS activity during voiding (relative PEP change from baseline to during voiding was more than 3 times lower in our healthy controls while remaining almost unchanged in children with BBD).  As described earlier in previous chapter, normal micturition is comprised of two phases (Figure 1.1 and Figure 1.2 ): 1) storage phase (controlled centrally by L-center within the rostral pons): during which the thoracolumbar SNS outflow (arising from T10 to L2 segments of the spinal cord), via hypogastric nerve, becomes dominant and inhibits detrusor muscle contraction via β3 receptors while simultaneously stimulating α-adrenergic receptors at bladder neck causing an increase in outlet resistance. This is reinforced by an excitatory pudendal (somatic) outflow to the external urethral sphincter and a direct inhibition of detrusor motor neurons by sacral spinal cord, 2) voiding phase (controlled centrally by M-center within the rostral pons): during which 34  the sacral PNS outflow (arising from S2, S3, and S4 segments of the spinal cord), via pelvic nerve, becomes dominant and acts mainly via M3 receptors on detrusor muscle, SNS and pudendal nerves are inhibited and voiding takes place as a result of the coordination between the detrusor contraction and internal and external sphincters relaxation.(32, 37, 87, 88) All of the above mentioned processes involve inhibitory or acceleratory inputs from cerebral circuits high above pons in the cortex to assess the appropriateness of voiding based on social norms or situation.   Neural excitatory transmission occurs in the bladder through different types of receptors that are expressed in the detrusor muscle of which M3 subtype is the most important one.(89) While efferent neural pathways bring action commands from higher centers in the brain to the bladder, information and sensations from urinary tract is also carried in afferent axons to higher nervous system centers through 3 sets of nerves (PNS via pelvic nerve, SNS via hypogastric nerve, and somatic via pudendal nerve).(90) These afferent axons consists of A-delta (thinly myelinated) and C fibers (unmyelinated) with the latter comprising 2/3 of bladder afferent nerves and dispersed more widely in the detrusor and suburothelium.(37) A-delta fibers respond to passive distention and active contractions of the bladder wall whereas C fibers are insensitive to physiological filling, and respond to noxious stimuli, or changes in the temperature and pH. These fibers (C fibers) are thought to be important in the pathogenesis of urgency, and OAB.(91)  Based on the above mentioned description of normal micturition physiology (SNS dominance during storage phase and PNS dominance during voiding), one might think that children with BBD should present with a higher PNS activity during storage phase (at rest) due to the overactivity of the detrusor muscle mainly causing symptoms of frequency and incontinence, 35  however, the results of the current study are in contrast with this idea and show a significantly lower overall HRV with a lower PNS activity during storage phase in children with BBD compared to healthy controls (Table 2.2). Below I have listed what I believe are the reasons behind the seemingly counter-intuitive findings of the current study (i.e. significantly lower HRV and PNS at rest in BBD patients):  1) There is a difference in the methods used for measuring the ANS activity between the current study (measurement of the ANS activity at the heart level) and the studies demonstrating the neural pathways during normal micturition (ANS measurement at the bladder, spinal cord, and brain level).   2) The same pattern described for the normal neurophysiological process during storage and voiding phases of bladder function may not necessarily hold at times when there is a urinary dysfunction (i.e. in case of BBD).   3) The well-recognized crucial role of urothelium in neural communication within the different layers of the bladder wall as a dynamic surface between urine and underlying nerves, connective tissue, cells, and muscles(92) should also be taken into account when investigating the source of voiding dysfunction in BBD. It is shown that stimulation of urothelium by wall stretch, inflammation, acidity, and temperature change can cause local release of neuropeptides (such as ATP, substance P, nerve growth factor, and neurokinin A) with paracrine actions resulting in modulation of the activity of afferent fibers (C fiber in this case) which can lead to increased bladder excitability, hypersensitivity, and 36  ultimately symptoms of bladder overactivity (OAB).(91, 93, 94) On the other hand, other experts believe the primary inciting events in BBD may originate from abnormal detrusor activity.(15, 77{Sillen, 1992 #3260, 95) The controversy continues in this regard as the cause of BBD is multifaceted and not completely understood.  4) It has been shown that stimulation of mesenteric region and abdominal visceral afferent nerves activates spinal pathways which results in reflex cardiovascular excitation causing increased heart rate, myocardial contractility, and arteriolar constriction in several regional vascular structures including the coronary system.(96) Other studies in humans and animals have also shown that bladder stimulation through distention of the bladder wall, via afferent SNS and predominantly PNS pathways, can cause reflex vasoconstriction, increase in blood pressure, and heart rate.(97-100) Given the above mentioned, I theorize that the overactivity of the bladder, possibly originating from a primary defect within the urothelium or the detrusor, causes an increase in the firing of the afferent C fibers to the spinal cord and higher micturition centers. Subsequently, the brain interprets this as hyperactivity of the PNS in the pelvic region and creates a simultaneous non-specific neural response via PNS efferent neurons to lower the pelvic PNS output. This non-specific response then leads to a reflex increase in heart rate and a decrease in HRV and cardiac PNS activity.  We used state-of-the-art technology to non-invasively capture and analyze HRV under controlled conditions and following strict study procedure protocol. This methodology has been accepted as a standard method for recording and analyzing HRV for the assessment of the ANS and vastly 37  used by researchers and organizations around the globe who investigate the stationary activity of the ANS in health as well as in the context of different disorders. One limitation of this study was our inability to measure the overall activity of the ANS and in specific the PNS during voiding due to the requirement of recording at least 1 minute(53) of the heart activity. Despite these circumstances, we were able to indirectly measure cardiac ANS activity during voiding by measuring the relative change in our ICG parameters of interest (i.e. PEP and HR) from baseline to during voiding. I also acknowledge that the measurement of the cardiac ANS under controlled conditions might not present the everyday ANS status of the participants in this study, however, since both BBD and control groups were assessed in the same setting this will not affect the overall results of the study. HRV and uroflow measurements in the current study were performed while participants were in a seated position. Studies in adults and children have established a close association between body posture and a change in ANS function measured via HRV (standing associated with a significantly higher HR, lower TP and HF compared to sitting)(101, 102). Given the above mentioned and the fact that there might have been males in the current study who preferred standing while voiding, I expect to have observed different HRV results during voiding had I asked the patients to stand while voiding. However, I do not think this phenomenon has biased the study results since we asked everyone (boys and girls across both groups) to be seated during all assessments.   The above findings show that children with BBD also present with a concomitant cardiac autonomic dysregulation during both phases of micturition. Similar ANS profile (reduced HRV and HF band in particular) as marker of chronic stress, is also associated with increased inflammation(34, 60, 103-106) and found in several major chronic diseases such as diabetes(61, 38  62) and cardiovascular disease risk factors(63, 64). In addition, it is also worth noting that the stimulation of afferent C fibers, thought to play a crucial role in the pathogenesis of symptoms of urgency and bladder overactivity, has shown to cause an inflammatory response likely through activation of the SNS.(60, 107-111)   This study is important in the context of the important role of the ANS in normal bladder function and investigation of the underlying cause of voiding disorders in children. Future studies with large sample sizes are needed to test the development of more effective treatment strategies for controlling LUTS by using HRV/ICG parameters as surrogate and objective treatment targets.  39  Chapter 3: Biofeedback for Bladder and Bowel Dysfunction in Children: A Systematic Review and Meta-analysis of Randomized Controlled Trials2  3.1 Synopsis Background: Biofeedback is a noninvasive therapy that has been used as an adjunctive treatment for children with bladder and bowel dysfunction not responding to standard urotherapy alone. It has been shown that biofeedback (used in other fields) can enhance the activity of the ANS in healthy individuals. However, the evidence for the efficacy and safety of this treatment is limited and based on small studies. We conducted a systematic review of the literature (SLR) to assess the efficacy of biofeedback as adjunctive therapy for symptoms of Bladder and Bowel Dysfunction (previously known as nonneuropathic daytime voiding disorders or NVD) in children up to age 18 years. Methods: Using the OvidSP® platform we searched MEDLINE®, Excerpta Medica Database (Embase), and Cochrane Central Register of Controlled Trials (CENTRAL). In addition, we searched conference proceedings for randomized controlled trials presented at scientific conventions, symposia, and workshops through August 13, 2013. Hand searches of reference lists of retrieved articles and previously published systematic literature reviews were also performed. The quality of the selected studies was evaluated using Cochrane Collaboration’s tool for assessment of risk of bias. In addition, the Grades of Recommendation, Assessment,                                                  2 A version of this chapter has been published as Fazeli MS, Lin Y, Nikoo N, Jaggumantri S, Collet JP, Afshar K. Biofeedback for nonneuropathic daytime voiding disorders in children: a systematic review and meta-analysis of randomized controlled trials. J Urol. 2015 Jan; 193(1):274-9. 40  Development and Evaluation (GRADE) criteria was used for grading the quality of evidence for the outcomes of interest across included studies. Results: A total of 1195 citations were identified from the literature searches, of which 27 were included for full-text screening. Ultimately, 5 studies were eligible for inclusion in the systematic review, of which 4 (382 participants) were pooled in the meta-analysis based on available outcomes data and end-points. The biofeedback and control group were similar with regards to the overall proportion of patients who achieved resolution of incontinence at month 6: odds ratio (OR) 1.37 (95% confidence interval [CI] 0.64 to 2.93), risk difference (RD) 0.07 (95% CI -0.09, 0.23). There was also no significant difference in mean peak urinary flow rate (mean difference 0.50 ml/s, 95% CI -0.56 to 1.55) or likelihood of urinary tract infection (OR 1.30 [95% CI 0.65 to 2.58]). Safety outcomes and health related quality of life were not reported across trials.  Conclusion: The results of the current systematic literature review do not support the efficacy of biofeedback as an adjunctive treatment in the management of children with bladder and bowel dysfunction. More high quality and well-designed randomized controlled trials with larger sample size and objective measures of outcomes are needed to better evaluate the efficacy and safety of biofeedback.   41  3.2 Background and Objectives This chapter follows the earlier description of down-regulated ANS in children with BBD (Chapter 2), which opens the door to consider using interventions with capabilities of modulating ANS to control symptoms of BBD. In this chapter, I will describe the systematic literature review and meta-analysis I conducted to assess the effects of biofeedback BBD. This chapter is the first one related to interventions that have the potential to improve symptoms of BBD through ANS modulation. The next chapters will describe the effect of anti-muscarinic agents (Chapter 4) and the effect of diaphragmatic breathing (Chapter 5) on the activity of the ANS. Dysfunction of the LUT in the pediatric population can be classified as organic with an underlying disease process that can be neurogenic or structural, or without anatomical or neurological lesions, i.e. Bladder and Bowel Dysfunction (BBD).(2, 4) BBD consist of a heterogeneous and inter-related group of syndromes and account for a significant number of outpatient referrals to pediatric urologists.(3) BBD mainly causes symptoms of LUT with or without urinary incontinence. In a 6-year period, spanning from 2002 to 2007, almost 2.6 million hospital or physician visits with diagnoses of pediatric urinary incontinence were documented in the Medicare/Medicaid system in the United States.(14) According to cross-sectional studies, the prevalence of daytime wetting (as the main symptoms of BBD) for all children at age 7 years is approximately 4.5% (95% CI 3.2 to 6.7) with girls being approximately 1.5 times more likely to have incontinence at age 7 and 5 to 10 times more likely at age 16.(13, 79) It has been shown through a number of observational studies that children with some of the subtypes of BBD, such as OAB, may carry this problem into their adulthood.(15-17) Typically, the treatment of BBD starts with urotherapy, which consists of providing the child and their family with necessary 42  educational materials as well as supporting and training them towards having healthy bladder and bowel habits. Lifestyle and diet modifications as well as bladder diaries are used as part of urotherapy. In addition to standard urotherapy, adjunctive therapies with anti-muscarinic agents, alpha-adrenergic blockers, neuromodulation, pelvic floor muscle retraining (biofeedback) or injection of intravesical botulinum toxin A may be used.(112)  Biofeedback is a training technique used in various fields and can be used to  influence body functions and improve health and wellness.(113) The benefits of biofeedback are achieved through gaining greater awareness of the physiological systems by using instruments that provide feedback on the activity of those systems. In the context of BBD, biofeedback is practiced with the main objective of gaining a better voluntary control and relaxation of the pelvic floor muscles during voiding. This is performed by using audio and visual aids that will assist the patient with learning about the physiological activity occurring during the voiding and filling phases.(114) There is substantial evidence that biofeedback (used in fields other than urology) can modify the activity of the ANS as measured through HRV in healthy individuals.(115-118) In one study healthy male athletes were subject to receiving either a biofeedback–based stress management tool or a control group and the results showed statistically significant improvement in the HRV indices in the biofeedback group compared to the control.(117) In another study on otherwise healthy manufacturing operators, participants were randomized to either a resonant breathing biofeedback group or control. The biofeedback group showed a significant increase in LF activity from baseline to after intervention compared to no change in the control group. This provides further support that biofeedback can affect the ANS activity.(118) 43  Given the harmless nature of biofeedback, its positive effect on the ANS activity, and the fact that in our first study (Chapter 2) I found that children with BBD had significantly lower overall cardiac ANS activity compared to otherwise healthy children, I considered using biofeedback as a potentially beneficial treatment for children with BBD. However, the published literature on the efficacy of biofeedback for resolution of incontinence as the main symptom of BBD had provided conflicting results.(26) During the preliminary literature search, I found two systematic reviews on the role of biofeedback in pediatric daytime incontinence.(119, 120) However, the authors of one of the reviews(119) included only 1 RCT with biofeedback and were unable to perform a meta-analysis and the other review included  a single RCT with the rest of studies being case series.(120) In addition, due to different outcome measurements, there was significant heterogeneity in the studies in both reviews. Therefore, due to inconsistency in the available evidence on the clinical efficacy of biofeedback coupled with a lack of up-to-date systematic reviews concerning this important topic, I aimed to systematically review the literature to address the question – What is the relative efficacy of biofeedback, as an adjunctive treatment for children with BBD, compared to control interventions in resolving the main clinical symptom of BBD? In doing so, I conducted a systematic review of the current scientific literature on the relative efficacy of biofeedback as one of the non-invasive and widely used adjunctive treatment modalities in resolution of clinical symptoms of BBD in children up to age 18 years.  3.3 Methods A protocol was developed based on Cochrane collaboration’s recommendations for conducting systematic literature reviews using Review Manager software version 5.2 (RevMan 5.2).(121) Criteria for selection of studies for this review was formed in Participants, Interventions, 44  Comparisons, and Outcomes (PICO) format (Table 3.1 ). The study participants were children up to 18 years of age diagnosed with BBD. The main intervention of interest was biofeedback as an add-on treatment to standard urotherapy and the comparators were standard urotherapy alone or combined with either pharmacotherapy or neuro-modulation. Outcomes of interest included resolution of incontinence (defined as achieving complete dryness), decreased incidence of UTIs, post-void residual volume (PVR), average urinary flow rate (Q-Average), mean peak urinary flow rate (Q-Max), adverse events and quality of life. The search included randomized controlled trials using an RCT search filter adapted from the Scottish Intercollegiate Guidelines Network (SIGN). (122). Studies published in any languages were included. Table 3.1 Study selection criteria for systematic literature review (PICO)  Patients   Children up to the age 18 years with Bladder and Bowel Dysfunction   Intervention   Standard Urotherapy + Biofeedback   Comparator   Standard Urotherapy alone or together with either pharmacotherapy or neuromodulation   Outcomes   Primary = Resolution of incontinence  Secondary = Improvement of recurrent urinary tract infection, uro-flowmetric parameters (PVR, Q-Average and Q-Max), all adverse events, and quality of life improvement   45  Search was carried out via the OvidSP® platform in databases including MEDLINE® (1946 to August 13th, 2013), Embase (1974 to August 13th, 2013), and CENTRAL (1991 to August 13th, 2013). The search strategies and keywords used for each database can be found in Appendix D. We also performed a grey literature search by searching “PapersFirst” and “ProceedingsFirst” databases for papers presented in symposia, conferences, workshops and meetings that were received by The British Library Document Supply Centre from 1993 to September 28th, 2013. Reference lists of all primary studies and review articles were also screened for additional potentially eligible studies and when necessary, authors were contacted. To avoid missing the most recently published articles in this field, we also hand-searched the latest 5 issues of “Pediatric Journal of Urology” and "The Journal of Urology”. All titles and abstracts identified through the literature searches were screened by two reviewers independently and in duplicate to assess eligibility (MSF and YL). Once title and abstract screening was complete, the investigators reconciled any discrepancies between studies selected as eligible as well as reasons for exclusion. If a consensus was not reached, a third reviewer provided arbitration (KA). The full texts for all articles deemed eligible for inclusion at the title and abstract screening phase were then screened, and the investigators reconciled any discrepancies between included studies as well as reasons for exclusion. If a consensus was not reached, a third investigator provided arbitration. This resulted in the final list of included studies that proceeded to the data extraction phase. Data was extracted independently by two investigators (NN and SJ) including details on main trial characteristics, patient characteristics, outcomes of interest, and safety outcomes using standardized forms tailored specifically to meet criteria outlined by the PICO for this review (see 46  Table 3.1 ). Any disagreement between the reviewers was resolved by discussion, or where necessary, by our senior scientific advisor (KA). All relevant data was entered into the Review Manager Software version 5.2 (RevMan 5.2).  The quality of included studies was assessed by two independent reviewers (MSF and YL) by using the Risk of Bias instrument, endorsed by the Cochrane Collaboration.(123) This instrument is used to evaluate 6 key domains: selection bias (sequence generation and allocation concealment), performance bias (blinding of participants and personnel), detection bias (blinding of outcome assessors), attrition bias (incomplete outcome data), reporting bias (selective outcome reporting), and other sources of bias. In addition to the 6 key components, an assessment of the study designs of the included trials was also conducted to determine whether additional quality components should be included in the bias risk assessment. The Risk of Bias instrument was used to assign summary assessments of within study bias; low risk of bias, high risk of bias, or unclear risk of bias (in instances when there is lack of information or uncertainty over the potential for bias). Any disagreements between the reviewers were resolved by discussion, or where necessary, by arbitration of our senior scientific advisor (KA). The quality of evidence for outcomes of interest across included studies was assessed by downgrading or upgrading evidence using GRADEpro GDT online software application (http://gradepro.org) according to the GRADE guidelines for rating the quality of evidence.(124, 125) To be eligible for this grading, the outcomes of interest must have been reported in at least 2 studies (i.e. in the case of this review the outcomes included resolution of incontinence, reduction in the incidence of UTIs, and Q-max). Outcomes were initially scored based on the methodology of the study; if an outcome came from an RCT it would be given a score of +4 47  indicating high quality of evidence. However, this score could be downgraded by up to 3 points if significant issues were present. Issues considered were: (1) design and implementation of available studies (e.g. lack of allocation concealment and blinding, a large loss to follow-up, selective reporting of outcomes), (2) indirectness of evidence (e.g. no direct comparisons between groups or indirect population, intervention, control, outcomes), (3) unexplained heterogeneity or inconsistency of results (including subgroup analyses), (4) imprecision of results (wide confidence intervals), and (5) high probability of publication bias. An overall GRADE score would then be assigned to each outcome: at least 4 points overall indicated high quality of evidence, 3 points indicated moderate quality of evidence, 2 points indicated low quality of evidence, and one or less points indicated very low quality of evidence. The definitions of the four categories are listed below(126): • High — We are very confident that the true effect lies close to that of the estimate of the effect • Moderate — We are moderately confident in the effect estimate: The true effect is likely to be close to the estimate of the effect, but there is a possibility that it is substantially different • Low — Our confidence in the effect estimate is limited: The true effect may be substantially different from the estimate of the effect • Very low — We have very little confidence in the effect estimate: The true effect is likely to be substantially different from the estimate of effect Odds ratio (OR) as well as Risk Difference (RD) for dichotomous outcomes and mean difference (MD) for continuous outcomes with 95% Confidence Intervals (CI) were also calculated. 48  Heterogeneity between studies was assessed by critical review of the methodology in each study, visual inspection of the forest plots as well as statistically via Chi-square test. The extent of heterogeneity among studies was expressed with I-squared statistic (I2).(127) Fixed effect model was applied for calculating summary estimates. 3.4 Results A total of 1195 citations were identified through our searches in main bibliographic databases and grey literature, 27 were eligible for full text review, of which 5 were included in this review. Four of these trials were included in the meta-analysis for a total of 382 participants.(128-131) The remaining trial could not be pooled in our meta-analysis due to separating the main population into two different groups based on diagnosis (OAB and dysfunctional voiding group), then stratifying randomization based on participating center, sex, age, UTI and VUR status and finally having a significantly different length of follow-up compared to the rest of the included studies 12 months after treatment termination.(132) The excluded studies at the abstract or full text screening stage either had a different population of patients, did not include the intervention of interest, were not RCTs, or measured different outcomes as outlined by PICO for this review (see Table 3.1 ). For a list of excluded studies at full-text screening stage as well as the reason for their exclusion, please see Table D.4. A PRISMA flow diagram showing the study selection procedure for this systematic review is presented in Figure 3.1 .   49  Figure 3.1 Modified PRISMA flow chart of search results and publication selection.                      Citations identified through database searching  (n = 1194) • Embase (n = 843) • MEDLINE (n = 272) • Cochrane (n = 79)  Screening Included Eligibility Identification Citations screened  (n = 1195) Citations excluded  (n = 1168) Full-text citations assessed for eligibility (including duplicates)  (n = 27) Full-text citations excluded, with reasons  (n = 12) • Population (n = 1) • Study Design (n = 10) • Outcome (n = 1)  Citations included in the systematic review  (n = 5) Citations included in the meta-analysis  (n = 4) Excluded due to different length of follow-up (n = 1) Citations identified through other sources  (n = 1) Duplicate publications (n = 10)  Full-text citations assessed for eligibility  (n = 17) 50  Table 3.2  outlines characteristics of included studies comprising the year of publication, country in which the study was designed and conducted, study design, different types of interventions, inclusion/exclusion criteria, outcomes investigated in each study, and the number of participants according to the interventions assigned. In summary, all included trials were open-label RCTs from different parts of the world published between years 2006 to 2014. All trials had standard urotherapy as the main component of treatment for their patients with different adjunctive therapies added to urotherapy via randomization. The age limitation for the inclusion criteria of the included trials was mostly above 5 years of age except for Klijn et al. 2006(129) in which this criteria was set between the ages of 6 to 16 years (6 < age < 16), and van Gool et al. 2014(132) in which the eligibility criteria for participation into the study was set between the ages of 6 to 12 years (6 < age < 12). The reported outcomes also varied between studies but mainly focused on relief in symptoms of urinary incontinence.    51  Table 3.2 Characteristics of included studies. First Author Year of publication/Country  Study Design  Intervention  Inclusion/Exclusion Criteria  Outcome  No. patients (Biofeedback)  No. patients (Control)  Kajbafzadeh(128) 2011/  Iran  Randomized open label study  Group A: Behavioral Modification + Pelvic floor muscle exercises + Animated Biofeedback  Group B: Behavioral modifications alone  Inclusion: Age > 5 years, simultaneous constipation and/or fecal soiling and voiding dysfunction.  Exclusion: Children whose physical examination, KUB or history suggested an occult neurological defect  Daytime wetting, nocturnal enuresis, urgency, constipation, fecal soiling and Dysfunctional Voiding Symptom Score (DVSS), uroflowmetry results, voiding pattern, pelvic floor activity during void, UTI, bladder capacity and PVR  10  11  Klijn(129) 2006/The Netherlands  Randomized open label parallel group trial  1 - Standard Outpatient Therapy  2 - Standard Outpatient Therapy + Personalized Video instructions  3 - Standard Outpatient Therapy + home uroflowmetry  Inclusion: 6 < Age < 16. Clear peaks and declines in urinary flow rate found in at least 2 flow registrations containing 100cc or more. Residual urine after voiding of at least 10% found on at least 2 ultrasound assessments. Present history of recurrent UTIs with or without daytime Relief of symptoms: infection and incontinence  33  30  52  First Author Year of publication/Country  Study Design  Intervention  Inclusion/Exclusion Criteria  Outcome  No. patients (Biofeedback)  No. patients (Control)  urinary incontinence.  Exclusion: Anatomical urological disorders except vesicoureteral reflux, Neurological disorders of bladder/sphincter function. Previous cognitive bladder training. Insufficient cognitive capacity, operationalized as: Ability to read the clock, Sufficient understanding of the problem as estimated by the therapist  Vasconcelos(130) 2006/Brazil  Randomized open label trial  Group 1: Standard therapy + pelvic training  Group 2: Standard therapy + pelvic training + biofeedback  Inclusion: Age > 5 years, failure to respond to previous treatment, willingness to undergo the training sessions, and informed consent of parents or guardians.  Frequency of incontinence episodes (diurnal and nocturnal), Urinary tract infections, uroflowmetric parameters, Presence of urge incontinence, 20  21  53  First Author Year of publication/Country  Study Design  Intervention  Inclusion/Exclusion Criteria  Outcome  No. patients (Biofeedback)  No. patients (Control)  Exclusion: Occult neurological lesions, complex urinary tract abnormalities [other than Vesico-ureteral reflux (VUR)], attention deficit hyperactivity disorder, and primary mono symptomatic nocturnal enuresis.  constipation, fecal soiling, pelvic floor contraction and relaxation  Kibar(131) 2010/Turkey  Prospective open label trial  Group 1: Standard urotherapy + biofeedback (voiding biofeedback with urodynamics)  Group 2: Standard urotherapy alone  Inclusion: Age>5, PVR>20ml, Abnormal voiding pattern (staccato), good motivation and willingness to undergo biofeedback treatment  Exclusion: Family or psychosocial problems and any neurologic abnormality  Voiding pattern, uroflow-electromyography, urinary tract infection (UTI), and PVR  62  32  *van Gool(132) 2014/Germany  Randomized multi-center open label trial with parallel groups  Branch I: Children with OAB:  Inclusion:  Branch I: urge incontinence, un-inhibitable urge to void, and numerous Day time in continence with/without UTI, fecal incontinence and bowel 55  50  54  First Author Year of publication/Country  Study Design  Intervention  Inclusion/Exclusion Criteria  Outcome  No. patients (Biofeedback)  No. patients (Control)  1) Cognitive treatment + oxybutynin  2) Cognitive treatment + Placebo  3) Cognitive treatment + bladder training  Branch II: Children with Dysfunctional voiding:  1) Cognitive treatment alone  2) Cognitive treatment + pelvic floor training with online feedback  small voidings per day (OAB), with/without history of UTI and/or VUR  Branch II: urinary incontinence and staccato or fractionated voiding (DV), with/without history of UTI and/or VUR  Exclusion: Renal or urinary tract disease other than UTI with/without VUR; preceding urinary tract surgery (including urethral dilatation); mono-symptomatic nocturnal enuresis; known neurological disorder; informed consent refused; inability to follow protocol; known adverse reactions to anticholinergics; age < 6 or age > 12.  movements, Achenbach’s Child Behavior Checklist  * Not included in the meta-analysis.55  Risk of Bias in Included Studies  Risk of bias in included studies were assessed using Cochrane Collaboration’s risk of bias instrument.(123) Four(128-130, 132) out of the five included studies had low risk of selection bias with regards to random sequence generation and one study(131) had unclear risk due to incomplete information on the randomization procedure. Three studies(128, 129, 132) had a low risk for allocation concealment while in 2 studies(130, 131) the risk was unclear. All 5 studies had high risk for performance bias, because the personnel and participants were not blinded. Four studies(128, 130-132) had low risk of attrition bias because of adequate follow-up information. One study(129) had 10% loss to follow-up without explanation. Details regarding possible adverse events due to use of biofeedback was not clearly described in any of the included studies. Figure 3.2  and Figure 3.3  illustrate the summary details of the risk of bias assessment in the included studies. We did not assess publication bias by funnel plot method due to the limited number of studies in this review. Figure 3.2 Review authors' judgments about each risk of bias item presented as percentages across all included studies.  56  Figure 3.3 Review authors' judgements about each risk of bias item for each included study. Green = low risk, yellow = unclear risk, red = high risk.  Quality Assessment of Outcomes in Included Studies As mentioned earlier in the methods section of this chapter, GRADE guidelines (133) was used to assess the quality of evidence for outcomes of interest across included studies. The overall rating of all the outcomes of interest across included studies was scored as low quality of evidence (Table 3.3 ). Risk of bias for all outcomes was scored as serious due to the open label design of the studies.(134) Inconsistency was considered not serious for all outcomes as the p-values of the Chi-squared test for heterogeneity were all > 0.05; in addition, the I2 yielded 0% for 57  all outcomes.(133) Indirectness was considered not serious as all the studies assessed considered similar population, interventions, and outcomes of interest.(135) Imprecision was considered serious in resolution of incontinence and reduction in the incidence of UTIs outcomes as the 95% CI of the pooled OR's included an OR of 1.0; as well, the pooled sample size was less than the optimal information size (OIS) of approximately 2,000 patients (estimated assuming a control event rate of 0.2 and a relative risk reduction [RRR] of 25%).(136) Imprecision for the continuous outcome of Q-max was considered serious as well as the mean difference 95% CI included a mean difference of 0.0 and the pooled sample size was less than 400.(136) Other considerations, such as selective reporting of outcomes, were not detected and therefore scored as not serious.58  Table 3.3 Summary of findings. Quality assessment № of patients Effect Quality Importance № of studies Study design Risk of bias Inconsistency Indirectness Imprecision Other considerations biofeedback comparator (standard urotherapy [SU] alone, SU + pharmacotherapy, or SU + neuromodulation) Relative (95% CI) Absolute (95% CI) Resolution of incontinence 3  randomised trials  serious 1 not serious  not serious  serious 2 none  45/63 (71.4%)  40/62 (64.5%)  OR 1.37 (0.64 to 2.93)  68 more per 1,000 (from 107 fewer to 197 more)  ⨁⨁◯◯ LOW  CRITICAL  Reduction in the incidence of UTI 4  randomised trials  serious 1 not serious  not serious  serious 2 none  61/85 (71.8%)  52/78 (66.7%)  OR 1.30 (0.65 to 2.58)  56 more per 1,000 (from 101 fewer to 171 more)  ⨁⨁◯◯ LOW  IMPORTANT  Q-max 2  randomised trials  serious 1 not serious  not serious  serious 3 none  102  72  -  MD 0.5 higher (0.56 lower to 1.55 higher)  ⨁⨁◯◯ LOW  IMPORTANT  CI: Confidence interval; OR: Odds ratio; MD: Mean difference. 1: Open label studies; 2: 95% CI of the pooled OR's included an OR 1.0. Pooled sample size lower than optimal information size (OIS); 3: 95% CI of pooled MD includes an MD of 0.0. Pooled sample size lower than 400 patients. 59  Effects of Interventions Resolution of incontinence (achieving complete dryness) Four out of the five included studies reported resolution of incontinence as their primary outcome.(128-130, 132) One of the studies assessed outcomes at 6 months only.(130) Two studies reported them at 6 and 12 months(128, 129) and one study assessed the outcomes at 18 months.(132) To ensure homogeneity, we pooled the results at 6 months. The result of our meta-analysis shows that the proportion of children with resolved incontinence and difference in events rate (RD) did not differ significantly between the groups (OR 1.37 [CI 0.64 to 2.93], I2 0% and RD 0.07 [−0.09, 0.23], Figure 3.4 and Figure 3.5 ). Figure 3.4 Forest plot of comparison between biofeedback and standard treatment, outcome resolved incontinence (OR). M-H, Mantel-Haenszel test.  Figure 3.5 Forest plot of comparison between biofeedback and standard treatment, outcome resolved incontinence (RD). M-H, Mantel-Haenszel test.  60   Reduction in incidence of urinary tract infection  Recurrent UTI was reported in four studies. Two of these studies(130, 131) reported the rate of UTI at 6 months while the other two studies(128, 129) described results at 6 and 12 months. For consistency, the data were pooled at the end point of 6 months. The meta-analysis did not show a statistically significant difference between biofeedback and control groups for reduction in incidence of UTI (OR 1.30 [95% CI 0.65 to 2.58], I2 0%, Figure 3.6 ). Figure 3.6 Forest plot of comparison between biofeedback and standard treatment, outcome reduction in incidence of UTI. M-H, Mantel-Haenszel test. Post-void residual volumes PVR was reported in three studies(128, 130, 131) of which only two of them reported PVR as a continuous variable.(128, 131) However, we were unable to combine the data from these studies due to structural error in the reporting of mean PVR in one of the studies.(131) For this reason, we contacted the authors multiple times to obtain more information; however, we did not receive a response. The third study reported PVR as a dichotomous variable (improved/not improved).(130)    61  Average urinary flow rate Only one study reported Q-average.(128) The authors reported an increase in Q-average from 9.2 to 10.9 ml per second (p = 0.03) in the biofeedback group and from 7.5 to 8 ml per second (p = 0.6) in the control group at 1 year of follow-up. Mean peak urinary flow rate Q-max was only reported in two studies.(128, 131) Our meta-analysis did not show a significant difference in pooled mean difference between biofeedback and control groups for Q-max (pooled mean difference 0.50, 95% CI −0.56 to 1.55 ml per second, I2 0%, Figure 3.7 ). Figure 3.7 Forest plot of comparison between biofeedback and standard treatment, outcome Q-max. M-H, Mantel-Haenszel test.  Adverse events None of the studies reported any adverse events related to use of biofeedback or urotherapy. Health related quality of life outcomes were also not reported. 3.5 Discussion As my first study (Chapter 2) showed, children with BBD had significantly lower overall cardiac ANS activity as measured by parameters of HRV. Given the limited number of treatment options available for these children, the seemingly non-invasive nature of biofeedback training, and the evidence showing biofeedback’s capability in up-regulating the activity of the ANS, I initially decided to propose (as part of my dissertation) testing the cardiac ANS activity before and after 62  administration of biofeedback in children with BBD to whom biofeedback was to be prescribed as part of routine therapeutic management of their voiding dysfunction. However, after reading a few articles I found that there is inconsistency in the current evidence regarding the clinical efficacy of biofeedback in children with BBD. This made me hesitant in pursuing my research with this approach. To ensure I was on the right path, I decided to conduct a systematic literature review to investigate the efficacy of biofeedback on the clinical symptoms of BBD.  In this systematic literature review we evaluated the effect of biofeedback as an adjunctive treatment in children with BBD. Some studies have previously reported benefits by using biofeedback in addition to urotherapy.(114, 137, 138) However, other studies have shown no benefit.(132) During our initial literature search, we found two previously published systematic reviews on pediatric daytime incontinence(119, 120). In one of the reviews(119), published in 2003, the eligible population for the review consisted of children with daytime urinary incontinence with interventions including imipramine, alarm system, tolterodine and biofeedback. Of the four studies included only 1 RCT was biofeedback related; therefore, the investigators decided not to pool the results across the included studies due to high level of heterogeneity. The second review, published in 2011, comprised of patients with dysfunctional elimination syndrome. This review included 27 studies of which only 1 study was an RCT while the rest were case-series.(120) There was significant heterogeneity between the studies included in this review mostly due to different methods of outcome assessment and intervention (I2 for improvement of daytime incontinence was 77%). Overestimation of treatment effectiveness is also a well-known fact in non-controlled, non-randomized clinical trials.(139, 140) Finally, the 63  authors of this review concluded that biofeedback is an effective treatment for dysfunctional elimination syndrome but also acknowledged that the included studies were of low quality.  In this systematic review, we combined data at 6 months of follow-up for the primary outcome of interest, which was available in only 3 of the 5 included studies.(128-130) van Gool et al reported the outcome of interest at 12 months after termination of the treatment which was 18 months after randomization.(132) It has been shown that up to 20% of children with BBD may have spontaneous improvement in their symptoms of incontinence during 6-month period with no intervention (141); therefore, we did not include the data from van Gool et al. study in our meta-analysis since the longer follow-up may have modified the outcome independent of the intervention. In addition, this study demonstrates no benefit of biofeedback when added to cognitive therapy. The cure rate was reported 49% for patients in the biofeedback arm and 52% for those undergoing cognitive therapy alone (p >0.05).(132) Therefore adding this study to the meta-analysis would have introduced unwarranted heterogeneity and without changing the final results. The result of the current systematic literature review reveals no evidence of benefit in using biofeedback for resolving daytime incontinence. In addition, having biofeedback as an adjunctive treatment to urotherapy was associated with no change in other outcomes such as the rate of recurrent UTI or uroflowmetric parameters. The act of randomization in RCTs eliminates selection bias thus providing sound evidence of the effects of interventions. Most of the included studies in this review were shown to have low risk of bias for randomization and allocation concealment which adds to the quality. However, four of five included studies were small, single centered and probably under powered to detect small 64  differences between intervention groups. Another limitation was that the frequency of wetting episodes was measured subjectively across the studies; hence, wetting episodes were considered as a binary outcome. In addition, we found inadequate evidence regarding measured uroflowmetric parameters and no evidence for health-related quality of life in these children. None of the studies reported adverse events although it seems unlikely that biofeedback may cause any harm.  Overall there is a gap in the current evidence supporting use of biofeedback, mainly due to the limitations of available studies. Biofeedback is noninvasive, especially compared to other recommended ancillary treatment options. However, its use is associated with costs to patients and the health care system.  Moreover, BBD comprises a heterogeneous group of disorders, which is also evident from the characteristics of the included studies (Table 3.2 ). For instance, different inclusion criteria were used to define LUT dysfunction such as increased PVR and presence of abnormal pelvic floor electromyography. We also found diversity in the nature of the interventions and duration of the studies which adds to the existing heterogeneity among the studies. For the above-mentioned reasons, the current systematic literature review does not support the use of biofeedback in children with BBD. Nevertheless, one should not interpret lack of evidence of effect as evidence of no effect.(142) There might be subgroups of children with BBD who may benefit from use of biofeedback as an adjunctive treatment and future studies should focus on identifying these subgroups of children.  Finally, the results of the current systematic literature review do not support evidence of efficacy of biofeedback as an adjunctive treatment for children with BBD. Nonetheless, the available 65  evidence is limited and not of high quality. Well designed, RCTs with adequate power and objective measurements of outcomes are required to elucidate the efficacy of biofeedback in children with BBD. Other important outcomes such as economic impact and health related quality of life changes should also be investigated.  66  Chapter 4: The Effects of Anti-muscarinic Agents on the Activity of the Cardiac Autonomic Nervous System in Children with Functional Overactive Bladder (A Subtype of BBD)  4.1 Synopsis Background: Functional overactive bladder (OAB) in children is one of the most prevalent sub-types of BBD causing symptoms of urinary urgency accompanied by frequency and nocturia, with or without urinary incontinence. The autonomic nervous system (ANS) plays a key role in normal bladder function. The results of my first study (Chapter 2) showed a significantly lower overall ANS, and lower cardiac PNS activity in children with BBD compared to healthy controls. Given that anti-muscarinic agents are considered the gold standard treatment for children with OAB and in the absence of evidence evaluating the effect of these medications on the already low PNS profile of these children I assessed cardiac ANS activity, measured by spectral analysis of heart rate variability (HRV) and impedance cardiography (ICG), in children with OAB before and 3 months after receiving oxybutynin (a frequently used anti-muscarinic agent).  Methods: This was an extension of the study previously described in Chapter 2 conducted at the department of Urology in the Children's & Women's Health Centre of British Columbia between October 2013 and April 2016 involving a follow-up of 10 children diagnosed with OAB who were prescribed a daily regimen of oxybutynin in combination with standard urotherapy as part of the normal practice of the clinic. Cardiac autonomic activity was assessed via spectral analysis of HRV at baseline and during voiding during the first visit and then approximately at 3 months after receiving the treatment. The primary outcome measure was high frequency (HF) parameter 67  of the HRV indicative of the pure activity of the PNS. The other main parameters of HRV/ICG, including total power (TP), pre-ejection period (PEP) and heart rate (HR) were also measured. Additionally, uroflow study outcomes including peak urinary flow rate, average urinary flow rate, volume voided, voiding time, pattern of voiding during urination into an uroflowmeter, and post-void residual (PVR) were investigated during voiding. Questionnaires were also used to assess the severity of LUT symptoms (BBD questionnaire) and quality of life (PinQ questionnaire) in children with BBD. Results: Ten children diagnosed with OAB (3 boys and 7 girls with median age of 10 years, range 6 to 14) were followed for median treatment duration of 11.8 weeks (range: 6 – 19.4 weeks). Comparison of outcomes during the storage phase before and after treatment with oxybutynin showed a significant reduction in the HF component of HRV after treatment with oxybutynin (median relative HF change from baseline to post-treatment = -24.17%, p = 0.047). Participants also showed a reduction in median TP (median relative TP change from baseline to post-treatment = -13%, p = 0.333) and an increase of +5.31% in their median HR (p=0.139) with no change in PEP after treatment. During voiding, PEP and HR did not change from before to after treatment with oxybutynin. Participants also showed trend indicative of a reduction in the median relative change from baseline for the main uroflow parameters of peak urinary flow rate (-36.1%, p = 0.508), average urinary flow rate (-41.2%, p = 0.139), and voided volume (-58.2%, p = 0.646) after receiving oxybutynin. PVR was developed de novo after treatment in 3 patients and increased significantly in another 2 patients. Use of medication did not significantly bring a complete resolution in any of symptoms reported by the patients and was associated with 68  commonly reported side-effects of dry mouth, and dry eyes which were reduced after the first month of medication consumption. Conclusion: The results of this study demonstrate that the use of oxybutynin (an anti-muscarinic agent) is associated with further reduction in the activity of the cardiac PNS in OAB children who already have significantly lower PNS activity compared to otherwise healthy children. The findings of this study, in the context of the crucial role of the ANS in normal bladder function, opens the door to the possibility of considering new treatment modalities aimed at modulation of the ANS. As well and after establishing a possible positive effect, the assessment of the ANS activity could be tested as an objective surrogate outcome for monitoring the effects of current and proposed interventions in the setting of future research studies in children with OAB. Finally, further studies with larger sample sizes are needed to verify the results of this study and test the development of more effective treatment strategies for managing OAB.            69  4.2 Background and Objectives Overactive bladder (OAB) in children might have neurological, anatomical, or functional origin.(9) In this dissertation, I will be discussing functional OAB, also referred to as “urge syndrome”, which is one of the most prevalent subtypes of BBD. OAB is characterized by phasic involuntary contractions of the bladder detrusor muscle during the storage phase of bladder function causing symptoms of urinary urgency accompanied by frequency and nocturia, with or without urinary incontinence.(1, 5) Children with OAB usually experience small quantities of urine loss often happening during the afternoon when the child is pre-occupied with either playing or doing activities and is not alert enough to contract the pelvic floor in response to the urge sensation.(1) These children have small bladder capacity for their age and usually choose to drink very little water to escape the leakage and a consequent social embarrassment. This low fluid intake together with the habitual pelvic floor contraction to counteract the urgency to void may often lead to inappropriate postponement of defecation which can result in constipation or fecal soiling.(6, 7, 77) The low intake of water in these children also helps mask incontinence as the primary presenting symptoms.(143)  Epidemiology: the prevalence of OAB has been reported by several cross-sectional studies. From these studies a large cross-sectional study on 16,516 children in Korea between 5-13 years of age reported an overall prevalence of 16.59% for OAB.(144) Another large survey on 5,282 Japanese schoolchildren between 7-12 years of age reported an overall prevalence of OAB to be 17.8% (19.1% in boys and 16.6% in girls) with increased daytime frequency comprising the highest prevalence among other symptoms of OAB (73.8%) next to urge incontinence (33.8%), constipation (20.4%), nighttime bedwetting (13.3%), and increased daytime frequency together 70  with urgency incontinence (7.5%).(145) Studies using urodynamic evaluations have also documented urge syndrome to be the predominant urodynamic finding (52-58%) in children with voiding dysfunction.(10, 146) It has been reported that the prevalence of OAB gradually decreases with increasing age in both genders(144, 145) with one study showing an overall prevalence of OAB significantly decreasing with age from 22.89% at the age of 5 years to 12.16% at the age of 13 years.(144) Diagnosis: as already discussed in Chapter 1, the diagnostic evaluation of children with BBD in general and OAB in particular, consists of a thorough history taking and physical examination combined with a documentation of the bladder and bowel function through the use of validated questionnaires quantifying severity of LUT and bowel symptoms(20, 21), as well as bladder and bowel diaries.(5, 19) In addition, uroflow study is also used to facilitate the diagnosis and screening of these children by measuring the rate, volume voided, voiding time, pattern of voiding during urination into an uroflowmeter, and post-void residual (PVR).(19, 23, 24)  Etiology: the exact pathogenesis of OAB in children is yet to be known. Several different theories have been proposed regarding the development of OAB in children. These theories have already been described in Chapter 1 and vary from neurocentric to vesico-centric theories including: 1) immature CNS control mechanisms; 2) low levels of inhibitory vasoactive intestinal peptide in detrusor muscle; 3) abnormal enkephalin-mediated inhibitory neurotransmission; 4) denervation hypersensitivity; and 5) change in the properties of the detrusor muscle cells (myogenic basis)(1, 91, 147-149)  71  Management: Urotherapy stands as the basic nonsurgical nonpharmacological conservative treatment strategy for children with OAB based on the concept of educating the patients and their families on the normal and abnormal LUT and bowel functions and training them on proper voiding and bowel habits as well as modifying their lifestyle by changing their diet and fluid intake.(29) Other treatment modalities may also be used in addition to standard urotherapy including pelvic floor muscle retraining (biofeedback),(150) pharmacological treatment including anti-muscarinic agents (oxybutynin, propiverine, tolterodin, and solifenacin),(151, 152) transcutaneous neuromodulation,(153, 154) and injection of Botulin Toxin A (BTA) into the detrusor muscle(155, 156) as an off-label treatment in children with OAB. Anti-muscarinics: these agents are the most frequently used pharmacological agents and the gold standard treatment for children with OAB.(1, 157) They work by blocking muscarinic receptors in detrusor muscle (mainly M3 and M1) that bind to acetylcholine therefore reducing the frequency and intensity of involuntary bladder contractions which results in an increased functional capacity of the bladder.(1, 91) From this class of drugs, oxybutynin (anti-muscarinic muscle relaxant with anesthetic properties) has traditionally been the drug of choice.(29, 77) Use of anti-muscarinic agents and in particular oxybutynin comes with the drawback of frequently occurring side effects causing up to 10% of children to discontinue treatment.(158) These side effects are mostly due to non-selective blockage of muscarinic receptors that are localized outside of the bladder.(29, 159) The common side effects of using anti-muscarinic agents include oral (reduction in saliva production which may cause dry mouth and tooth decay), visual (dry eyes and blurred vision), and gastrointestinal (constipation and distention) symptoms(143) which are reported to occur in up to 74% of patients.(160, 161) Due to the specific property of 72  oxybutynin in crossing blood-brain barrier,(162) its use has also been linked to central nervous system (CNS) symptoms.(143) A review of the United States Food and Drug Administration data regarding reported CNS adverse effects associated with the use of oxybutynin in children has revealed CNS symptoms such as dizziness or psychological problems (hallucination, agitation, sedation, confusion, amnesia, and nightmares) occurring in up to 31% of young children who were prescribed with this drug.(163) Side effects caused by oxybutynin have also been shown to occur 3-4 times more frequently in children than in adults.(163, 164)  A multi-center randomized controlled trial comparing a 6-month-long regimen of oxybutynin to 6-12 sessions of bladder training or placebo, all in addition to standard urotherapy for the treatment of urge syndrome did not find a significant difference in improvement of the symptoms between oxybutynin (43% cure rate), bladder training (44% cure rate) and placebo (39% cure rate) 12 months after completion of the treatment.(132) Retrospective and prospective observational studies have also shown non-satisfactory improvement of OAB symptoms after long-term follow-ups post-antimuscarinic agents.(165, 166) From these studies, administration of anti-muscarinic agents in addition to standard urotherapy after an average follow-up of 3.15 (± 1.92) years has been shown to bring about a complete recovery from all symptoms of OAB in only 45.7% of girl and 50% of boys with a 75% success rate in resolution of daytime incontinence.(166)   Normal bladder function has already been well-described and discussed in previous chapters with the main components of the ANS (i.e. PNS and SNS) having a key role in regulating the storage and voiding phases of micturition process (please see section 1.2). However, there is a gap in the evidence with regards to the ANS activity in children with BBD. The results of my 73  first study (Chapter 2) showed that children with BBD (with the majority of population comprising of patients with OAB) at storage phase had significantly lower HRV with significantly lower PNS activity compared to healthy controls. This seemed to be in contrast with the idea of higher PNS activity during normal voiding since these children were mostly suffering from symptoms of incontinence, urgency, and detrusor overactivity although we need to acknowledge that OAB occurs during the storage phase while normally the higher PNS activity happens during normal voiding phase. In the discussion section of Chapter 1, I described and justified the results of our first study which may seem to be in contrast with what we might have intuitively expected to find which was the overactivity of the PNS in children with OAB (please see Section 2.5).  While children with OAB compared to healthy controls have a significantly lower overall ANS function with lower PNS activity,(167) the most widely accepted adjunctive treatment modality for children with OAB is the use of anti-muscarinic agents. The rationale behind the use of anti-muscarinics for the treatment of OAB has so far been focused only on local blockade of muscarinic receptors in bladder detrusor muscle (mainly M3 receptor) without further investigating the overall effects of these medications on the ANS activity.(1) In light of our results showing an already lower PNS activity in children with OAB and the absence of evidence showing the effects of such treatment on the overall activity of the cardiac ANS and in specific the PNS component, I decided to follow a subset of population from our first study (Chapter 2) who were diagnosed with OAB and received anti-muscarinic agents to address the question - How does the use of anti-muscarinic agents, as one of the widely recommended adjunctive 74  treatments for children with OAB (a subtype of BBD), influence the autonomic activity as captured by spectral analysis of the HRV? The primary objective of this study was to investigate the effects of anti-muscarinic agents, used in management of children with OAB (a subtype of BBD), on the cardiac PNS activity. I hypothesized to find a significant reduction in the PNS activity of these children measured by HF after 3 months of using anti-muscarinics. The secondary objectives of this study were to investigate the effects of anti-muscarinic agents on the overall activity of the ANS, uroflowmetric variables, and severity of BBD symptoms and quality of life as measured by BBD and PinQ questionnaires, respectively.  4.3 Methods This was an extension to my first study (details of the original study are described in Chapter 2) conducted between October 2013 and April 2016 to assess the cardiac ANS activity in children with OAB (a common subtype of BBD) before and after receiving anti-muscarinic agents as part of their treatment.  The study involved following 10 children diagnosed with OAB who were prescribed a daily regimen of 0.4 mg/kg of oxybutynin(29) in combination with standard urotherapy as part of the normal practice of the clinic. In addition, in case of constipation the patients were also prescribed daily consumption of stool softeners (Polyethylene (PEG) 3350 at 0.5 to 1.5 g/kg daily). Inclusion criteria for this study has already been described in chapter 2 section 2.3 but in specific the children were between the ages of 5 to 18 and diagnosed with OAB who received prescription for anti-muscarinic agents in addition to standard urotherapy on the first assessment visit. Diagnosis was made by our attending pediatric urologists (Drs. KA and AEM) via a 75  combination of thorough clinical history, physical examination, validated questionnaire,(21) uroflowmetric evaluation, as well as voiding and stool diary. Exclusion criteria consisted of any anatomical or neurological lower urinary tract abnormalities such as posterior urethral valve, urethral obstruction or stricture, ectopic ureters, and congenital abnormalities of the spinal cord, as well as the current use of medication or treatments which were known to affect bladder function or the activity of the ANS. Children with psychological/behavioral abnormalities known to affect the activity of the ANS (such as ADHD, or severe anxiety disorder) or prevent the cooperation of the child with the study coordinator or the urologist were also excluded from participating into this study.  After a full discussion of the study procedure and consent/assent obtainment, anthropometric data including weight and height were collected (please see Chapter 2 section 2.3 for full details regarding baseline measurement). Subsequently, all participants were asked to fill out validated questionnaires already described in Chapter 2. Briefly, the Dysfunctional Voiding Questionnaire (or BBD questionnaire) which measures the frequency and severity of BBD symptoms (Appendix A)(21), and the Pediatric Incontinence Quality of Life Questionnaire (PinQ) which measures the impact of the symptoms on the patient’s quality of life (Appendix B).(22)  The cardiac ANS activity was measured in a controlled environment and according to a protocol for capturing and analyzing HRV and ICG as described in Chapter 2 (see also Appendix C). Data were collected by Biopac MP150 data acquisition system using 6 electrodes on the skin surface of the chest, neck, and lower back. Signals were sampled at 500 Hz throughout all the testing. At first, participants were asked to sit comfortably on a chair for 5 minutes prior to HRV data recording. After the 5-minute adaptation period, baseline data recording was performed for 76  5 minutes according to the standard recommended length of recording for a short-term spectral analysis of HRV.(53) Consequently, the participants were asked to sit on a uroflowmetry unit and urinate. The main uroflow parameters measured were average and peak urinary flow rate, and voided volume.(80)  Uroflow and HRV/ICG data were recorded during 2 visits (before and after using anti-muscarinics) approximately 3 months apart from each other. HRV/ICG data recording consisted of recording at baseline rest when participants were seated on a comfortable chair as well as during the voiding period. At the end of urination, the participants were asked to lie down on a bed so an attending urologist could measure their urinary residual volume (PVR) using a hand-held ultrasound device.  After completion of the data collection and manual review and analysis of the captured HRV/ICG data via AcqKnowledge® software version 4.2, HRV/ICG and uroflow variables already described in Chapter 2 were reported in tables of results.  Descriptive statistics were used to report the results of this study at baseline and after approximately 3 months of treatment with anti-muscarinic agents: median value and quartiles are reported for each parameter and changes over time. To control for the baseline differences between participants, relative percentage change from baseline rest to the voiding period was calculated. For the before-after comparison of the ANS and uroflowmetric profile, Wilcoxon signed-rank test was used as a non-parametric approach. McNemar’s test was also used for paired nominal variables (presence of symptoms reported through BBD questionnaire).   For all statistical tests, a P value of < 0.05 was considered as being statistically significant. Statistical analyses were performed using SPSS® v.22.0. 77  4.4 Results A total of 10 children diagnosed with OAB (3 boys and 7 girls with median age of 10 years, range 6 to 14) were recruited. Participants were followed for median treatment duration of 11.8 weeks (range: 6 – 19.4 weeks) (Table 4.1 ). The most commonly reported symptoms according to patients’ response to specific items on BBD questionnaire(21) were urgency, constipation, incontinence, nocturia, frequency, interrupted pattern of urination, enuresis, encopresis, and dysuria which were reported in 10, 10, 9, 8, 7, 7, 5, 4, and 3 patients, respectively. Please see Table 4.2  for the list of common symptoms reported at baseline and post-treatment in the participants of this study.  Table 4.1 Table of baseline characteristics Variables OAB (n = 10) Gender (% female) 70 Median age, year (range) 10 (6 - 14) Median BMI, kg/m2 (range) 16.7 (14.5 – 22.4) Median follow-up duration in weeks (range) 11.8 (6 – 19.4)     78  Table 4.2 Symptoms reported by BBD questionnaire at baseline and post-treatment. Symptoms Baseline (Visit 1) Post-treatment (Visit 2) p value Urgency, n  10 10 N/A Constipation, n 10 10 N/A Incontinence, n 9 8 1.000 Nocturia, n 8 6 1.000 Increased Voiding Frequency, n 7 4 0.250 Interrupted urinary pattern, n 7 7 0.500 Enuresis, n 5 4 1.000 Encopresis, n 4 4 1.000 Dysuria, n 3 2 1.000 p values reported using McNemar’s test (binomial distribution used). Values are number of patients. N/A, Not Applicable  Comparison of the ANS and uroflow profile before and after treatment with anti-muscarinics: Comparison of cardiac ANS profile at the filling phase before and after using anti-muscarinic agents revealed a significant reduction in the HF component of HRV after treatment with oxybutynin (median relative HF change from baseline to post-treatment = -24.17%, p = 0.047). Participants also had median reduction of 13% in TP component after treatment however this was not statistically significant (p = 0.333). Consistent with the reduction in the HF component, median HR also increased from 78.61 to 84.44 beats/min after treatment (median relative HR change from baseline to post-treatment = +5.31%, p = 0.139). PEP did not change after treatment with oxybutynin (Table 4.3 ). Trend-wise, the main uroflow parameters were also reduced after receiving treatment: peak urinary flow rate, average urinary flow rate, and voided volume median relative changes from 79  baseline were -36.1% (p = 0.508), -41.2% (p = 0.139), and -58.2% (p = 0.646), respectively (Table 4.3 ). During baseline assessment (before treatment initiation) eight patients did not have PVR post-urination; after receiving oxybutynin, 3 of these patients developed PVRs of 60 ml, 80 ml, and 20 ml. The two patients who already had PVR of 60 ml and 55 ml during their baseline assessment also showed an increase in their PVR to 238 ml and 200 ml during their post-treatment assessment.  Comparison of the relative change from baseline to the voiding period was only performed for variables extracted from ICG data analysis (PEP and HR) because in order to assess the HF and TP component of the HRV a recording of a minimum of 1 and 2 minutes is required, respectively.(53) PEP and HR relative change from baseline to during voiding did not change significantly from before treatment to after treatment (Table 4.4 ). Use of oxybutynin was not associated with complete resolution (defined as 100% reduction(5)) in any specific symptoms (Table 4.2 ), however, a significant reduction in the overall BBD score, indicative of the severity of the symptoms, was observed after treatment by oxybutynin (BBD score 23 vs. 18 after treatment, p = 0.034). PinQ score indicative of the impact of the symptoms on the quality of life in these children did not change from before to after treatment (Table 4.3 ).  Use of medication was also associated with commonly reported side-effects of dry mouth, dry eyes, and loose stool which were reduced after the first month of medication consumption.  80  Table 4.3 Autonomic and uroflowmetric profile at baseline and post-treatment. Autonomic Variables Baseline (Visit 1) Post-treatment (Visit 2) Relative change from baseline to post-treatment (median %) p value HF (ms2/Hz) 672.58 (303.74 to 1053.63) 540.09 (283.54 to 671.67) -24.17 (-55.37 to -10.74) 0.047 TP (ms2/Hz) 1159.81 (767.35 to 1560.37) 1106.96 (868.53 to 1238.00) -13.00 (-40.00 to 12.25) 0.333 LF (ms2/Hz)  301.83 (132.12 to 438.91) 301.14 (161.99 to 494.99) -4.17 (-39.88 to 63.52) 0.959 HFnu 63.24 (54.75 to 72.36) 60.93 (47.69 to 65.90) -9.61 (-26.67 to 6.36) 0.139 LF/HF ratio 0.59 (0.39 to 0.83) 0.64 (0.52 to 1.12) 47.37 (-14.85 to 142.52) 0.203 PEP (msec) 112.12 (107.42 to 115.35) 112.35 (105.06 to 121.11) 1.19 (-4.94 to 5.61) 0.767 HR (beats/min) 78.61 (74.89 to 83.65) 84.44 (73.09 to 87.18) 5.31 (-0.50 to 9.23) 0.139 Uroflowmetric Variables Baseline Post-treatment Relative change from baseline to post-treatment (median %) p value Peak urinary flow rate (mL/sec)  11.4 (5.2 to 22.2) 10.8 (5.6 to 15.1) -36.1 (-57.2 to 183.9) 0.508 Average urinary flow rate (ml/sec) 4.4 (2.2 to 9.8) 2.5 (1.2 to 5.0) -41.2 (-82.7 to 87.8) 0.139 Voided volume (ml)  82.5 (27.6 to 128.6) 45.6 (25.3 to 92.4) -58.2 (-71.3 to 199.2) 0.646 Uroflow curve (n abnormal) 5/9 8/8 - N/A BBD score 23 (19 to 31) 18 (14 to 22) - 0.034 PinQ score 37 (26 to 49) 35 (13 to 48) - 0.206 p values reported using Wilcoxon signed-rank test. Values are median (IQR).    81  Table 4.4 Relative change in autonomic profile from filling to voiding phase during baseline and after receiving full treatment of oxybutynin. Autonomic Variables Relative change from pre-voiding to voiding (%) p value Baseline (Visit 1) Post-treatment (Visit 2) PEP (msec)  -0.74 (-3.62 to 1.97) -0.79 (-5.54 to 0.59) 0.374 HR (beats/min) 6.34 (0.66 to 13.60) 8.43 (-6.04 to 13.34) 0.678 p values reported using Wilcoxon signed-rank test. Values are median (IQR).   4.5 Discussion The current evidence does not establish a solid efficacy profile in treatment modalities for BBD.(31) Nevertheless, anti-muscarinic agents, which are yet to be proven highly efficacious,(132, 165, 166) are the most widely accepted adjunctive treatment for children with OAB.(1, 157) The rationale for the use of anti-muscarinics has so far been focused only on local blockade of muscarinic receptors in bladder detrusor muscle (mainly M3 receptor) without further investigation into the effects of these medications on the overall ANS activity.(1) To my knowledge there is no study that has previously assessed the effect of anti-muscarinics on the activity of the ANS through analysis of HRV in children with OAB. Since it is known that PNS is more active during the voiding phase, the assumption could be that children with OAB might have in general a higher PNS activity than otherwise healthy children. However, the results of our first study (Chapter 2) proved that these children, in fact, present with a significantly different ANS profile comprising of significantly lower HRV and in particular PNS component compared to healthy controls.  To my knowledge, this is the first study that objectively assesses the activity of the cardiac ANS in children with OAB after having received a daily regimen of anti-muscarinics for 82  approximately 3 months compared to the baseline assessment (before treatment) to assess the effect of the treatment on the activity of the ANS in these children.  The results of the current study provide remarkable information suggesting that the use of oxybutynin (an anti-muscarinic agent) is associated with even further reduction in the activity of the cardiac PNS in children who already are significantly lower in their PNS activity compared to otherwise healthy children (relative HF change from baseline to post-treatment = -24.17%, p = 0.047). Although this level of significance could merely be due to the fact that we did not calculate power for this study and the sample size was small. There also was a reduction in the median total HRV (TP) of -13% (IQR = -40.00 to 12.25) after treatment with oxybutynin, however, this change was not statistically significant. The overall severity of BBD symptoms was decreased significantly; however, we did not observe a significant complete resolution in any of the individual OAB symptoms recorded through BBD questionnaire after the treatment. These symptoms included urgency, constipation, incontinence, nocturia, frequency, interrupted pattern of urination, enuresis, encopresis, and dysuria (Table 4.2 ). It is worth noting that although bowel symptoms such as constipation and encopresis may co-exist with one another(168) and with LUT symptoms in BBD patients,(6, 7) since OAB treatment with anti-muscarinics have been shown to worsen constipation,(169) clinicians are advised to consider treating constipation before starting anti-muscarinic treatment for OAB.(170) In the case of this study, as already mentioned in the methods section, our clinicians treated underlying constipation with stool softeners simultaneously while treating OAB. This could mostly be due to avoiding multiple patient trips to the clinic for the sake of patient’s convenience and preference. 83  There was a reduction in the main uroflow parameters after receiving treatment, however, none of the changes were statistically significant: peak urinary flow rate, average urinary flow rate, and voided volume relative changes from baseline were -36.1% (p = 0.508), -41.2% (p = 0.139), and -58.2% (p = 0.646), respectively (Table 4.3 ). PVR was also developed de novo in 3 of the patients and increased in other 2 patients whom had reported a PVR of greater than zero during their baseline uroflowmetric assessment. A decrease in detrusor pressure at maximal capacity as well as an overall increase in bladder capacity, and PVR after antimuscarinic therapy has already been shown in studies(171, 172) and some even consider a high PVR at baseline as a contraindication for the use of oxybutynin.(173) It is also worth noting that elevated PVR has been proven to be associated with UTIs irrespective of gender and age.(174, 175)  Proper management of OAB is important since if untreated, it can cause complications such as UTIs, VUR, and upper urinary tract problems.(8, 9) These problems can also be carried into the adulthood in the form of voiding or sexual dysfunctions.(15, 16) The results of this study show that not only the use of oxybutynin was inefficacious in this cohort of patients with OAB to completely resolve the main symptoms of these patients but that its use was also associated with a significant reduction in the HRV parameters. This reduction is possibly due to the non-selective properties of oxybutynin which can interfere with the muscarinic receptors that are localized outside of the bladder.(29, 159) The non-selectiveness action of oxybutynin is also deemed as the responsible pathway for the multiple side-effects that occur in up to 74% of patients.(160, 161)  The further reduction in the PNS component and total regulatory power of the ANS of these children post-treatment with oxybutynin may become clinically important since similar ANS 84  profile (depressed HRV and HF band in particular) as marker of chronic stress, has been shown to be associated with increased inflammation(34, 60, 103-106) and found in several major chronic diseases such as diabetes(61, 62) and cardiovascular disease risk factors.(63, 64) Nonetheless, the reader should be cautious and fair in interpreting the results of this study and making clinical connection between the HRV profile of the population in this study and the population of patients with chronic diseases in the above mentioned studies since oxybutynin is usually not used as a long-term treatment for OAB patients. Therefore, the results of the current study should not be interpreted as evidence that the observed HRV changes are clinically significant after oxybutynin use. In addition, it should be noted that there is no solid efficacy profile for treatment of OAB with oxybutynin with available evidence consisting of published studies and unpublished first-hand physician experience showing both efficacious and non-efficacious properties of this medication in resolving clinical symptoms of OAB.         The current study is important in the context of the crucial role of the ANS in normal bladder function and further investigation into the possible role of the ANS as an objective surrogate treatment target in the investigation of the efficacy of the current and proposed future interventions for the management of children with OAB. Future studies with large sample size are needed to verify the results of this study as well as to test the development of more effective treatment strategies for controlling LUTS by using HRV/ICG parameters as surrogate and objective treatment targets.     85  Chapter 5: Diaphragmatic Breathing as an Adjunctive Therapy in the Management of Children with Bladder and Bowel Disorders: A Pilot Randomized Clinical Trial  5.1 Synopsis Background: Diaphragmatic Breathing (DB) is a type of breathing technique that is performed by a noticeable expansion of the abdomen rather than the chest cavity during inspiration. The role of DB in up-regulation of the ANS activity and its clinical efficacy in reducing symptoms caused by stress and several major disorders including chronic pulmonary diseases, and type II diabetes has previously been established. Given the results of my previous study (Chapter 2) showing a significantly down-regulated ANS activity in children with BBD, the low level of clinical efficacy of the current adjunctive treatments available for this condition, the harmless nature of DB, and the paucity of studies investigating DB as an adjunctive therapy in the management of the children with BBD, I conducted a pilot randomized controlled trial (RCT) to investigate the feasibility of implementing this intervention, assess the processes that are necessary in conducting a large RCT, and to explore scientific evaluation of the treatment effect of DB as a potential adjunctive therapy candidate in the management of children with BBD.  Methods: A pilot randomized controlled trial was conducted at the department of Urology in the Children's & Women's Health Centre of British Columbia between January 2015 and June 2016. A total of 23 children clinically diagnosed with BBD were randomly assigned to standard urotherapy plus a thrice daily practice of DB (Group A, n = 11) or standard urotherapy alone (Group B, n = 12). Feasibility of processes necessary for conducting a larger RCT was assessed. 86  Number of wetting episodes, main parameters of HRV/ICG (i.e. TP, HF, PEP, and HR), uroflow outcomes (volume voided, average and peak urinary flow rate, and post-void residual volume), severity of BBD symptoms and quality of life questionnaires were measured at baseline, and at approximately 3 months after receiving the treatment. Exploratory intention-to-treat (ITT) and per-protocol (PP) analysis were performed.      Results: Eligible participants were recruited at a recruitment rate of 85%. The main reasons for participation refusal were pressure of time, and unwillingness to commit to the treatment regimen and follow-up. Group A and B each had 1 and 5 participants who were lost to follow-up which resulted in retention rates of 91% and 58%, respectively. The average rate of adherence to DB practice was 35% (95% CI: 14-56%). The main reason for this low adherence to DB was noted by the participants as being “boring”. The average overall score for treatment acceptability was 30 (95% CI: 25-35) from patients’ perspective and 41 (95% CI: 37-45) from parents’ perspective indicative of overall good acceptance of DB treatment from both patients and their parents. Participants in both groups had similar characteristics at baseline. Proportion of participants with resolved incontinence at the end of the follow-up period adjusted for age, gender, BMI, and baseline values (values at visit 1) were similar in the two groups in both the ITT and the PP populations. However, average BBD score was significantly reduced in Group A after treatment with DB while remaining unchanged in Group B in PP population (average BBD score before treatment: 25 vs. 20 after receiving DB, p = 0.011 and 20 vs. 21 after receiving urotherapy alone, p = 0.172). HRV/ICG parameters were also similar in both groups for both ITT and PP populations at the end of the trial. Comparison of the two groups’ ANS response to voiding at the end of the trial showed a trend of larger overall decrease in PEP (increase in SNS 87  activity) and an increase in HR during voiding in the control group for ITT and PP populations (average HR change from filling to during voiding at the end of the trial: -2.68% for Group A vs. 10.18% for Group B, p = 0.047 in ITT population| -3.61 for Group A vs. 11.33 for Group B, p = 0.228 in PP population). Uroflow parameters were similar in both groups at the end of the trial for both ITT and PP populations with Group A showing a rising trend while Group B showing a falling trend in average urinary flow rate and PVR for PP population (median PVR: 6 ml vs. 20 ml after receiving DB, p = 0.080 and 20 ml vs 15 ml after receiving urotherapy, p = 0.343). There was a positive correlation between the number of DB practiced and the relative change in the main parameters of HRV (r = 0.58, p = 0.082 and r = 0.69, p = 0.029 for TP and HF relative change from visit 1 to visit 2, respectively). As well, a negative correlation was observed between the number of DB practices and the relative change in the number of wetting episodes per day (r = -0.54, p = 0.215), BBD score (r = -0.56, p = 0.093), and a significantly high positive correlation with PVR change after DB practice (r = 0.72, p = 0.029).  Conclusion: The results of this pilot trial suggest low overall feasibility for proposing DB as a non-invasive intervention in controlling symptoms of BBD due to low practical adherence to DB practice during an approximately 3 months of follow-up regardless of highly positive correlation between DB practice and resolution of clinical symptoms, improvement in the autonomic balance and function, and PVR. More studies with large sample size, and more sophisticated tools in persuading adherence to treatment are warranted to better investigate the efficacy of DB as an adjunctive therapy in the management of children with BBD.  88  5.2 Background and Objectives BBD in children has already been described in previous chapters (Chapter 2 in specific) as a common functional disorder of the lower urinary tract and bowel with heterogeneous group of syndromes.(2, 4) Common presenting symptoms in children with BBD include LUT or bowel symptoms varying from increased voiding frequency, incontinence, nocturia, and enuresis to decreased voiding frequency, hesitancy, straining, intermittency, dysuria, feeling of incomplete emptying, constipation, and post-micturition dribbling.(5) The prevalence of daytime wetting as the most common reason for referral to a pediatric urology department in children with BBD has been reported in a number of cross-sectional studies and varies from 1.8% in a random sample of 7-year-old children(79) to 6% among females and 3.8% among males at the age of 7 years.(13) BBD symptoms seem to increase between the ages of 6 to 9 years and decrease towards puberty with an estimated spontaneous cure rate of 14% per year in daytime wetting.(77) An association between childhood daytime wetting and overactive bladder (OAB), which is the most common subtype of BBD, on the one hand and adulthood urinary incontinence and sexual dysfunction on the other has also been established via observational studies(15-17) The etiopathogenesis of BBD is still unclear. With the ANS function having an active role in normal storage and voiding phases of the bladder(32), little is known of the ANS’ role in the etiopathogenesis of BBD. Changes in cardiac ANS activity, which is a proxy for changes in the overall ANS activity, are explored by assessing the cardiac rhythm with appropriate non-invasive method of spectral analysis of the beat-to-beat cardiac signal variations (HRV/ICG). This method is considered to be the most precise non-invasive procedure to investigate the status of the ANS.(53) Details regarding the ANS activity during normal bladder function as well as the 89  measurement of the ANS activity by means of spectral analysis of HRV and ICG have been described in Chapter 1 sections 1.2 and 1.2.1. Through the first study of my PhD (Chapter 2) I was able to show an association between BBD and a dysfunction of the ANS presented by a significantly lower total HRV and in specific the PNS activity of children with BBD compared to otherwise healthy controls.(167) Diagnostic evaluation of children with BBD consists of a thorough history taking and physical examination combined with a documentation of the bladder and bowel function through the use of validated questionnaires(20, 21), as well as bladder and bowel diaries.(5, 19) Uroflow study is also used to facilitate the diagnosis and screening of these children by measuring the rate, the volume voided during urination into an uroflowmeter, and post-void residual (PVR).(19, 23, 24) It has been shown that symptoms of BBD decrease towards puberty in both genders.(77) Also, the prevalence of OAB (one of the most prevalent subtypes of BBD) has been reported  to gradually decrease with increasing age(144, 145) with one study reporting the overall prevalence of OAB to significantly decrease with age from 22.89% at the age of 5 years to 12.16% at the age of 13 years.(144) Regardless of the spontaneous cure rate of some of the main symptoms of BBD with age, the proper management of BBD symptoms is still necessary due to the importance of avoiding future complications such as UTIs, VUR, or urinary/sexual problems that can be carried into the adulthood resulting from untreated BBD symptoms. The basic conservative treatment strategy for children with BBD is urotherapy. This nonpharmacological treatment consists of five standard steps as follows(5):  90  1) Information and demystification: during which the physician will educate the child and his/her family on normal LUT and bowel function, voiding habits and proper voiding posture and how they might deviate from normality.  2) Instruction: during which the physician will train the child and family on how to resolve the problem through altering behavior with regular voiding and bowel habits, adapting a proper voiding posture, and avoiding holding maneuvers. 3) Lifestyle advice: during which the physician will recommend a lifestyle modification to maintain balanced diet and fluid intake, regular voiding and bowel emptying habits to avoid constipation. 4) Registration: during which the physician will provide the child and family with proper tools such as bladder and bowel diaries to record voiding habits and frequency of symptoms.  5) Support and Encouragement: during which the patient will be booked for regular follow-up appointments with the caregiver. It has been shown through retrospective analysis of children with daytime incontinence that treatment with standard urotherapy alone can bring about 55% cure rate in daytime wetting in these children.(176) However, studies suggest that treatment compliance, although playing a key role in improvement of symptoms,(177) is also not high for standard urotherapy alone.(178) Other adjunctive treatment modalities may also be used in addition to standard urotherapy including biofeedback,(150) anti-muscarinic agents,(151, 152) transcutaneous 91  neuromodulation,(153, 154) and injection of Botulin Toxin A (BTA) into detrusor muscle(155, 156) as an off-label treatment in children with OAB.  As mentioned earlier in the introduction chapter of this dissertation, observational studies analyzing large databases regarding health services utilization trends in outpatient children with functional urinary incontinence in Germany report an estimated 78% of the population under investigation not receiving any specific treatments for their urinary incontinence.(31) The results of such studies show that the current treatment modalities only partially comply with the available current practices for treating children with BBD implying a questionable efficacy of current treatment modalities for urinary incontinence in childcare. Earlier in chapter 2 of this dissertation, an association between BBD and a dysfunction of the ANS was established in children with BBD. In chapter 3, I further investigated through systematic literature review and meta-analysis of randomized controlled trials the relative clinical efficacy of biofeedback as a potential non-invasive adjunctive treatment candidate with ANS enhancing properties for children with BBD. However, the results of this review did not support a positive clinical efficacy for biofeedback. Later in chapter 4, I showed that the use of oxybutynin, a popular FDA approved anti-muscarinic agent used as part of OAB management, is associated with further reduction in the PNS activity, in OAB children who were already significantly lower in their PNS activity compared to otherwise healthy children. The results of this study further questioned the already debatable efficacy(132) and safety profile of using oxybutynin as an adjunctive treatment modality in children with OAB.  Considering the above mentioned together with the available evidence, I believe there is room for improvement in current adjunctive treatments available for BBD. This would open the door 92  to the possibility of investigating the efficacy and compliance of new non-invasive add-on treatment strategies with the ANS function up-regulating capabilities (diaphragmatic breathing being one of them).   Diaphragmatic breathing and the Autonomic Nervous System: The diaphragm is the most efficient muscle involved with breathing, and also happens to be the largest muscle in our body which is in direct contact with the ANS fibers through the vagus and the phrenic nerves.(32) Diaphragmatic or Deep Breathing (DB) is a breathing technique that is performed by a marked expansion of the abdomen (contracting diaphragm) rather than the chest cavity during inspiration. DB has been considered a healthier way to breathe and a useful form of complementary and alternative treatment for stress and several major disorders improving pulmonary function tests in chronic obstructive pulmonary disease (COPD) and asthma, as well as achieving better glycemic control in type II diabetes.(179-183)  The effect of DB on the ANS has been well established through a number of studies. Martarelli et al.(182) in a study on healthy subjects showed that performing 40 minutes of DB exercise 10 minutes after having a meal rich in carbohydrates compared with a control group spending 40 minutes reading a journal significantly increases insulin, and reduces glycemia as well as free radical production. The authors associated the results of this study to being likely due to the activation of the PNS. It is worth to note that the role of ANS and in specific the PNS in increasing insulin secretion from pancreatic cells and regulation of blood glucose has previously been well-established in a number of publications and that hyperglycemia is associated with reduced HRV.(184-186)  93  In another study,(183) the effect of DB as an add-on therapy to standard care (diet and exercise) was compared with standard care alone in 123 patients with type 2 diabetes mellitus. The results showed that the addition of practicing DB twice daily for three months compared to standard care only resulted in a significant reduction in the following outcomes: BMI, waist-hip ratio, fasting and post-prandial plasma glucose, HbA1c (indicator of the average blood sugar levels for a period of approximately 3 months), and biomarkers of cellular oxidative stress (malondialdehyde, superoxide dismutase, improvement in glutathione and vitamin C). The authors concluded that DB can be employed as an effective therapy in reducing oxidative stress, which plays a key role in the development of complications caused by diabetes,(187) while improving the anthropometric and glycemic parameters in type 2 diabetes.(183) Considering that close association between depressed overall ANS, and in particular PNS activity, have been shown in diabetic patients(61, 62) it is possible that the mechanism by which DB renders its therapeutic effects is via upregulation of the ANS activity.  Kulur et al.(188) showed in a study on 145 randomly selected male patients with ischemic heart disease (IHD), IHD and diabetes (IHD-DM), and IHD with diabetic neuropathy (IHD-DN), that practicing DB for 10-15 minutes twice daily was associated with a significant increase in HRV time domain indices at the three- and twelve-month follow-up in all 3 groups. This phenomenon was observed while all non-compliant control groups with the same conditions (IHD, IHD-DM, and IHD-DN) did not experience a significant change at the three-month follow-up and had a significant decrease in their HRV at the one-year follow-up. The authors also reported a significant decrease in HbA1c and blood glucose levels after 3 and 12 months following routine daily DB exercise in all compliant groups while non-compliant groups who did not practice DB experienced 94  no change after 3 months and a significant increase in HbA1c levels occurred in non-compliant IHD-DN group after one year of follow-up.  In another study, 160 healthy volunteers with the mean age of 19 were randomly assigned into two comparative groups where each participant was asked to participate in 4 different exercises presented in a counterbalanced format as follows: (1) sitting quietly with their eyes closed (baseline), (2) engaging in DB, (3) using autogenic relaxation training technique, and (4) counting their pulse (concentration task) while their ECG was monitored for 7 minutes during each exercise. Authors were able to show that both DB and autogenic training produced significantly lower heart rate which was replicable in both groups. They also found DB to be associated with a significant increase in the HF component of the HRV (increased PNS tone) which was different than the effect of other 3 groups.(189) DB has also been found to reduce systolic (SBP) and diastolic blood pressure (DPB) in clinically healthy normotensive subjects in a single-arm clinical trial following three deep DBs performed for 2 weeks at about 2-hr intervals while awake (reduction of 5.6 ± 0.8 mmHg in SBP [P < 0.001] and 1.4 ± 0.8 mmHg in diastolic DBP [P < 0.005]).(190) It is also worth noting that the role of the ANS, and in particular the SNS, in regulating blood pressure has already been well established.(191) This role could explain the mechanism by which DB can reduce blood pressure.  Diaphragmatic breathing and urinary incontinence in children: DB together with pelvic floor muscle retraining (PFM or biofeedback) as an add-on treatment to standard urotherapy has been shown to be beneficial in treatment of urinary incontinence, nocturnal enuresis, and UTIs as well as normalizing urinary function in children diagnosed with dysfunctional voiding (a subtype of BBD presented by habitual contraction of pelvic floor leading 95  to staccato pattern with or without interrupted flow on repeat uroflow).(192, 193) Through an RCT on 86 children diagnosed with dysfunctional voiding (DV) and randomly assigned to standard urotherapy plus DB and PFM (Group A) or standard urotherapy only (Group B), Zivkovic et al.(192) showed that adding DB and PFM to standard urotherapy was associated with significantly larger number of resolution in urinary incontinence (number of resolved incontinence: 20/24 in Group A vs. 2/18 in Group B, p<0.0001), and nocturnal enuresis (number of resolved enuresis: 14/21 in Group A vs. 5/15 in Group B, p<0.0001) in subjects 12 months post-treatment. Authors also reported a larger number of children with resolved UTI and constipation in Group A compared to Group B; however, the difference between the two groups was not statistically significant (UTI and constipation resolution: 13/19 and 15/15 in Group A vs. 6/15 and 6/10 in Group B, respectively). The authors also found a significant improvement in post-treatment uroflow parameters and curve patterns in Group A. One obvious limitation in this study was that the authors were not able to distinguish between DB and PFM (biofeedback) effect. However, as already described in chapter 3,(194) since my systematic review of RCTs on the effect of biofeedback for BBD found the evidence of the efficacy to be insignificant, therefore, I believe that the effect shown in the above trials was mostly due to DB rather than PFM. In another study, 72 children between the ages of 4-18 diagnosed with chronic functional constipation were randomized into physiotherapy (laxatives + isometric training of the abdominal muscles + DB exercise + abdominal massage) or medical treatment (only laxative) groups and the results showed the frequency of bowel movements to be significantly higher in the physiotherapy group than in the medical treatment group 6 weeks post-treatment.(195)  96  There have also been studies investigating the coordination and co-activation of abdominal and pelvic floor muscles using Electromyography (EMG) and real-time dynamic magnetic resonance imaging (MRI). Using anal and vaginal EMG techniques, studies in healthy women with no history of stress urinary incontinence (please see glossary of terms) or lower back pain have shown that there is a programmed co-activation between the pelvic floor and abdominal muscles after a voluntary muscular contraction.(196, 197) Based on the above findings, trunk stabilization rehabilitation programs which use abdominal muscle actions to initiate tonic pelvic floor muscle activity is proposed in women with stress urinary incontinence.(198) Through the use of real-time dynamic MRI, Talasz et al.(199) were able to show that in healthy women, there is a parallel concomitant cranio-caudal movement of the diaphragm and the pelvic floor muscles (PFM) during breathing and coughing with significantly larger mean vertical displacement of the right and left diaphragmatic dome and PFM during forceful breathing and coughing compared to quiet breathing (respective mean amplitudes of cranial movement of the right and left diaphragmatic copulae were 15±6 and 9±7 mm during quiet breathing; 32±15 and 28±16 mm during deep breathing; and 32±13 and 28±7 mm during coughing). The findings of the two above studies supports the previously known correlation between breathing patterns (measured through lung function tests) and PFM contractions(200) making DB a good candidate for the synchronous activation of the pelvic floor muscles.  Continence and micturition is achieved by a tight coordination and balance between urethral and detrusor muscle activity. A study by Sapford et al.(201) has shown that the urethral pressure is under the influence of specific voluntary abdominal actions. The authors described that voluntary abdominal bulging (which is part of what happens during inspiration phase of practicing DB) 97  significantly decreases the urethral pressure. This maneuver might have possible implications for rehabilitation of voiding dysfunction in patients who do not relax the urethral sphincter for voiding (which is part of the pathophysiological mechanism that is described for some of the subtypes of BBD such as dysfunctional voiding).  The above studies and findings explore the possibility that pelvic floor muscles can be reached and exercised indirectly through activation of the abdominal muscles by non-invasive techniques such as DB. The results of my first study (Chapter 2) showed a dysregulated overall ANS function with significantly lower power in the PNS component in children with BBD compared to the healthy controls. As already described in previous chapters there is the cost, invasiveness, partial ineffectiveness, and possible side-effects as well as the complexity that is associated with the prescription and administration of current adjunctive treatment paradigms for BBD. DB exercise on the other hand is a form of non-invasive and peaceful therapy that can be taught and learned quickly at almost no cost that is also shown to be effective in increasing the power in the PNS component towards a better balance in the ANS.  Given the harmless nature of DB, its ease of use and administration, and the paucity of studies investigating this non-invasive complementary and alternative intervention as an adjunctive therapy in the management of the children with BBD and in specific the possibility of such intervention creating a positive change in the ANS activity and clinical symptoms of these patients, I conducted a pilot randomized controlled trial to address the question - Is it feasible to conduct an RCT and investigate the effect of DB as a proposed add-on therapy on the clinical symptoms and ANS activity of children with BBD? This pilot study and our approach of 98  investigating ANS changes overtime in these patients provides imperative feasibility information and a better insight on the real effect of DB on the ANS activity and BBD symptoms, which has never been studied before. The primary objective of this study was to collect useful information on the feasibility of implementing this intervention by assessing the processes that are crucial to the success of a larger trial. These processes include recruitment/refusal rates, retention rates, (non)compliance or adherence rates, eligibility criteria (is it obvious to the recruiting healthcare practitioners who meets and who does not meet this criteria), and practicality of delivering the intervention in the proposed setting. Additional processes also include patients’ understanding of study questionnaires or data collection tools (for instance, do subjects provide no answers, multiple answers or unanticipated answers on questionnaires), length of time to fill out the study forms, and acceptability of the intervention and randomization from patients’ perspectives. Due to DB’s completely non-invasive and peaceful nature as well as its ease of administration and practice, I expected to find that conducting a larger trial was deemed feasible by showing a high recruitment/follow-up rate, practicality of delivering the intervention in the proposed setting, and high acceptability of the intervention and randomization from patients’ perspectives.  The secondary objectives were: 1) To estimate the proportion of participants with resolved incontinence at the end of the trial in the group undergoing standard urotherapy while practicing diaphragmatic breathing (SU+DB) as well as the control group undergoing standard urotherapy only (SU only). I hypothesized to find both groups achieve improvement in their overall clinical symptoms of BBD with the SU+DB group 99  achieving a higher proportion of participants with resolved incontinence compared to the SU only group.  2) To determine whether there is difference in the change in ANS profile following 3 months of practicing DB daily in group SU+DB versus SU only and to see if it correlates with clinical symptoms and uroflowmetric parameters. I hypothesized to find both groups achieve a higher overall ANS power and PNS tone up-regulation at the end of the trial represented by a higher TP and HF values with the SU+DB group achieving higher overall ANS power and PNS activity compared to the SU only group. In addition, I expected to find a positive correlation between intensity of practicing DB and parameters of HRV and average flow rate and post-void residual volume.    5.3 Methods After receiving an institutional research ethics approval certificate from the University of British Columbia’s Children and Women’s Research Ethics Board, I conducted a pilot randomized controlled trial at the department of urology in the Children's & Women's Health Centre of British Columbia between January 2015 and June 2016. The main goal of this study was to assess the feasibility, clinical efficacy, and the ANS activity in children with BBD randomized to a treatment regimen of either “SU+DB” or “SU only” for a period of approximately 3 months. The follow-up period was chosen to be 3 months long in this study because of previous studies showing efficacy of DB practice in type II diabetic patients with or without cardiac ischemia after 3 months(183, 188) and because the routine practice of this clinic was to visit patients after 100  approximately 3 months in order to evaluate the effectiveness of the prescribed initial treatment by the pediatric urologist. This trial was registered at ClinicalTrials.gov registry with the trial identifier NCT02597764.  I used the same eligibility criteria described previously in Chapter 2. In short, inclusion criteria consisted of the children between the ages of 5 to 18 and diagnosed with BBD by our attending pediatric urologists (Drs. KA and AEM) via a combination of thorough clinical history, physical examination, validated BBD questionnaire,(21) uroflowmetric evaluation, as well as voiding and stool diary. Exclusion criteria consisted of any anatomical or neurological lower urinary tract abnormalities such as posterior urethral valve, urethral obstruction or stricture, ectopic ureters, and congenital abnormalities of the spinal cord, as well as the current use of medication or treatments which are known to affect bladder function or the activity of the ANS. Children with psychological/behavioral abnormalities known to affect the activity of the ANS (such as ADHD or severe forms of anxiety disorder) or prevent the cooperation of the child with the study coordinator or the urologist were also excluded from participating into this study.  I decided to recruit 20 children with for this pilot study, a sample size large enough to assess feasibility and to show trends of effect when comparing the two groups. In order to do so, I approached eligible patients for recruitment by attending BC Children’s hospital urology clinic days three times a week for a span of 18 months. After a full discussion of the study procedure and obtaining participant/parents signatures on the informed consent/assent forms, participants were randomized (randomization was concealed) using an online computer software program that generated random sequence (https://www.random.org) into one of these two treatment groups: 101  (1) Group A (receiving standard urotherapy + daily practice of diaphragmatic breathing [SU+DB])  (2) Group B (receiving standard urotherapy only [SU only]).  During the first visit, baseline measurements of outcome variables were made for all participants. For the second visit, which took place approximately 3 months following the first, all participants were assessed for the outcomes of interest. The visits took place as follows:  Visit 1: during the first hospital visit, participant’s anthropometric data including weight and height was measured. Subsequently, each participant was asked to fill out BBD and PinQ questionnaires for severity of BBD symptoms and quality of life, respectively. Details about these questionnaires have already been described in Chapter 2 (Appendix A and B).(21) This was ensued with the measurement of the ANS activity by following a protocol for capturing and analyzing HRV and ICG at rest and during voiding (the same procedure already described in Chapters 2 and 4, Appendix C). In short, HRV data was recorded on all patients for a period of 5 minutes while sitting on a comfortable chair and then consequently during voiding while their main uroflow parameters (average and peak urinary flow rate, and voided volume) were also being measured simultaneously by an uroflow machine. At the end of urination, the participants were asked to lie down on a bed so our attending urologist could measure their PVR volume via a hand-held ultrasound device. For full details and descriptions of HRV/ICG variables and uroflow parameters please see Chapter 2.  After completion of the data collection and manual review and analysis of the captured HRV/ICG data via AcqKnowledge® software version 4.2, the same HRV/ICG and uroflow variables described in Chapter 2 were chosen and reported in tables of results.  102  At the end of visit 1, based on the treatment group where the participant had been randomized to, SU and DB practice were instructed to the participants as follows: Standard Urotherapy: SU was taught to all participants in both groups by our pediatric urologists (Drs. KA and AEM) following the core principles already mentioned in the background section of this chapter.  Diaphragmatic Breathing: Participants randomized to group A were given specific instructions consisted of practicing DB for 10 minutes 3 times daily for a period of 90 days (or until next visit). I taught practice of DB to the participants and their parents randomized to Group A by using pamphlets with illustrations created specifically for this study (Appendix E, Figures E.1 and E.2), video tutorial, and performing real-time practice together with the patient. In addition, I instructed the patients and their families to use “Breathe2Relax” mobile application for the purpose of learning re-enforcement and practicing of DB (http://t2health.dcoe.mil/apps/breathe2relax). Breathe2Relax mobile application is a free and portable tool that uses top notch graphics, animation, narration, and videos to teach and track practice of DB in users. This application is developed and maintained by the Defense Centers of Excellence for Psychological Health and Traumatic Brain Injury (DCoE) National Center for Telehealth & Technology (T2), however, the research features on the application (such as sending the data on the user’s practice to the researcher via email) is blocked by T2. I enabled these features by providing T2 with the ethics approval certificate of the study. No funding for this project was provided by T2 nor did they provide any participants for this trial. T2 only assisted me in using a research-enabled version of the ‘Breathe2Relax’ mobile application (app) in order to collect data from participants. We 103  provided each participant in Group A with an android tablet on which I already had installed a research-enabled version of “Breathe2Relax” application. Any patient-related information was de-identified as per guidelines provided and approved by ethics committee for the purpose of privacy protection and data security. During the participation period, the device was responsible for tracking each participant’s usage of the mobile app, including: •    time and date the mobile app was accessed, •    frequency of use, •    duration of time (in seconds) spent in each area of the mobile app, •    specific features of the app that are accessed (e.g., assessments, tutorial videos, tools) The summary of data recorded onto each participant’s mobile electronic device was supposed to be sent in an excel file to my organizational email address each time the participants practiced DB. Unfortunately, a technical error happened with the research-enabled component of the application during the follow-up of our fifth participant assigned to receive treatment with DB. This participant was not able to send information using the research-enabled component of the application. I tried to resolve the issue by contacting T2. They pinpointed the problem to the technical issues with the application for the android platform and were able to fix the issue the first time via an update to the application, however, the problem repeatedly occurred during the study for this participant as well as the next ones and T2 was not responsive ever since. Therefore, I continued obtaining information on the number of times the participant practiced 104  DB by providing participants with a diary where they could record every day the number of times they practiced DB and bring the form to the clinic during their second visit. In the meanwhile, I continued to provide the remainder of the participants with tablets where they could still use non-research features of “Breathe2Relax” application including video tutorial on how DB is performed as well as receive assistance from the application to practice DB.  Resolution of incontinence and information on bowel movements were also measured using 2-day voiding diaries at the outset of the trial before starting treatment (visit 1) as well as at the end (visit 2). These diaries were provided to the participants on the day of recruitment along with envelopes with the printed return address and stamps as well as information on their booked appointments. Telephone follow-up: all patients were followed via a phone call at approximately 6 weeks following recruitment to ensure proper compliance of the proposed treatment plan and to ensure all questions and concerns have been addressed.   Visit 2: during the final hospital visit, the same procedures in visit 1 were repeated for the assessment of the outcomes after receiving the treatment regimen for the comparison of before to after differences. Subsequently, the participants and their parents in Group A were asked to fill out a standard validated treatment acceptability questionnaire (TAQ)(202, 203) modified to capture treatment acceptability of DB from the patient and their parents’ perspective (Appendix F). Children’s TAQ consists of 6-items measure on a 7-point Likert scale focusing on the acceptability of treatment, ethics of procedures, perceived effectiveness of the treatment, harmful side-effects, knowledge and trustworthiness of the research therapist. In the modified version of children’s TAQ, for this study, I had difficulty explaining the word “ethical” to our patients who 105  were mostly young children and also because DB is a harmless treatment therefore I removed one of the items asking the patients on “how ethical” they think DB might be. I also added 2 items with “Yes/No” answers at the end of the child’s questionnaire in order to get an overall opinion of the child on whether they would recommend DB to other children with similar conditions and if practicing DB has been associated with any possible unpleasant experiences for them. Possible scores for this questionnaire could range from 5 to 35; however, each individual item of the questionnaire can also be interpreted by itself. On the other hand, the parents’ TAQ consisted of 8-items measure on a 6-point Likert scale focusing on the acceptability of treatment, its perceived effectiveness, justification of using it in the context of this disorder, willingness to continue this treatment for their child after completion of the trial, harmful side-effects, whether parents like this treatment, and whether the proposed treatment is able to handle their child’s problem. Possible scores for this questionnaire could range from 8 to 48 with a suggested overall cut-off score of 28 indicating an acceptable treatment.(204)    Descriptive statistics on baseline characteristics and outcome parameters in both groups were used to report the results of this study. Feasibility of the processes that need to take place as part of pursuing with larger randomized trial were assessed and reported according to Thabane et al.’s tutorial(205) on pilot studies. The scientific exploratory results of this pilot study were reported by following Consolidated Standards of Reporting Trials (CONSORT) guidelines.(206)  Relative percentage change from baseline to the voiding period was also calculated in order to control for the baseline differences between participants. Wilcoxon signed-rank test was used as a non-parametric approach to test the within-group difference in the ANS variables, uroflowmetric parameters, as well as BBD and PinQ scores. For between-groups comparison of 106  the ANS and uroflowmetric profile, analysis of covariance test was used adjusting for age, gender, BMI, and baseline values as co-variates.  Analysis of the results was performed using both ITT and PP analysis methods for clinical trials as follows: Intention-to-treat Population: The ITT population was defined as all patients who were randomized and received specific instructions for the treatment arm to which they were assigned. In order to include missing data in ITT for participants who were lost to follow-up or had incomplete assessments, two methods of imputation were used as follows: - For missing outcome data at visit 2, imputation by “Last-Observation-Carried-Forward (LOCF)” method was used in which the missing outcome data were replaced by the values observed at visit 1.  - For missing outcome data at visit 1 or both visits, imputation by “Mean substitution” method was used in which the missing outcome data were replaced by the adjusted mean of that variable (adjusted for age, sex, and BMI) obtained from all other cases at the according visit.   Per-Protocol Population (PP): The PP population was the subset of ITT patients who had no major protocol deviations affecting the primary endpoint measure or the treatment compliance.     For all statistical tests, a P value of < 0.05 was considered as being statistically significant. Statistical analyses were performed using SPSS® v.22.0. 107  5.4 Results The process of recruitment and follow-up is described in study flow diagram (Figure 5.1 ). Between January 2015 and June 2016, a total of 144 children with BBD were identified and approached for enrollment out of which 27 met the inclusion criteria specific for this study and 23 were recruited and randomized to the proposed treatments. Characteristics of study participants at baseline are described in Table 5.1 . Of the total 23 participants randomized, 11 were assigned to receive treatment regimen in Group A (SU + DB) (6 boys and 5 girls with median age of 6.5 years, range: 5.5 to 10) and 12 to treatment regimen in Group B (SU only) (5 boys and 7 girls with median age of 8 years, range: 7 to 14). The 2 groups were fairly similar with regards to their anthropometric, HRV/ICG, uroflow parameters, and measures of the BBD symptom severity and patients’ quality of life at baseline with all population showing a similar HRV profile to other BBD patients recruited for the first study described earlier in Chapter 2. One participant from Group A and one from Group B could not complete the 5-minute baseline assessment necessary for analysis of HRV variables during visit 1 due to experiencing urgency to void at the time of baseline testing. Also, one participant in Group B could not urinate during the voiding test at visit 1 and thus was missed in the reporting of voiding parameters during baseline assessment. We did not receive the first voiding diaries for 3 participants from Group A. Upon follow-up with 2 of these participants, the parents claimed that they sent us the diaries in the mail but we never received them. The other participant was lost to follow-up and never responded to my follow-up calls. I also did not receive the first voiding diaries for 3 participants from Group B, of which 2 of them were lost to follow-up and one child’s parents claimed that they sent the first diary to us via mail which again I never 108  received. The parents of this child also forgot to bring the second voiding diary to their follow-up visit. I provided the parents of this child with a stamped envelope and asked them to mail the diary to us after returning home from the follow-up visit but I never received it. I also tried contacting them via phone but they were not responsive. The median number of wetting episodes at baseline were 2.8 (range: 0.5 – 4.5) for Group A and 1 (range: 0 – 4.5) for Group B. Both groups were also similar to each other as well as to the BBD cohort studied in Chapter 2 with regards to their BBD and PinQ scores at baseline (median BBD score for Group A: 20 (range: 10 – 30), Group B: 20 (range: 10 – 29), median PinQ score for Group A: 30 (range: 7 – 57), Group B: 27 (range: 8 – 50)).   109  Table 5.1 Table of baseline characteristics. Demographics Group A (n = 11) Group B (n = 12) Female, n (%) 5 (46) 7 (58) Median age, year (range) 6.5 (5.5 – 10.0) 8.0 (7.0 – 14.0) Median BMI, kg/m2 (range) 14.7 (13.6 – 22.5) 16.2 (13.8 – 24.0) Median follow-up duration in weeks (range) 12.6 (10.4 – 20.0) 10.1 (8.0 – 15.0) HRV/ICG variables Group A (n = 10) Group B (n = 11)  TP, ms2/Hz 980.56 (786.00 – 1209.20) 1239.53 (628.07 – 2015.17) HF, ms2/Hz 410.25 (288.94 – 695.97) 680.78 (296.39 – 1408.32) LF, ms2/Hz 350.52 (299.48 – 493.29) 232.48 (179.33 – 685.39) LF/HF ratio 0.91 (0.53 – 1.52) 0.72 (0.43 – 1.12) HFnu, % 52.62 (40.07 – 65.51) 58.02 (47.07 – 69.93) PEP, msec 109.90 (104.13 – 116.68) * 104.36 (97.40 – 116.09) HR, beats/min 83.48 (66.44 – 90.41) * 79.05 (75.89 – 85.19) Relative change (baseline rest to voiding) Group A (n = 11) Group B (n = 11) PEP, % -1.32 (-3.66 – 1.70) -2.59 (-3.67 – 0.94) HR, % 2.22 (-1.40 – 6.88) 5.09 (-4.13 – 14.22) Uroflowmetric variables Group A (n = 11) Group B (n = 11) Peak urinary flow rate, mL/sec  10.4 (3.5 – 15.4) 18.0 (10.1 – 23.3) Average urinary flow rate, ml/sec 4.2 (1.7 – 9.3) 4.9 (1.7 – 7.1) Voided volume, ml  51.8 (23.0 – 157.0) 96.0 (68.0 – 178.0) Post-void residual, ml 15.0 (0.0 – 35.0) 20.0 (0.0 – 40.0) Uroflow curve, n abnormal (%) 8 (73) 10 (83) Voiding diary Group A (n = 8) Group B (n = 9) Median number of wetting episodes per day (range) 2.8 (0.5 – 4.5) 1 (0 – 4.5) Questionnaires Group A (n = 11) Group B (n = 12) Median BBD score (range) 20 (10 – 30) 20 (10 – 29) Median PinQ (range) 30 (7 – 57) 27 (8 – 50) Values are median (IQR) unless specified otherwise. * n = 11. BMI, body mass index; TP, total power; HF, high frequency; LF, low frequency; nu, normalized units; PEP, pre-ejection period; HR, heart rate.  110  Feasibility outcomes  Recruitment/Refusal rate: Of the total of 144 children identified with BBD, initially 27 of them met the eligibility criteria to be enrolled. We were able to recruit and randomize 23 of the 27 which led to a recruitment rate of 85% (Figure 5.1 ). Eligible candidates who did not participate in the study refused either because they were not able to commit to the time needed for each visit (the research portion of each visit took approximately 1 to 1.5 hours to complete), they were unwilling to commit to the treatment regimen and follow-up, or they opted for more rapid treatment options such as the use of anti-muscarinic agents at the discretion of the treating urologist. The other BBD patients who did not meet the eligibility criteria were excluded from enrolment mainly because they were receiving any of the standard treatments including but not limited to SU, or had concurrent psychological/behavioral abnormalities such as attention deficit hyperactive disorder, or autism that were already known to affect the activity of the ANS or could prevent their cooperation with me or our attending urologist.    111  Figure 5.1 Study flow diagram   Retention rate: participants were followed for overall median treatment duration of 12.3 weeks (range: 8 – 20.0 weeks). Out of 11 participants who were randomly assigned to Group A (SU + DB), one child was lost to follow-up by giving the reason “travelling” who also did not practice DB. This led to a retention rate of 91% for Group A. In addition, another 2 patients did not practice DB but showed up for their follow-up appointment and assessment. The reason provided 112  by these patients on why they did not practice DB was because they found the exercise “boring”. Of 12 patients randomly assigned to Group B (SU only), 5 patients were lost to follow-up without notifying us or providing any reasons as to why they decided to drop out of the study. Several attempts were made for the phone follow-up as well as to remind them about their second follow-up visit, however, they did not respond to any of the phone calls. Neither did they show up for their second appointment at the urology clinic. This led to a retention rate of 58% for SU only treatment arm. Adherence/Compliance rate: The average rate of adherence to (or compliance with) DB practice in Group A, calculated as the percentage of the recommended number of DB exercises actually practiced by participants over the follow-up period(207) using below formula specific to this study [𝐴𝑑ℎ𝑒𝑟𝑒𝑛𝑐𝑒 𝑡𝑜 𝑝𝑟𝑒𝑠𝑐𝑟𝑖𝑏𝑒𝑑 𝐷𝐵 𝑟𝑒𝑔𝑖𝑚𝑒𝑛 =Total number of DB practiced by the patient(Patient′s followup duration (in days)𝑥 3)𝑥 100] was 35% (95% CI: 14-56%) (Table 5.2 ) with a minimum adherence of 0% observed in 3 participants and a maximum adherence of 81%. The rate of treatment adherence for Group B was not calculated since part of the treatment involved educating the child and their parents on the disease pathways as well as providing them with proper diet and liquid intake restrictions for which the adherence was hard to measure. In addition, it was not known whether the patients who did not complete the trial decided to withdraw because of resolution of the voiding problems or aggravation of the symptoms.   113  Table 5.2 Table of study treatment adherence and follow-up Indicators of treatment adherence Total (N=23) Group A (SU + DB) (N=11) Group B (SU only) (N=12) p-value* Study treatment intake       Discontinued treatment 8 (34.8%) 3 (27.3%) 5 (41.7%)        Average rate of adherence (95% CI) --- 35% (14-56%) ---  Patient follow-up        Completed the study (Retention rate) 17 (73.9%) 10 (90.9%) 7 (58.3%) 0.155        Lost to follow-up 6 (26.1%) 1 (9.1%) 5 (41.7%)     * P value is reported using Pearson’s Chi-squared test  Obviousness of the eligibility criteria: one of the feasibility outcomes was to assess if the study eligibility criteria was obvious to the healthcare practitioner. We used the same eligibility criteria described in Chapter 2. The eligibility determination was done in a multi-staged process as follows: Step 1) during which I reviewed the list of clinic patients with their reason for referral before the clinic started and made note of potentially eligible patients. I then checked the patients’ charts and history of illness and presented them to our attending urologists (Drs. Afshar and MacNeily). Step 2) during which our attending urologists met the patients and clinically assess them as routine practice of the clinic and simultaneously check them against our inclusion/exclusion criteria for this study. Step 3) once eligibility of a patient has been confirmed, the attending urologist notified me and I discussed the study procedure with the patients and their families through routine ethical consent/assent obtaining procedure. While doing so, I double checked the eligibility criteria to make sure the attending urologist and myself had an agreement over the decision on enrollment of this patient into the study. In all cases, the attending urologist and I had reached a consensus on determining the eligibility criteria showing the obviousness of this eligibility requirement for this trial. 114  Practicality of delivering the intervention in the proposed setting: throughout the study recruitment procedure, I did not encounter any problems while explaining and delivering DB training to the patients and their families. As already described in the methods section of this chapter, I ensured proper learning of the DB practice by our patients via live training sessions using one of the clinic’s rooms and rehearsing DB practice in front of the patient by myself first and then asking the patient to lie on the bed and teaching them to practice exercising DB. In addition, pamphlets with illustrations (Appendix E, Figures E.1 and E.2) were used and provided to the patients. As well, we watched “Breathe2Relax” video tutorial at the clinic together and I suggested the application to re-enforce DB practice at home.  Patients’ understanding of study questionnaires or data collection tools: the validity and reliability of all questionnaires in the study (BBD, PinQ, and TAQ) had already been tested via studies.(21, 22, 202, 208) Regardless, in order to check the patients’ understanding of study questionnaires or data collection tools, I sat with each individual and observed them while they went through the questionnaire items answering each item. In most cases, our participants were able to easily respond to the items of the questionnaires and rarely were they hesitant between 2 answers in which case, they would ask a question from me and after my description they were able to answer the question definitely.  Length of time for filling out the study forms: the length of time spent to fill out study forms depending on how fast the participants and their families were able to go through the consent/assent forms, and other study forms and questionnaires ranged between 30 to 45 minutes.     115  Randomization and Treatment acceptability: the patients and their parents were positive with regards to accepting randomization. I did not encounter any refusals to participation due to being randomized to either of the intervention arms. In order to make the randomization more acceptable, I promised the patients and their families randomized to Group B that they would receive the same training procedure and materials for DB practice at the end of the second visit. The acceptability of the intervention from patients’ and parents’ perspective randomized to Group A were assessed at the end of the follow-up visit by asking them to fill out TAQ questionnaire modified to capture treatment acceptability of DB (Appendix F). I did not obtain an overall treatment acceptability from patients’ perspective since I had modified some of the items to fit the context of this study and there was no previous published study that measured the validity of this questionnaire for DB practice. Having said that, I report below a description of all participants’ response to each item of this questionnaire:   For item 1 corresponding directly to DB’s acceptability, out of 10 participants completing the study, four gave this item the highest score as being “very acceptable” and two participants scored the lowest describing DB as being “very unacceptable”. These were the same two participants whom described the nature of DB practice as being “boring”.  For item 2, corresponding to how helpful the participants think DB might be, six participants gave this item the highest score as being “very helpful” while one participant scored the lowest describing DB as being “very unhelpful”.    For item 3 corresponding to the likelihood of DB causing any harm, all participants agreed that it is “very unlikely” for DB to cause any harms.  116  For item 4 corresponding to how knowledgeable the participants think the researchers are with regards to recommending DB as an intervention for this study, six participants gave this item the highest score as being “very knowledgeable” while one participant scored the lowest and another participant scored low for “not knowledgeable”.   For item 5 corresponding to how trustworthy the participants think the researchers are, nine participants gave this item the highest score as being “very trustworthy” while one participant scored the lowest describing me as being “very untrustworthy”.  All but one participant responded “Yes” to a question asking whether they would suggest such treatments to other children with the same condition.  Also, eight participants responded “No” to the question asking whether they experienced any unpleasant or negative feelings while practicing DB. Two participants recalled unpleasant experience with practicing DB; one recalled DB practice making them “feel sleepy”, and the other recalled practicing DB to interfere with their play time.     It is worth to note that the participants whom answered negative to most of the items on the TAQ questionnaire was the same person for whom a technical error happened with the research-enabled component of the “Breathe2Relax” application during the follow-up period and also described practicing DB as being “boring”. Participants also did not report of any adverse events or side-effects associated with practicing DB. The mean overall score for treatment acceptability from parents’ perspective was 41 (95% CI: 37-45) indicative of a high level of overall acceptability for this treatment from parents’ view. All but one parent rated item 1 corresponding directly to DB’s acceptability, as “very 117  acceptable”. The reason why this parent rated DB as an unacceptable intervention, according to her, was because she had to put a lot of effort to manage her schedule and adhere to the recommended 3 times daily practice of DB. She also described that this became even more difficult as her child did not have the personality of being told what to do. On average, the parents did not have any strong opinions with regards to whether they believe that DB is effective in changing their child’s bladder habit (item 2). Eight parents strongly agreed and one parent agreed that their child’s bladder problem is troublesome enough to justify the use of DB (item 3) while one parent stayed neutral. All parents but one (the parent of the same child described above whom scored low on acceptability of practicing DB) responded positively to the item assessing their willingness to continue DB treatment with their child (item 4). All parents believed in the safety of the treatment (item 5). All but one parent (same parent described above) liked the treatment (item 6) and all but 2 parents believed that DB might be a good way to handle their child’s problem (item 7). All parents but two agreed that the treatment would overall help their child (item 8).  Clinical efficacy outcomes    As already described in the methods section, the comparison of the variables between the two groups was done using both ITT and PP analysis methods as follows: ITT population:  The results of ITT analysis are summarized in Table 5.3 . Our main clinical secondary outcome “proportion of participants with resolved incontinence at the end of the trial” measured through the use of voiding diary and patients’ response to BBD questionnaire did not differ at visit 2 between the two groups with only 1 out of 11 patients 118  (9.1%) in Group A and 2 out of 12 patients (16.7%) in Group B achieving continence at the end of the trial. Patients in Group A and Group B also showed a mean of 2.2 and 1.8 wetting episodes per day after treatment, respectively (p = 0.398). Both groups also reported a similar average BBD score at visit 2. Comparison of the ANS profile at rest between Group A and B after receiving treatment: Comparison of the two groups at rest during visit 2, after imputation for missing values at both visits and adjusting for patients’ age, gender, BMI, and baseline value (i.e. values at visit 1) values as covariates, revealed no significant difference in ANS profile between the two groups with Group B showing a slightly higher TP, HF, and PEP after treatment compared to Group A.  Comparison of the between-groups difference for the relative change from rest to the voiding period (only performed for variables extracted from ICG data analysis i.e. PEP and HR), showed a significant difference between the two groups with Group A showing a decrease and group B showing an increase in their HR response to voiding after treatment (relative HR change (%) from rest to voiding = -2.68 % in Group A vs. 10.18 % in Group B, p = 0.047). Also, a larger decrease in PEP in response to voiding (although not statistically significant) was observed in Group B compared to Group A (relative PEP change (%) from rest to voiding = -0.72 % in Group A vs. -2.76 % in Group B, p = 0.293). The main uroflow parameters (peak and average urinary flow rate, voided volume, and PVR) were also similar in both groups after treatment. The adjusted mean score on PinQ questionnaire was also similar in both groups after receiving treatment. The exploratory within-group analysis of variables is also shown in Table 5.3  with most variables showing a non-significant change from before to after receiving treatment in both groups.  119  Table 5.3 Intention-to-treat analyses.   Group A (n = 11) Group B (n = 12) HRV variables Visit 1, Median (IQR) Visit 2, Median (IQR) P-value Visit 1, Median (IQR) Visit 2, Median (IQR) P-value   973.98 (798.84 – 1104.01) 871.30 (529.35 – 1458.54) 0.859 1413.98 (641.04 – 1972.14) 1213.77 (613.19 – 2668.66) 0.735 TP Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2       1041.19 (235.06)     1533.41 (222.60)   0.192*   421.63 (323.56 – 669.22) 388.22 (266.19 – 815.60) 0.790 723.91 (304.03 – 1257.69) 721.58 (305.37 – 1507.18) 0.735 HF Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2       496.83 (158.21)     845.08 (150.12)   0.164   345.69 (292.10 – 489.22) 241.65 (181.44 – 523.33) 0.424 289.46 (180.76 – 672.97) 280.10 (151.17 – 958.14) 0.499 LF Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2       345.36 (76.75)     548.23 (72.67)   0.105   1.07 (0.56 – 1.38) 0.57 (0.40 – 0.99) 0.594 0.75 (0.46 – 1.11) 0.63 (0.54 – 1.10) 0.499 LF/HF Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2       0.88 (0.25)     0.83 (0.24)   0.913   55.45 (42.01 – 64.12) 63.63 (50.37 – 71.49) 0.248 57.22 (47.46 – 68.59) 61.43 (47.73 – 64.78) 0.866 Hfnu Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2       61.97 (4.86)     57.23 (4.61)   0.525 ICG variables Visit 1, Median (IQR) Visit 2, Median (IQR) P-value Visit 1, Median (IQR) Visit 2, Median (IQR) P-value   109.90 (104.13 – 116.68) 97.89 (95.74 – 116.00) 0.214 104.36 (97.40 – 116.09) 109.81 (104.33 – 115.20) 0.063 PEP Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2       104.13 (2.10)     109.96 (1.99)   0.084   83.48 (66.44 – 90.41) 87.03 (74.60 – 94.24) 0.374 79.05 (75.89 – 85.19) 77.97 (73.92 – 85.15) 0.612 HR Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2       82.78 (3.02)     80.12 (2.86)   0.569 Relative change (baseline rest to voiding) Visit 1, Median % (IQR) Visit 2, Median % (IQR) P-value Visit 1, Median % (IQR) Visit 2, Median % (IQR) P-value PEP, % -1.32 (-3.66 – 1.70) 0.05 (-2.68 – 1.96) 0.441 -2.52 (-3.49 – 0.36) -1.65 (-2.92 – -0.14) 0.499 Adjusted mean % (SEM) at Visit 2     Adjusted mean % (SEM) at Visit 2     -0.72 (1.25)     -2.76 (1.19)   0.293 HR, % 2.22 (-1.40 – 6.88) -1.40 (-7.61 – 3.15) 0.314 4.60 (-2.53 – 12.93) 5.48 (-0.17 – 14.66) 0.063 Adjusted mean % (SEM) at Visit 2     Adjusted mean % (SEM) at Visit 2     -2.68 (3.99)     10.18 (3.78)   0.047 Uroflowmetric Variables Visit 1, Median (IQR) Visit 2, Median (IQR) P-value Visit 1, Median (IQR) Visit 2, Median (IQR) P-value   10.4 (3.5 – 15.4) 12.4 (11.0 – 16.3) 0.161 16.1 (10.3 – 22.8) 18.0 (10.5 – 28.8) 0.612 Peak urinary flow rate Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2       16.5 (3.6)     19.2 (3.4)   0.616   4.2 (1.7 – 9.3) 7.8 (4.3 – 9.3) 0.173 4.5 (1.8 – 7.1) 4.4 (2.2 – 8.3) 0.866 Average urinary flow rate Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2       6.2 (0.9)     6.2 (0.8)   0.974   51.8 (23.0 – 157.0) 87.0 (42.0 – 125.5) 0.173 91.3 (69.1 – 175.0) 89.0 (67.6 – 145.5) 0.499 Voided volume Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2       114.9 (21.6)     97.9 (20.5)   0.609   15.0 (0.0 – 35.0) 15.0 (10.0 – 35.0) 0.271 21.0 (0.0 – 37.5) 17.5 (0.0 – 35.5) 0.248 Post-void residual Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2       29.0 (8.3)     17.2 (7.9)   0.365 120  Voiding diary Group A (n = 11) Group B (n = 12) Resolved Incontinence Patients with resolved incontinence at visit 2, n (%)  Patients with resolved incontinence at visit 2, n (%)  P-value 1 (9.1) 2 (16.7) 0.590**  Visit 1, Median (IQR) Visit 2, Median (IQR) P-value Visit 1, Median (IQR) Visit 2, Median (IQR) P-value Number of wetting episodes per day  2.7 (1.7 – 3.3) 3.0 (1.0 – 3.5) 0.906 1.5 (0.5 – 2.4) 0.7 (0.1 – 2.1) 0.026 Adjusted mean (SEM) at Visit 2 Adjusted mean (SEM) at Visit 2 2.2 (0.3) 1.8 (0.3) 0.398 Questionnaires Visit 1, Median (IQR) Visit 2, Median (IQR) P-value Visit 1, Median (IQR) Visit 2, Median (IQR) P-value   20 (15 – 26) 18 (14 – 25) 0.168 20 (13 – 25) 19 (12 – 24) 0.172 BBD score Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2       18 (1)     19 (1)   0.617   30 (10 – 35) 17 (10 – 32) 0.137 27 (21 – 36) 25 (19 – 33) 0.028 PinQ Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2       23 (3)     24 (2)   0.910 * P-Values for between group comparisons using analysis of covariance adjusted for Visit 1, age, sex, and BMI. ** P-Value for between group comparisons using Pearson’s Chi-squared test. Values at Visit 1 and Visit 2 are median (interquartile range).  P-values for within group comparisons are reported using Wilcoxon signed-rank test. BMI, body mass index; TP, total power; HF, high frequency; LF, low frequency; nu, normalized units; PEP, pre-ejection period; HR, heart rate; SEM, standard error of the mean.  121  PP population: The results of PP analysis are summarized in Table 5.4 . The per-protocol analysis of the main clinical secondary objective “proportion of participants with resolved incontinence at the end of the trial” showed similar results in both groups at visit 2 with only 1 out of 5 patients (20%) in Group A and 2 out of 5 patients (40%) in Group B achieving continence at the end of the trial. Patients in Group A and Group B also showed a mean of 2.4 and 1.9 wetting episodes per day after treatment, respectively (p = 0.729). Both groups also reported a similar adjusted average BBD score at visit 2 with Group A showing a lower score at visit 2 (17 in Group A vs. 20 in Group B). Also, within-group analysis (exploratory analysis) showed a significant reduction of BBD score in Group A after receiving DB treatment (median BBD score in Group A at visit 1 = 25 (IQR: 16 – 27) vs. 20 (IQR: 10 – 25) at visit 2).  Comparison of the ANS profile at rest between Group A and B after receiving treatment: Comparison of the two groups at rest during visit 2, adjusting for patients’ age, gender, BMI, and visit 1 values as covariates, revealed no significant difference in ANS profile between the two groups with Group B showing a slightly higher TP, HF, and PEP after treatment compared to Group A.  Comparison of the between-groups difference for the relative change from rest to the voiding period (only performed for variables extracted from ICG data analysis i.e. PEP and HR), revealed some differences between the two groups with Group A showing a decrease and group B showing an increase in their HR response to voiding after treatment (relative HR change (%) from rest to voiding = -3.61 % in Group A vs. 11.33 % in Group B, p = 0.228). PEP in response 122  to voiding decreased in both groups with Group B showing a slightly higher decrease compared to Group A (relative PEP change (%) from rest to voiding = -1.36 % in Group A vs. -2.66 % in Group B, p = 0.694). The main uroflow parameters (peak and average urinary flow rate, voided volume, and PVR) were also similar in both groups after treatment. The adjusted mean score on PinQ questionnaire was also similar in both groups after receiving treatment. The exploratory within-group analysis of variables is also shown in Table 5.4  with most variables showing a non-significant change from before to after receiving treatment in both groups.                123  Table 5.4 Per-protocol analyses. HRV variables Group A (n = 8) Group B (n = 6) Visit 1, Median (IQR) Visit 2, Median (IQR) P-value Visit 1, Median (IQR) Visit 2, Median (IQR) P-value   925.80 (760.32 – 1399.77) 1161.13 (605.58 – 1672.96) 0.575 1239.53 (628.07 – 2015.17) 1213.77 (823.22 – 1981.17) 0.249 TP Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2       1207.56 (187.59)     1352.24 (225.41)   0.673*   410.25 (219.70 – 695.29) 459.66 (270.76 – 1015.01) 0.401 680.78 (296.39 – 1408.32) 721.58 (469.47 – 1055.98) 0.753 HF Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2       657.00 (153.17)     766.66 (182.70)   0.688   350.52 (312.18 – 475.51) 274.92 (184.32 – 555.30) 0.889 232.48 (179.33 – 685.39) 280.10 (170.88 – 868.23) 0.917 LF Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2       353.84 (96.25)     484.67 (115.30)   0.458   0.91 (0.61 – 1.73) 0.50 (0.32 – 0.92) 0.263 0.72 (0.43 – 1.12) 0.57 (0.30 – 0.76) 0.917 LF/HF Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2       0.73 (0.33)     0.70 (0.39)   0.965   52.62 (37.26 – 62.32) 66.90 (52.27 – 75.73) 0.093 58.02 (47.07 – 69.93) 63.53 (57.33 – 77.35) 0.753 Hfnu Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2       67.19 (6.67)     59.17 (7.96)   0.506 ICG variables Group A (n = 7) Group B (n = 7) Visit 1, Median (IQR) Visit 2, Median (IQR) P-value Visit 1, Median (IQR) Visit 2, Median (IQR) P-value   107.50 (96.00 – 116.57) 96.04 (93.98 – 115.56) 0.735 104.36 (97.40 – 116.09) 107.31 (106.21 – 113.05) 0.063 PEP Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2       103.45 (2.51)     107.95 (2.51)   0.290   83.81 (77.37 – 94.13) 88.05 (74.60 – 94.24) 0.866 79.05 (75.89 – 85.19) 79.04 (73.00 – 85.83) 0.612 HR Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2       82.98 (3.33) 80.86 (3.33) 0.695 Relative change (baseline rest to voiding) Group A (n = 7) Group B (n = 6) Visit 1, Median % (IQR) Visit 2, Median % (IQR) P-value Visit 1, Median % (IQR) Visit 2, Median % (IQR) P-value PEP, % -0.20 (-6.86 – 1.84) -1.58 (-5.37 – 1.96) 0.735 -2.59 (-3.67 – 0.94) -1.15 (-2.78 – 0.13) 0.345 Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2     -1.36 (1.94)     -2.66 (2.13)   0.694 HR, % 1.76 (-3.67 – 5.92) -4.33 (-7.61 – 4.03) 0.499 5.09 (-4.13 – 14.22) 4.14 (-2.33 – 14.80) 0.116 Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2     -3.61 (6.92)     11.33 (7.60)   0.228 Uroflowmetric Variables Group A (n = 7) Group B (n = 6) Visit 1, Median (IQR) Visit 2, Median (IQR) P-value Visit 1, Median (IQR) Visit 2, Median (IQR) P-value   11.7 (3.9 – 15.2) 12.3 (11.0 – 14.5) 0.345 18.0 (10.1 – 23.3) 17.7 (9.9 – 22.7) 0.917 Peak urinary flow rate Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2       12.0 (2.3)     15.8 (2.6)   0.365   4.0 (1.7 – 8.5) 6.5 (4.3 – 8.6) 0.128 4.9 (1.7 – 7.1) 4.4 (3.7 – 8.4) 0.600 Average urinary flow rate Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2       6.2 (1.2)     6.3 (1.3)   0.971  37.9 (22.3 – 99.0) 46.0 (33.0 – 114.0) 0.128 96.0 (68.0 – 178.0) 123.0 (66.0 – 146.0) 0.600 Voided volume Adjusted mean (SEM) at Visit 2 Adjusted mean (SEM) at Visit 2  109.6 (37.6) 89.1 (41.3) 0.748 124  Uroflowmetric Variables Group A (n = 7) Group B (n = 6) Visit 1, Median (IQR) Visit 2, Median (IQR) P-value Visit 1, Median (IQR) Visit 2, Median (IQR) P-value   6.0 (0.0 – 41.5) 20.0 (10.0 – 50.0) 0.080 20.0 (0.0 – 40.0) 15.0 (0.0 – 45.0) 0.343 Post-void residual Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2       34.7 (13.0)     20.9 (13.0)   0.518 Voiding diary Group A (n = 5) Group B (n = 5) Resolved Incontinence Patients with resolved incontinence at visit 2, n (%)  Patients with resolved incontinence at visit 2, n (%)  P-value 1 (20) 2 (40) 0.49**  Visit 1, Median (IQR) Visit 2, Median (IQR) P-value Visit 1, Median (IQR) Visit 2, Median (IQR) P-value Number of wetting episodes per day  2.5 (0.9 – 4.1) 3.0 (1.0 – 4.0) 0.715 1.0 (0.5 – 2.8) 1.0 (0.0 – 2.8) 0.102 Adjusted mean (SEM) at Visit 2 Adjusted mean (SEM) at Visit 2 2.4 (0.8) 1.9 (0.8) 0.729 Questionnaires Group A (n = 8) Group B (n = 7) Visit 1, Median (IQR) Visit 2, Median (IQR) P-value Visit 1, Median (IQR) Visit 2, Median (IQR) P-value   25 (16 – 27) 20 (10 – 25) 0.011 20 (13 – 25) 21 (13 – 24) 0.172 BBD score Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2       17 (1)     20 (1)   0.262   26 (10 – 34) 16 (10 – 24) 0.175 27 (21 – 36) 22 (8 – 25) 0.028 PinQ Adjusted mean (SEM) at Visit 2     Adjusted mean (SEM) at Visit 2       20 (3)     20 (3)   0.970 * P-Values for between group comparisons using analysis of covariance adjusted for Visit 1, age, sex, and BMI. ** P-Value for between group comparisons using Pearson’s Chi-squared test. Values at Visit 1 and Visit 2 are median (interquartile range).  P-values for within group comparisons are reported using Wilcoxon signed-rank test. BMI, body mass index; TP, total power; HF, high frequency; LF, low frequency; nu, normalized units; PEP, pre-ejection period; HR, heart rate; SEM, standard error of the mean.  The low average rate of 35% adherence to DB practice in Group A, already described earlier in this section, revealed a low number of patients following the recommendations provided to them for practicing DB. Given that I had the total number of DB practiced by each patient, in an exploratory attempt, I ran a correlation matrix using a non-parametric measure of rank correlation (Spearman’s rho) between the total number of DB practiced and the change from visit 1 to visit 2 in the other outcome measures. The results of this test are summarized in Table 5.5 . In short, I found significant positive correlation between the total number of DB practice and HF relative change (r = 0.69, p = 0.029), as well as absolute PVR change (r = 0.72, p = 0.029) from visit 1 to visit 2. Although not 125  statistically significant, I also found a trend with positive correlation between total number of DB practice and TP relative change (r = 0.58, p = 0.082), and a negative correlation with absolute change in BBD score (r = -0.56, p = 0.093).126  Table 5.5 Correlation between the total number of DB practice and the relative change (in % except otherwise specified) from Visit 1 to Visit 2 in the other outcome measures. Variable Correlation coefficient* P value HRV variables (n = 10) TP relative change 0.58 0.082 HF relative change 0.69 0.029 LF relative change 0.43 0.214 LF/HF ratio relative change -0.53 0.117 HFnu relative change 0.28 0.425 ICG variables prior to voiding (n = 9) PEP relative change 0.45 0.224 HR relative change -0.45 0.224 ICG baseline rest to voiding (n = 9) PEP absolute change -0.08 0.831 HR absolute change 0.20 0.606 Uroflowmetric variables (n = 9) Peak urinary flow rate relative change -0.15 0.700 Average urinary flow rate relative change -0.05 0.898 Voided volume relative change 0.05 0.898 Post-void residual absolute change 0.72 0.029 Voiding diary (n = 7) Number of wetting episodes per day relative change -0.54 0.215 Number of wetting episodes per day absolute change -0.25 0.589 Questionnaires (n =10)   BBD score absolute change -0.56 0.093 PinQ absolute change 0.52 0.122 * Spearman’s rho. 127  5.5 Discussion I already discussed in previous chapters the questionable efficacy of current treatment modalities for BBD in childcare and the fact that studies have shown more than two third of children with BBD not receiving any specific treatments for their urinary incontinence as the main presenting symptom of BBD.(31) This together with my findings of a dysfunctional autonomic profile in children with BBD, which has never been studies before, intrigued me to run a pilot randomized clinical study and explore the feasibility and scientific validity of DB training exercise (as an adjunctive therapy with ANS up-regulating properties) in resolution of BBD symptoms (mainly the urinary incontinence) in these children.   The results of the current study provide remarkable information describing the processes and setbacks encountered by myself while running this pilot trial as well as exploratory scientific evaluation of the treatment effect. Conducting pilot or vanguard studies like the one described in the current chapter, provides other colleagues in the medical research community with a unique opportunity to estimate the feasibility of conducting large full-scale studies investigating efficacy of similar interventions and increase the possibility of success in their future trials.  The results of the feasibility assessment of the processes that need to take place as part of pursuing with larger randomized trial showed a recruitment rate of 85% for this pilot trial. This high recruitment rate expresses a remarkable interest from patients and their families for participating in this trial. The reasons for participation refusal of the eligible candidates were mostly due to pressure of time and participants’ unwillingness to commit to the proposed intervention and follow-up and in one case it was having the option for other rapid treatments such as the use of add-on anti-muscarinic agents.  128  As already described in the results section of this chapter determination of eligibility criteria was done in multiple steps and our clinicians and I did not encounter any disagreement or problems determining the eligibility criteria for this study. I also did not encounter any issues with the training and delivery of DB practice to the patients at our hospital setting. The validity and reliability of our study questionnaires had already been tested, however, to ensure that our participants have a good understanding of the study questionnaires and data collection tools, I checked the answers on the questionnaires and in most cases, participants were able to easily respond to the items being rarely hesitant between 2 answers.  I did not assess multiple clinicians’ participation as part of the feasibility in this study since I already obtained verbal commitment from our 2 clinicians (Drs. K. Afshar, and AE, MacNeily) to help facilitate recruitment of patients for this trial. Nevertheless, our clinicians were easily able to fit checking patients for eligibility criteria and describing randomization procedure into the time allotted for the appointment.  This study had a retention rate of 91% (10 out of 11 participants retained) for the main intervention (Group A) and 58% (7 out of 12 participants retained) for the control (Group B) arm. The significant gap in retention between the two intervention groups in this study and the fact that the participants who were lost to follow-up in the control group did not provide us with any reasons as to why they left the study and were unresponsive to follow-up calls and emails is concerning. Retention is an important part of clinical trials since enrolled participants who do not complete the trial can compromise the internal and external validity of study findings by creating attrition bias which can be alarming especially in situations where participants are not lost at random but have specific characteristics that can result in better or worse outcomes in which case 129  even the ITT method of analysis cannot overcome this problem.(209, 210) Although there is no standard for loss to follow-up, some studies set a threshold of 20% attrition for a trial to be labelled as having attrition bias.(211)       Several different factors have been identified which can affect retention rate in clinical trials. These factors include(210):  - Study design: happening especially in open-label trials in which the participants may become reluctant to completing the trial after finding that they are assigned to the control arm thus increasing the chance of attrition. In our open-label trial, we tried to avoid loss to follow-up in the control arm by promising participants randomized to Group B that they will receive the same training for DB after their follow-up visit and by incentivizing them with gift cards for both visits.   - Visit reminders: in specific using “telephone reminders” as a common method to remind participants of impending visits. I implemented this method by setting up follow-up phone call dates and notifying the parents of this during their initial visit. As well, for the ones who missed out on my follow-up calls, I tried contacting them 1 week before their second visit to remind them of their follow-up appointment. Since the second follow-up visit is a routine practice of the clinic, we were initially optimistic that patients will not miss this appointment, however, I was not able to contact 5 of the patients in the control group who were lost to follow-up and the patients missed their second visit which led to a deep gap in retention between the two arms. 130  - Up-front scheduling: after the initial visit, we made sure all participants are scheduled a second appointment which was done by the clinic receptionist discussing possible dates with the parents and giving them written notes detailing the date and time of their second visit.   - Demographic and other factors: demographic features proposed as factors which can affect attrition include older age, male gender, poorer cognitive performance, lower verbal intelligence, and worse physical health. In the case of this study none of the above factors were detected in our cohort of participants. Therefore, these factors may have not been the reason why we observed a higher loss to follow-up in the control group.  - Transparency of the informed consent/assent documents and the strong relationship among the study coordinator, care provider, and participants are also listed as other determining factors affecting attrition in clinical trials. We did not notice any issues with regards to consenting procedures or problems with the relationship between the study coordinator and the participants.    Adherence to treatment is another important issue. As described in the results section of this chapter, one participant in Group A was lost to follow-up without practicing DB. Also, another 2 participants did not practice DB and described practice of DB as “boring” and that was the reason why they could not adhere to the recommended exercise. The rest of the participants in this group practiced DB but did not fully comply with the recommendations. This led to an overall average rate of 35% treatment adherence for the follow-up duration. Also, in Group B, 131  we lost 5 patients to follow-up. Also, it was not known whether the patients who did not complete the trial (especially the ones in Group B) decided to withdraw because of resolution of the voiding problems or aggravation of the symptoms. Adherence (synonym: compliance) is defined as “the extent to which a patient acts in accordance with the prescribed interval, and dose of a dosing regimen”.(212) Treatment adherence is a crucial part of clinical trials and a primary determining factor for treatment success with medication non-adherence resulting in worsening of the disease, increased healthcare costs, and in some instances even death.(212-216) By admitting the disadvantages of the indirect subjective measures of adherence, and the low overall rate of treatment adherence found in this study we should take cautionary steps when recommending such therapy for future trials. Common barriers to treatment adherence are usually under patients’ control therefore it is necessary to pay attention to these barriers in order to improve adherence. These common barriers which might have played a key role in the low adherence to DB in this study include forgetfulness, child or parent’s other priorities, decision of omitting sessions of treatment, lack of information, emotional factors (such as those arising from family conflicts) or even discontinuation without providing any specific reasons.  When it comes to interventions that involve learning new skills and behaviour, such as the case of DB in the current study, child and family’s knowledge of the disease and behavior becomes an important factor which requires proper education, lifestyle modification and at times a high degree of self-regulation. Family functioning and support as well as child’s environment has also been investigated as an important factor affecting adherence to treatment in different diseases such as Cystic Fibrosis,(217) and type 1 diabetes mellitus.(218). Education alone has shown to 132  be appropriate only for short-term medication regimens therefore it is advised that this form of training is coupled with written information on the treatment and instructions on how it is administered as well as having follow-up visits or telephone calls. These steps should be taken cautiously in children since the anxiety on how a complex task is performed as a treatment can also become a deterrent to adherence.(219)  In this study, in order to increase adherence in patients, after completion of the education and training on performing DB and a live practice session involving the child, I provided all children in Group A with pamphlets (Figure E.1 and Figure E.2 ) as well as computer tablets. I then walked them through features and tutorials of “Breathe2Relax” application already installed on the tablet. In order to ensure more adherence, I also scheduled telephone follow-up around half-way through completion of the treatment to talk to the parents and find out how the treatment is going. Unfortunately, most of my calls were left un-responded by the parents even after several attempts or leaving voice messages. In cases, where there was a response to my follow-up call, most things seemed to have been going well with DB practice. On average, through items on the TAQ questionnaire and verbal interview the parents did not have a strong opinion whether practice of DB is effective enough in changing their child’s bladder habit. One of the main concerns that was raised from one of the parents was the effort and time this parent had to put in order to convince the child to practice DB. The fact that “Breathe2Relax” application stopped sending research information properly in the middle of the recruitment was also another disadvantage which led to one of the children losing interest in practicing DB. Most of parents and children also had difficulty performing the second daily practice of DB since they were attending school at that time and it was difficult to perform the 133  technique at school. Once this was raised, I adjusted the treatment plan with every parent prompting them not to ask the child to perform the second session of the treatment at school but to practice it once they are back from school. Nevertheless, this might have also impacted the overall adherence since in my opinion the routine was broken which can lead to dis-organized practice and discontinuation in the long run.  Finally, the long follow-up period of 3 months might have also played a role in low adherence seen in both arms. Although the reason for justifying 3 months of treatment has already been described above, it is possible that this long period of time, with or without temporary resolution of the symptoms, be in itself a barrier to motivation and thus adherence to DB exercise due to intangible immediate effect of DB practicing. The problem of long follow-up becomes specifically more complicated for the control group as they might have felt being left out for a long time without particular attention and for this reason decided to drop out of the study. There is also the chance that the symptoms disappeared in the control group during the follow-up period thus they did not have enough incentive to continue with the follow-up visit.    Some of the strategies that can be used to help improve treatment adherence in children are described by Gardiner et al 2006(220) as follows: For physicians/researchers: - Medications/treatments should be given once or twice daily - Ask others (e.g. pharmacists/family physicians, family members) to reinforce the information - Provide information handouts - Discuss medication benefits and safety issues with parents 134   For patients/parents: - Incorporate dosing into daily routines and take medication at the same time each day (e.g. after brushing teeth, before meal) – In the case of this study, I advised patients to take the morning practice right after waking up in the morning and the night practice at bed before sleeping. - Keep a tally sheet of number of practices, mark a calendar or a diary – A diary was provided to each patient in this study to mark the number of DB practiced each day.  - Use visual reminders (e.g. notes on the refrigerator) - Ask a friend or a family member to remind you - Set an alarm on your clock or watch - Use a paging system with a beeper - Parents can offer children rewards (e.g. stickers, small toys) for remembering Treatment acceptability plays an important role in the conduct of clinical trials and management of disorders due to its influence on adherence to the treatment, outcomes, and satisfaction of the participants with the treatment.(221) In addition, a strong relationship between child’s home environment and in particular family support and treatment acceptability has been established which demonstrates the fact that when working with pediatric population in clinical trials, children should not be looked at as the sole entities in the trial.(222) In this study although there were a few instances where the child or their parents did not approve of DB for particular reasons already mentioned in the results section, the interpretation of each individual items in the 135  TAQ as well as the mean overall TAQ scores were suggestive of a good overall level of acceptability for DB from both affected children and their parents.  To conclude the discussion on the treatment adherence in this study, it is worth noting that numerous articles have recently shown the use of smartphone applications to be associated with the improvement in exercise adherence in adults and children in health as well as in the context of different disorders.(223-225) Use of social media and video games are also gaining popularity as high-tech tools that can motivate exercise and improve treatment adherence.(226) This opens the door to exploring more the use of applications with more gaming features for the purpose of practicing DB in young children in the future, however, use of such applications should be considered with caution as there are many features in the applications that need to be tested before it is labelled as “reliable for research purposes” and current studies show some exercise applications still need improvement in order to be able to increase adherence to expert-recommended guidelines and strategies.(227) One of the obstacles in the conduct of this study which can also be considered a limiting factor to feasibility was receiving completed voiding diaries from the patients. Although I did my best in terms of reminding the participants to mail the diaries after the first visit and even provided them with stamped envelopes, we had a number of diaries not being mailed back to us or cases where the participants forgot to bring the diaries to their second visit. This led to having missing information on one of the key outcomes which is the number of wetting episodes. One solution to avoid such issues in future trials where the patient is asked to send certain diary or information could be to familiarize patients with specific applications which can track and record number of wetting episodes and send de-identified information to the investigator via internet. Another 136  alternative would be to use Research Electronic Data Capture (REDCap) platform where the study coordinator can set up the diary as a survey queue so that the participants will receive a link via email to complete the diary at home.  Overall, the current evidence with regards to the feasibility of processes necessary for running a larger trial based on the results of the current pilot study may suggests low overall feasibility for proposing DB as a non-invasive intervention for controlling symptoms of BBD. This decision comes mainly due to low practical adherence to the main intervention during the follow-up period despite good acceptability of treatment from the children and their parents in theory. Moreover, the high attrition in the control intervention group may make it difficult for future studies to estimate true comparative treatment effect while running a randomized trial unless improvements are made to increase retention in the control arm. While stating the above, I try not to make a strong recommendation based on low feasibility since although mean adherence to DB was low, patients showed high retention and will to practice DB throughout the study period. Having said that, the main suggested modifications in order to make the processes more feasible in my opinion are as follows: - Providing the participants in both groups (especially the control group) with more incentives in order to persuade adherence and avoid discontinuation. One way to incentivize the control group could be to provide them with mobile applications with capacity to assess physiological features such as sleep duration and steps counting during the follow-up period in a fun and interactive way such as a game  137  - Shortening of the consent/assent and study description time by interviewing potential candidates prior to the main initial clinic visit  - Use of sophisticated and easy to use mobile applications (such as REDCap) to ensure recording and submitting of the results of voiding diary by participants to the study investigator   - Incorporating computer games into applications which promote DB practice and ensuring a safe, ethical transfer of DB practice data to the study investigator. As well, use of similar applications with game features that can be used to engage the control group and keep them in the trial  - Adjusting DB practice frequency to twice daily with higher intensity of practice in order to facilitate the practice and avoid discouragement due to missing some practices daily       Last but not least, as expected, practicing DB was not associated with any side-effects or unpleasant experiences. I used an online computer software for randomization and both the participants and I were not aware of the treatment assignments until after the initial assessment at baseline. This was done to eliminate any possibility of selection bias. Since DB is an exercise that needs to be taught and is structurally different than SU, plus there is no known sham for this type of treatment, it was not possible for us to blind participants and personnel to the treatments. As a result, this study is considered to have high risk of bias in blinding of participants, personnel, and outcome 138  assessors. Also, due to having missing data and unjustified dropouts the study was at high risk for attrition bias.  Since there was no formal power and sample size calculations for this trial, the scientific results regarding the efficacy of the treatments are performed and reported descriptively in an exploratory fashion and should be used cautiously as rough estimate of treatment effects and variance for future trials power and sample size calculations. The participants in both groups of treatment had similar characteristics at baseline with regards to their anthropometric, HRV/ICG, uroflow parameters, severity of BBD symptoms, and quality of life questionnaire scores. These characteristics were also similar to the characteristics of BBD population observed in the first study of this dissertation (Chapter 2) which confirms the validity of our patient selection procedure in this study.  As already described in the results section, due to having missing data, I performed both intention-to-treat (ITT) and per-protocol (PP) analysis for this trial in order to compare the outcomes of interest between the two intervention groups. Comparison of study outcomes using both methods of ITT and PP are discussed as follows:  Clinical outcomes: Comparison of the pooled mean for proportion of participants with resolved incontinence at the end of the follow-up period adjusted for age, gender, BMI, and values at visit 1 was similar in the two groups for both ITT and PP populations. However, the results of within-group comparison for PP population showed a significant reduction in the average BBD score in Group A after treatment with DB (average BBD score before treatment: 25 vs. 20 after receiving DB, p = 0.011). The significant change in BBD score was observed in the main treatment group while BBD score remained unchanged after SU only in Group B (average BBD score before 139  treatment: 20 vs. 21 after receiving SU, p = 0.172). This established a stronger effect of DB on reducing overall severity of BBD symptoms compared to the control group, however, the between-group difference was not statistically significant when Group A was compared to the control group probably because of the small sample size.  HRV outcomes: Comparison of the pooled mean for all HRV/ICG variables at the end of the trial adjusted for age, gender, BMI, and values at baseline value (i.e. values at visit 1) showed similarity in response to the two interventions between groups after performing both ITT and PP analysis. It is difficult to make in-depth interpretations based on the results of this analysis due to the small sample size and the low and scattered adherence to DB reported for participants in Group A. Nevertheless, I found a highly positive correlation between the relative change in TP and in specific the HF component of the HRV from before to after receiving DB and the total number of DB practice (Table 5.5 ) indicative of a positive effect of DB practice, if done properly and consistently, on increasing the total variability and in specific the PNS activity towards a healthier ANS status.  When comparing pooled adjusted mean of the relative change in PEP and HR from filling to voiding phase at the end of the trial, an overall trend showing a larger decrease in PEP was observed in the control group for both ITT and PP populations indicating a larger increase in SNS activity during voiding after receiving SU only treatment in Group B. This was confirmed by HR change during voiding in the two intervention arms with participants in the SU + DB arm having a slight decrease and SU only arm an increase in their adjusted HR mean during voiding phase after receiving the treatment (adjusted HR mean at the end of the trial: -2.68% for Group A vs. 10.18% for Group B, p = 0.047 in ITT population| -3.61 for Group A vs. 11.33 for Group B, 140  p = 0.228 in PP population). The increased HR during voiding in the control group was observed while children in DB group experienced an overall improvement in their relative change of HR in response to voiding after receiving DB more towards HR reduction in response to voiding. This is in line with an observed inverse correlation between the number of DB practice and HR relative change from filling to voiding phase described in Table 5.5 which is a natural phenomenon expected to be seen in healthy individuals in response to voiding explained by a dominance of the PNS during voiding. These findings should be cautiously interpreted since they could merely be due to the fact that we did not calculate power for this study and the sample size was small.  Uroflow outcomes: The adjusted pooled mean of uroflow parameters at the end of the trial was similar in the two groups for both ITT and PP populations with an observed trend of an increase in average urinary flow rate for both ITT and PP populations and an increase in PVR of PP population in Group A (median PVR before DB: 6 ml vs. 20 ml after receiving DB, p = 0.080). This was in line with highly positive correlation (r = 0.72, p = 0.029) found between the changes in PVR and the total number of DB practiced (Table 5.5 ) indicating greater efficiency in bladder emptying after receiving DB therapy in Group A.  The impact of the BBD symptoms on the quality of life in these children, measured through response to PinQ questionnaire was also similar in both groups at the end of the trial for ITT and PP populations with both groups showing a trend with a reduced overall PinQ score indicative of a reduced impact of the BBD symptoms on the quality of life.  The above findings on the effect of DB on HRV parameters coupled with a negative correlation between number of DB practices and the number of wetting episodes per day as the main clinical 141  outcomes of this study (r = -0.54, p = 0.215), severity of BBD symptoms captured through BBD questionnaire (r = -0.56, p = 0.093), and a significantly high positive correlation with PVR change post-treatment (r = 0.72, p = 0.029) is indicative of a possible therapeutic effect of DB practice as a potential add-on treatment candidate in the management of BBD symptoms. However, for DB to become such a candidate the main barriers of feasibility faced in this study must be overcome. Finally, more studies with large sample size and sophisticated study designs are needed to better investigate the efficacy of DB as an adjunctive therapy in the management of children with BBD and the results of the current study play a crucial role in helping with better design, planning, and conduct of these future trials.   142  Chapter 6: Conclusions  6.1 Summary of Findings and Contributions to the Scientific Knowledge Although the ANS is well known to play a key role in regulating normal bladder and bowel function, there is a gap in the scientific literature on the ANS involvement in the pathogenesis of BBD. The overall goal of this dissertation was thus set to investigate the activity of the cardiac ANS via spectral analysis of HRV, as a proxy of the overall ANS activity, in children with BBD.  In the study presented in Chapter 2, I showed that children with BBD at rest, compared to healthy controls, had significantly lower overall ANS activity (TP) with a significantly lower PNS power (HF), and a higher heart rate. By conducting this study; I was able to contribute to the research on BBD in children by focusing on a structural physiological aspect of normal bladder and bowel function that was not previously investigated during a dysfunctional state. The results of this study have been presented at the Society for Pediatric Urology 63rd Annual Meeting and published in the Journal of Urology.(167)  The findings of Chapter 2, together with the partial effectiveness of the current treatment modalities for BBD as well as the availability of interventions that have been shown to  manipulate ANS activity, led me to investigate the use of interventions that have shown to improve BBD symptoms and influence the activity of the ANS. Looking at the current widely used treatment options available for children with BBD, I was initially interested in testing the effect of pelvic floor muscle retraining (a form of biofeedback) on the ANS activity of these children since biofeedback is generally known to augment the activity of the ANS in other fields. Nevertheless, due to inconsistency in the available evidence and the lack of up-to-date systematic 143  literature reviews on the clinical efficacy of biofeedback, I decided to conduct a systematic literature review to assess the relative efficacy of biofeedback in the management of BBD (Chapter 3). The result of this systematic literature review failed to support positive relative efficacy of biofeedback in resolution of urinary incontinence as the main clinical outcome of interest in children with BBD. By conducting this review, my contribution to the scientific knowledge was to synthesize current evidence regarding clinical efficacy of biofeedback and create more awareness among healthcare professionals and decision makers regarding the necessity of well-designed systematic literature reviews to help derive therapeutic decisions in children with BBD. The results of this study have been published in the Journal of Urology.(194) Furthermore, in Chapter 4, in the absence of evidence evaluating the effect of anti-muscarinic agents on the already low PNS profile of children with BBD (shown in Chapter 2), I assessed the activity of the cardiac ANS in a subset of population with OAB (a subtype of BBD) who participated in the first study before and 3 months after receiving oxybutynin (a frequently used anti-muscarinic agent). The results of this study showed not only that the use of oxybutynin did not bring a significant resolution in OAB symptoms, but also that it caused a further reduction in the cardiac PNS activity of these children whom had already shown to be significantly lower in their PNS activity compared to healthy controls. Through the findings of this study, I contributed to the scientific knowledge, regarding therapeutic management of BBD, by describing the changes that can happen in the functioning of the ANS following the use of antimuscarinic agents. This provides key information that may be used in future studies investigating the effects of antimuscarinics on the activity of the ANS. Furthermore, the results of this study may provide clinicians with more information that can be used in making decisions on choosing anti-144  muscarinic agents as part of the treatment plan in patients with OAB by weighing the clinical efficacy associated with the usage of anti-muscarinics while bearing in mind the potential effects of these agents on the ANS activity and the body as a whole. Finally, given the significantly down-regulated ANS activity in children with BBD (Chapter 2), the evidence showing low level of clinical efficacy of the current adjunctive treatments available for this condition (Chapters 3 and 4), non-invasive nature of diaphragmatic breathing, evidence supporting ANS up-regulating capabilities of DB, and the lack of studies investigating DB as an adjunctive therapy in the management of the children with BBD, I decided to conduct a pilot trial to test the feasibility of conducting a large clinical trial to investigate the effect of DB as an adjunctive therapy in the management of children with BBD (Chapter 5). The results of this pilot trial suggested low overall feasibility for proposing DB as a non-invasive intervention in controlling symptoms of BBD in these children mainly due to low practical adherence to DB practice during the proposed follow-up period (approximately 3 months) despite the positive correlation found between DB practice and the resolution of clinical symptoms, and the improvement in the autonomic function, and uroflow parameters. These results will provide investigators with an insight to the challenges that they may encounter when conducting larger clinical trials and help them make more informed decisions on the proper design of future clinical trials, with the aim of assessing the clinical efficacy of DB in children with BBD.   6.2 Strengths and Limitations I used state-of-the art non-invasive technology and followed a strict study protocol to capture and analyze HRV. This procedure was done under controlled conditions to eliminate noise in our data as well as guarantee high quality of recording. The methodology that I used is accepted as a 145  standard approach for recording and analyzing HRV in the assessment of the ANS and is widely used by researchers and organizations around the globe investigating the stationary activity of the ANS.(53) The recorded HRV parameters were objective which increased the validity of my findings. Other strengths of the studies in this dissertation included the use of validated questionnaires to quantify severity of LUTS and bowel symptoms (BBD questionnaire), the emotional impact of BBD on the child’s quality of life (PinQ questionnaire), and treatment acceptability of DB from the patient and their families’ perspective (TAQ questionnaire). The validity and reliability of these questionnaires has already been confirmed by significant psychometric property testing.(21, 22, 202, 208) Nonetheless, there may still be some individual differences in the way patients responded to these questions that could have created recall bias. However, due to the simplicity of the questions and the fact that the questionnaire items do not ask the patient to recall events at a specific time point, but rather ask about the overall aspects of their symptoms and treatment, I do not think that a possible recall bias is associated with a major threat to this study.   In order to investigate the relative efficacy of biofeedback compared to other standard add-on treatments for BBD, I used systematic literature review and meta-analysis of randomized controlled trials methodology which is a well-known research methodology that holds the highest place in the “hierarchy of evidence” for grading healthcare recommendations and decision making.(228) Use of an RCT design (Chapter 5) also allowed a rigorous evaluation of the effect of DB versus conventional conservative treatment of SU only. Proper randomization and prospective nature of this design also eliminated the presence of multiple different possible biases for this study.  146  One limitation of the studies in this dissertation was the inability to measure the overall activity of the ANS in specific the PNS during voiding due to the requirement of recording at least 1 minute(53) of the heart activity during voiding in order to obtain the main HRV parameters of interest (HF and TP). However, we were able to indirectly measure cardiac ANS activity during voiding by measuring the relative change in PEP and HR from baseline to during voiding. Another limitation was the fact that our measurements of HRV were performed under controlled conditions which might not present the everyday ANS status of the participants. The laboratory context may have been source of stress, especially for the healthy controls that came to the hospital for the first time. This factor may explain the increase of SNS during micturition while we expected a decrease. This might also limit generalizability of the results to the regular daily living conditions of these patients, especially regarding the HRV raw values captured. However, since both BBD and the healthy control groups were assessed in the same setting this will not affect the overall results of the study comparing HRV parameters between BBD and control groups, before-after treatment, or intervention arms (DB+SU vs. SU).  A small sample size especially in the final RCT was also another restrictive factor that could have lead to lower precision of the estimate of the effect size and thus limiting the generalizability of the results.(229) Having said that, given the innovative nature of these projects, I was not able to calculate power and sample size for this study and the primary outcomes of interest focused on the feasibility aspects while scientific evaluations were performed as secondary objective of this research and the reader is encouraged to use caution when interpreting the results regarding efficacy of the interventions.  147  6.3 Significance and Future Research Directions The work I performed for this dissertation is important considering the high prevalence of BBD among children, paucity of evidence investigating possible role of the ANS in etiopathogenesis of BBD, complications and co-morbidities (both physical and psychological) that result from lingering of this condition which may even be carried into adulthood, and questionable efficacy of current treatment modalities for BBD. These results are particularly important given the psychological consequences and financial burden of BBD and its complications on patients, their families and the health care system at large.  Throughout my dissertation research, I strived to emphasize on translating my work for the scientific community by publishing the studies of this dissertation in peer-reviewed journals and presenting them in relevant national and international conferences (e.g. American Urological Association (AUA)), and Northwest Urological Society (NWUS)).  In Chapter 2, although the association between BBD and imbalanced autonomic tone was established, I was not able to comment on causality. However, the results of this study open a new horizon into further investigation of the role of the ANS in regulation of the lower urinary tract function in BBD in specific, further research on a possible cause-and-effect relationship. In addition, the association between imbalanced ANS activity and BBD can be used for the purpose of testing new treatment strategies known to modulate the ANS regulatory capabilities such as massage therapy, diaphragmatic breathing and exercise.(84, 182, 230) Furthermore, HRV/ICG parameters can be used in future trials as surrogate and objective treatment targets to investigate the efficacy of current or proposed interventions for controlling LUTS.  148  The result of the SLR conducted in Chapter 3 was not able to support biofeedback as an efficacious adjunctive treatment for children with BBD. Conduction and publication of this SLR is of high value to the scientific society as it brings greater awareness among healthcare professionals and decision makers regarding the current evidence on the efficacy of biofeedback as one of widely used adjunctive treatment modalities for BBD. Moreover, it necessitates reliance on well-designed systematic literature reviews rather than isolated clinical trials in order to derive therapeutic decisions in these children. Additionally, the limited number of clinical trials in the current evidence-base generates the need for well designed, RCTs with adequate power and objective measurements of outcomes to illuminate the efficacy of biofeedback in children with BBD.  The research I conducted in Chapter 4 provides new ideas for testing the use of HRV/ICG parameters as surrogate and objective treatment targets when investigating precise effects of anti-muscarinics on the ANS activity of children with BBD. This may become clinically important as the further reduction, after receiving treatment with oxybutynin, in the PNS component of children with OAB and total regulatory power of the ANS resembles the ANS profile (depressed HRV and HF band in particular) shown to be indicative of chronic stress associated with increased inflammation(34, 60, 103-106) and found in several major chronic diseases such as diabetes(61, 62) and cardiovascular disease risk factors.(63, 64) Although, as described earlier one should be cautious not to the interpret these results as making connections with chronic diseases as the current study had small sample size with no power calculation and antimuscarinics are often not used as long-term treatment options for children with OAB. Additionally, this novel approach of using HRV/ICG parameters as surrogate and objective 149  treatment targets will need to be verified through future studies with large sample sizes with the goal of developing more effective treatment strategies for controlling LUTS. Finally, the results of the pilot RCT (Chapter 5) showing a low overall feasibility for proposing DB as a non-invasive intervention in controlling symptoms of BBD will provide future investigators with insight to the possible challenges they may encounter when conducting larger clinical trials investigating new novel interventions (DB in specific) that can work through augmentation of the ANS activity. Findings of Chapter 5 will help these investigators make better informed decisions mainly with respect to the trial design and strategies that can help increase treatment adherence to both the main intervention of interest as well as the control group when deciding on proper design of their clinical trials.   6.4 Concluding Remarks  The work presented in this dissertation represents a primary yet novel step in investigation of the children with BBD. The ideas researched in this dissertation are innovative as that they revolve around the key roles the ANS can play as both a potential marker for detection/confirmation of the BBD and the severity of this dysfunction as well as a target that can be manipulated for treatment purposes.   In summary, the results of the studies in this dissertation demonstrate that children with BBD compared to healthy controls have a significantly lower overall HRV, and a lower cardiac PNS activity. While biofeedback is used as one of the acceptable adjunctive treatments for the management of children with BBD in current practice, the systematic literature review was not able to support its relative treatment efficacy. Additionally, the use of oxybutynin as other recommended adjunctive therapy in the management of overactive bladder was shown to be 150  associated with further reduction in the cardiac PNS activity in OAB children who had already shown to be significantly lower in their PNS activity compared to otherwise healthy children. Finally, the results of the pilot trial suggested low overall feasibility for proposing DB as a non-invasive intervention in controlling symptoms of BBD mainly due to low practical adherence to DB practice during an approximately 3 months of follow-up. This was despite the observed highly positive correlation between DB practice and resolution of clinical symptoms, improvement in the autonomic balance and function, and uroflow parameters.  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J Urol. 2005;174(5):1994-7; discussion 7-8. 239. Shei Dei Yang S, Wang CC. Outpatient biofeedback relaxation of the pelvic floor in treating pediatric dysfunctional voiding: a short-course program is effective. Urologia internationalis. 2005;74(2):118-22. 240. Zeng F, Chen H, Qi L, Zhang X, Li Y. Comparative Study of Pelvic Floor Biofeedback Training and Tolterodine for Treatment of Detrusor After-contraction in Posturination Dribbling in Children. Journal of International Medical Research. 2012;40(6):2305-10.  166  Appendices Dysfunctional Voiding Questionnaire Dysfunctional Voiding Questionnaire(21): ID:                                          Date   /  /    Please check/cross the option that best describes your current situation.    1. I pee in my underwear during the day: □                          □                          □                          □                          □ Never                   1                          2-3                       4-5                       Everyday                       Day a week           Days a week         Days a week  2. When I pee in my underwear, they are: □                          □                          □                          □                          □ I don't pee in      Almost dry         Damp                    Wet                    Soaked                                    my underwear                                     3. In a normal day I go to the washroom to pee: □                          □                          □                          □                          □ 1-2 times             3-4times              5-6 times             7-8 times        more than 8 times  4. I feel that I have to rush to the washroom to pee: □                          □                          □                          □                          □ Never                 less than half         half                     more than             Everyday                            of the time           of the time         half of the time  5. I hold my pee by crossing my legs or sitting down: □                          □                          □                          □                          □ Never                 less than half         half                     more than             Everyday                            of the time           of the time         half of the time  6. It hurts when I pee: □                          □                          □                          □                          □ Never                 less than half         half                     more than             Everyday                            of the time           of the time         half of the time  7. I wet my bed at night: □                          □                          □                          □                          □ Never                        3-4 nights            1-2 nights              4-5 nights               Every night                                   per month             per week              per week  8. I wake up to pee at night: □                          □                          □                          □                          □ Never                        3-4 nights            1-2 nights              4-5 nights               Every night                                   per month             per week              per week  167  9. When I pee, it stops and starts: □                          □                          □                          □                          □ Never                 less than half         half                     more than             Everyday                            of the time           of the time         half of the time  10.  I have to push or wait to start my pee: □                          □                          □                          □                          □ Never                 less than half         half                     more than             Everyday                            of the time           of the time         half of the time  11. I have bowel movements (poop): □                          □                          □                          □                          □        More than once     every day       every other day         every 3 days         more than         per day                          every 3 days                            12. My stool (poop) is hard: □                          □                          □                          □                          □ Never                 less than half         half                     more than             Everyday                            of the time           of the time         half of the time  13.  I have bowel (poop) accidents in my underwear: □                          □                          □                          □                          □ Never                        1-2times             3 times            4-5 times          Everyday                                    per week           per week          per week  14. How easy was to answer these questions? □                          □                          □                          □                          □ Very easy              easy                        neither easy         difficult               very difficult                                                              nor difficult     168  Pediatric Incontinence Quality of Life Questionnaire (PinQ) Pediatric Incontinence Quality of Life Questionnaire (PinQ):  The Pediatric Incontinence Quality of Life Questionnaire (PinQ) is comprised of 20 items which measure quality of life in children with bladder dysfunction. This scale has undergone rigorous reliability and validity testing.(22, 208)  Please check/cross the option that best describes your current situation.   ID:                                          Date   /  /    1. I get shy because of my bladder problem   NO    HARDLY EVER    SOMETIMES   OFTEN    ALL THE TIME  2. People in my family treat me in a different way because of my bladder problem   NO    HARDLY EVER    SOMETIMES   OFTEN    ALL THE TIME  3. I am worried that people might think my clothes smell of wee   NO    HARDLY EVER    SOMETIMES   OFTEN    ALL THE TIME  4. I think that my bladder problem won’t get better   NO    HARDLY EVER    SOMETIMES  OFTEN    ALL THE TIME  5. Mum and dad worry about me because of my bladder problem  NO    HARDLY EVER    SOMETIMES  OFTEN    ALL THE TIME  6. I would feel better about myself if I didn’t have a bladder problem   NO     MAYBE     PROBABLY    YES    DEFINITELY  7. My bladder problem makes me feel nervous   NO    HARDLY EVER    SOMETIMES  OFTEN    ALL THE TIME  8. Mum or dad sometimes seem a bit cranky because of my bladder problem   NO    HARDLY EVER    SOMETIMES  OFTEN    ALL THE TIME    169  9. My bladder problem stops me going on sleep-overs or holidays   NO    HARDLY EVER    SOMETIMES  OFTEN    ALL THE TIME  10. My bladder problem makes me feel bad about myself   NO    HARDLY EVER    SOMETIMES  OFTEN    ALL THE TIME  11. I wake up during my sleep because of my bladder problem   NO    HARDLY EVER    SOMETIMES  OFTEN    ALL THE TIME   12. I miss out on doing things because of my bladder problem   NO    HARDLY EVER    SOMETIMES  OFTEN    ALL THE TIME  13. I feel unhappy because of my bladder problem   NO    HARDLY EVER    SOMETIMES  OFTEN    ALL THE TIME  14. My bladder problem makes me feel sad   NO    HARDLY EVER    SOMETIMES  OFTEN    ALL THE TIME  15. I think about m y  bladder problem w h e n  choosing which sport to play   NO    HARDLY EVER    SOMETIMES  OFTEN    ALL THE TIME  16. I have to go to the toilet when I’m watching a movie   NO    HARDLY EVER     SOMETIMES   OFTEN     ALL THE TIME  17. If my bladder problem was fixed I would invite more friends to my house         NO    MAYBE    PROBABLY    YES     DEFINITELY  18. I choose   hobbies that won’t be spoiled   by stopping to go to the toilet   NO    HARDLY EVER    SOMETIMES    OFTEN    ALL THE TIME  19. My bladder problem makes me feel different to other people   NO    HARDLY EVER    SOMETIMES  OFTEN    ALL THE TIME  20. I miss out on being with friends because of my bladder problem   NO    HARDLY EVER    SOMETIMES  OFTEN    ALL THE TIME 170  Protocol for Recording Heart Rate Variability Protocol for recording heart rate variability Preparation of the examination room: • The EKG Amplifier is a very sensitive machine capable of capturing very small electrical signals generated by other devices in the examination room such as cell phones, computer monitors, radio transmitting devices and other medical devices. Therefore, the device should be kept at least 3 feet (approximately 1 meter) away from any electronic equipment (including monitors, fluorescent, halogen or neon lights) and 10 feet (approximately 3 meters) away from any radio transmitting devices. • All unused sensors not connected to the patient must be disconnected from the device because they may act as antennas and capture unwanted signals which would corrupt our data.  • In order to prevent static discharge from damaging the sensor and the data, antistatic mats or sprays or a humidifier producing hot, dry air should be used in the working area. • A private room or office that is relatively quiet and free of noise or other distractions is the suitable location for the examination to take place.  • Small or empty offices or areas should be avoided as they can induce a claustrophobic feeling in some patients.  Preparation of the patient: • The whole procedure must be explained to the patient briefly. • Patient preparation: 171  - The patient should be seated in a comfortable chair with a backrest which extends high enough to provide support for the head and neck and is tilted at a slight angle (≤ 15⁰) - just enough to relax the weight of the head so the neck can relax, but not so much as to encourage drowsiness. - The patient should be asked to keep quiet without talking or falling asleep, crossing legs or making unnecessary movements with their eyes open and their mind not engaged in intense mental activity. - The patient’s feet should be able to reach the floor comfortably or be supported with a footrest. - The patient should not be able to see the computer monitor during the baseline recording. - The patient should not have had heavy exercise for at least 24 hours before the procedure neither should they have consumed coffee, tea or other caffeinated beverages within 3 hours before the baseline measurement. The patient should not have smoked a cigarette for at least 30 minutes before the procedure and it is best to wait at least an hour and a half after a heavy meal to take the measurement. Also, if the patient is on any kind of medications, it should be noted in their profile and stopped for 5 half-lives before the procedure. - Anticholinergic (including antidepressant, antihistamine, and over-the-counter cough and cold medication) and sympathomimetic (α- and β-agonists) and parasympathomimetic agents are avoided for 48 hours. Short-acting α- and β-172  antagonists are discontinued for 24 hours and long-acting ones for 48 hours, at the discretion of the referring physician. Analgesics, including opioids, are avoided the day of the test.  - The patient should not have had an acute illness in within the previous 48 hours. - Compressive clothing including Jobst stockings and corsets, are not worn the morning of the test. • Skin preparation: - For the sake of signal quality, a good skin preparation should be performed prior to attaching the surface electrodes with the purpose of avoiding artifacts as much as possible. - Make sure the skin is dry and clean before attaching the surface electrodes by rubbing it with alcohol pad or shaving excess hair or using abrasive creams if necessary. • Attachment of the electrodes to the skin: - Application of conductive gel or paste is recommended to the center of the electrodes for the purpose of having a better signal quality. - To further improve the signal quality, we suggest using pre-gelled electrodes. - In order to prevent artifacts due to the movement of the electrodes, the use of tape is recommended to attach the electrodes and it is also recommended to make a loop with the electrode lead cable under the tape to reduce the chance of displacement due to a sudden pull. 173  - A waiting time of at least 5 minutes has been suggested prior to the recording procedure in order for the gel to be absorbed by the skin for the purpose of reducing the impedance. HRV data artifacts and how to avoid them: • An artifact is as undesirable data within a signal which originates from other sources can distort the signal thus making the HRV analysis difficult or even impossible.  • HRV artifacts: - Appear as missed beats, extra beats or natural physiological events such as premature atrial or ventricular contraction (PAC or PVC) which distorts HRV analysis and should be corrected by the software. • EKG artifacts: - Line interference (50/60Hz noise): most common EKG artifact which comes from the power line and is transmitted by electrical devices (monitors, computers or etc.) near the EKG amplifier. On the computer screen the trace looks like a fuzzy line. It can be removed by the software’s special filter or avoided using proper skin preparation and gel usage on the electrodes or taking additional precautions such as keeping the device 3 feet (approximately 1 meter) away from any electronic equipment and 10 feet (approximately 3 meters) away from any radio transmitting devices and disconnecting all unused sensors not connected to the patient from the device.  174  - Muscle contraction artifacts: artifacts produced from the contraction of other muscles, particularly from chest and arm muscles. These artifacts can cause a lot of extra beats and are reduced by using a chest electrode instead of arm and by training the patients to relax their muscles, placing their arms on their knees and staying as quiet as possible. - Direct current (DC) offset artifacts: these artifacts can occur as a result of bad skin-electrode contact (for instance, in case of attaching electrodes to greasy or dirty skin or reusing electrodes for many times which distorts the quality of the conduction). In this case, a direct current offset will make the signal drift up or down on the scale due to high impedance between the skin and the electrodes which in some conditions can cause missed beats. These artifacts can be avoided by cleaning and preparing the skin prior to attaching the electrodes and by using new electrodes instead of reusing old ones. - Electrode movement artifacts: these artifacts can occur as a result of patients dragging the cables and pulling one of the electrodes off the skin which can be detected as very wide signal deviations which confuse the beat detection algorithm causing many extra beats. This can be avoided by making sure that the cables are loose enough to provide some freedom of motion and/or by using pre-gelled electrodes. - Electrode polarity artifacts: these artifacts can occur as a result of electrodes being misplaced which can be detected as reversed R-spikes and can be avoided by making 175  sure that electrodes are positioned in the correct location on the patient’s chest or arms.  HRV measurement: • Baseline resting HRV range measurement: 5 minutes recording of heart-rhythm data must be collected while the patient is sitting quietly. After that the session must proceed to the next test phase without a pause in recordings.     176  Search Strategies for the Systematic Review Table D.1 MEDLINE® search strategy for the systematic review and results. Database: Ovid MEDLINE(R) In-Process & Other Non-Indexed Citations and Ovid MEDLINE(R) <1946 to Present> Search Strategy: -------------------------------------------------------------------------------- 1     functional voiding disorder.mp. (2) 2     Urinary Incontinence/ (17680) 3     Enuresis/ (3843) 4     Urination Disorders/ (10263) 5     Urinary Bladder Diseases/ (9083) 6     Urodynamics/ (12934) 7     Constipation/ or Vesico-Ureteral Reflux/ (17663) 8     overactive bladder.mp. (3279) 9     Urinary Bladder, Overactive/ (2311) 10     Detrusor overactivity.mp. (1616) 11     Urinary Bladder/ (41649) 12     urge incontinence.mp. (2083) 13     Urinary Incontinence, Urge/ (533) 14     unstable bladder.mp. (238) 15     voiding postponement.mp. (20) 16     Urinary Tract Infections/ (31943) 17     Dysfunctional voiding.mp. (346) 18     fractionated voiding.mp. (1) 19     underactive bladder.mp. (32) 20     lazy bladder.mp. (22) 21     Hinman syndrome.mp. (32) 22     non-neurogenic neurogenic bladder.mp. (26) 23     vaginal reflux.mp. (20) 24     dysfunctional elimination syndrome.mp. (43) 25     Elimination Disorders/ (19) 26     1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18  or 19 or 20 or 21 or 22 or 23 or 24 or 25 (125528) 27     biofeedback.mp. (8006) 28     EMG.mp. (24014) 29     Biofeedback, Psychology/ (6276) 30     Electromyography/ (66688) 31     27 or 28 or 29 or 30 (78814) 32     Randomized Controlled Trials as Topic/ (100690) 33     randomized controlled trial/ (382293) 34     Random Allocation/ (80788) 35     Double Blind Method/ (129303) 36     Single Blind Method/ (19091) 177  37     clinical trial/ (499771) 38     clinical trial, phase i.pt. (15867) 39     clinical trial, phase ii.pt. (26422) 40     clinical trial, phase iii.pt. (9869) 41     clinical trial, phase iv.pt. (950) 42     controlled clinical trial.pt. (88866) 43     randomized controlled trial.pt. (382293) 44     multicenter study.pt. (178051) 45     clinical trial.pt. (499771) 46     exp Clinical Trials as topic/ (292298) 47     or/32-46 (1055436) 48    (clinical adj trial$).tw. (221103) 49    ((singl$ or doubl$ or treb$ or tripl$) adj (blind$3 or mask$3)).tw. (132020) 50     PLACEBOS/ (33365) 51     placebo$.tw. (165418) 52     randomly allocated.tw. (16873) 53    (allocated adj2 random$).tw. (19439) 54     or/48-53 (431872) 55     47 or 54 (1202182) 56     case report.tw. (199653) 57     letter/ (818501) 58     historical article/ (297722) 59     56 or 57 or 58 (1304577) 60     55 not 59 (1172299) 61     26 and 31 and 60 (272)     178  Table D.2 Embase search strategy for the systematic review and results. Database: Embase <1974 to 2013 August 12> Search Strategy: -------------------------------------------------------------------------------- 1     functional voiding disorder.mp. (4) 2     bladder dysfunction/ (4018) 3     urine incontinence/ (31370) 4     urodynamics/ (14597) 5     enuresis/ (4755) 6     constipation/ (53394) 7     Overactive Bladder.mp. (8356) 8     overactive bladder/ (7357) 9     Detrusor overactivity.mp. (2734) 10     urge incontinence.mp. (5394) 11     urge incontinence/ (4201) 12     urinary tract infection/ (67452) 13     unstable bladder.mp. (306) 14     bladder instability/ (575) 15     Voiding postponement.mp. (27) 16     lower urinary tract symptom/ (5125) 17     Dysfunctional voiding.mp. (500) 18     fractionated voiding.mp. (3) 19     bladder sphincter/ (3012) 20     underactive bladder.mp. (59) 21     overactive bladder/ (7357) 22     lazy bladder.mp. (35) 23     Hinman Syndrome.mp. (51) 24     bladder disease/ (7621) 25     Non-neurogenic neurogenic bladder.mp. (42) 26     Vaginal Reflux.mp. (26) 27     Dysfunctional Elimination Syndrome.mp. (66) 28     or/1-27 (179881) 29     biofeedback.mp. (7151) 30     feedback system/ (52981) 31     electromyogram/ (19751) 32     uroflowmetry/ (2458) 33     EMG.mp. (31343) 34     or/29-33 (97357) 35     Clinical trial/ (892126) 36     Randomized controlled trial/ (356328) 37     Randomization/ (63163) 38     Single blind procedure/ (18083) 39     Double blind procedure/ (119513) 40     Crossover procedure/ (38120) 179  41     placebo/ (236082) 42     Randomi?ed controlled trial$.tw. (92875) 43     Rct.tw. (12374) 44     Random allocation.tw. (1332) 45     Randomly allocated.tw. (19722) 46     Allocated randomly.tw. (1938) 47    (allocated adj2 random).tw. (812) 48     Single blind$.tw. (14054) 49     Double blind$.tw. (145888) 50    ((treble or triple) adj blind$).tw. (349) 51     Placebo$.tw. (199092) 52     Prospective study/ (246691) 53     or/35-52 (1386376) 54     case study/ (20766) 55     case report.tw. (259898) 56     abstract report/ (89581) 57     letter/ (811017) 58     or/54-57 (1175910) 59     53 not 58 (1349067) 60     28 and 34 and 59 (843)   180  Table D. 3 Cochrane Central Register of Controlled Trials (CENTRAL) search strategy for the systematic review and results. Database: EBM Reviews - Cochrane Central Register of Controlled Trials <July 2013> Search Strategy: -------------------------------------------------------------------------------- 1     Urination Disorders/ (415) 2     Urodynamics/ (785) 3     overactive bladder.mp. (521) 4     Urinary Bladder, Overactive/ (204) 5     Detrusor overactivity.mp. (178) 6     Urinary Incontinence/ (638) 7     urge incontinence.mp. (299) 8     Urinary Incontinence, Urge/ (55) 9     unstable bladder.mp. (33) 10     Urinary Bladder Diseases/ (145) 11     Dysfunctional voiding.mp. (23) 12     Urination Disorders/ (415) 13     Dysfunctional Elimination Syndrome.mp. (3) 14     or/1-13 (2308) 15     biofeedback.mp. (1418) 16     Biofeedback, Psychology/ (713) 17     EMG.mp. (1642) 18     Electromyography/ (2283) 19     15 or 16 or 17 or 18 (4038) 20     14 and 19 (79)   181  Table D.4 Excluded studies and reasons for exclusion. Study Reason for Exclusion Bael 2008(231) Study design Barroso 2006(232) Study design Combs 1998(233) Study design Hoebeke 2011(234) Study design Kibar 2007a(235) Study design Kibar 2007b(236) Study design McKenna 1999(237) Study design Yagci 2005(238) Study design Yamanishi 2000(150) Study design Yang 2004(239) Study design Zeng 2012(240) Population Zivkovic 2012(193) Study design     182  Diaphragmatic Breathing Figures Figure E.1 Diaphragmatic breathing.  Illustration by Armin Mortazavi. Used under a Creative Commons Attribution 4.0 International license.    183  Figure E.2 Diaphragmatic breathing – continued.  Illustration by Armin Mortazavi. Used under a Creative Commons Attribution 4.0 International license.     184  Treatment Acceptability Questionnaire (TAQ) Treatment Acceptability Questionnaire (TAQ): Study ID:__________ Date:___________ Please answer these questions that deal with your reactions to the proposed treatment. Cross the number that best describes your reactions. (Adapted with modifications from “Development of TAQ” by John Hunsley(202)) Participant’s Questionnaire  1. Overall, how acceptable do you find Diaphragmatic Breathing to be?   2. How helpful do you think Diaphragmatic Breathing might be?   3. How likely do you think it is that Diaphragmatic Breathing may harm you?   4. How knowledgeable do you think the researchers are in giving you this treatment?   5. How trustworthy do you think the researchers are?   6. Would you recommend Diaphragmatic Breathing to other children?   Yes/No 7. Did you have any unpleasant or negative experience related to practicing Diaphragmatic Breathing?   Yes/No  If yes, please describe your experience: -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------  Very Unacceptable 1 2 3 4 5 6 7 Very Acceptable Very Unhelpful 1 2 3 4 5 6 7 Very Helpful Very Unlikely 1 2 3 4 5 6 7 Very Likely Not Knowledgeable 1 2 3 4 5 6 7 Very Knowledgeable Very Untrustworthy 1 2 3 4 5 6 7 Very Trustworthy 185  Please indicate how much you agree or disagree with the following statements regarding Diaphragmatic Breathing exercise for your child. Cross the number that best describes your opinion. (Adapted with modifications from “The Role of Treatment Acceptability in the Initiation of Treatment for ADHD” by Amy Krain et al.(203)) Parents’ Questionnaire 1. This is an acceptable treatment for my child’s bladder problem.   2. This treatment should be effective in changing my child’s bladder habit.   3. My child’s bladder problem is troublesome enough to justify the use of this treatment.   4. I would be willing to continue using this treatment with my child.   5. This treatment would not have bad side effects for my child.   6. I like this treatment.    7. This treatment is a good way to handle my child’s problem.   8. Overall, this treatment would help my child.   Strongly disagree 1 2 3 4 5 6 Strongly agree Strongly disagree 1 2 3 4 5 6 Strongly agree Strongly disagree 1 2 3 4 5 6 Strongly agree Strongly disagree 1 2 3 4 5 6 Strongly agree Strongly disagree 1 2 3 4 5 6 Strongly agree Strongly disagree 1 2 3 4 5 6 Strongly agree Strongly disagree 1 2 3 4 5 6 Strongly agree Strongly disagree 1 2 3 4 5 6 Strongly agree 

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