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Longitudinal investigation of middle ear function using multi-frequency, multi-component tympanometry… Cai, Anika 2010

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LONGITUDINAL INVESTIGATION OF MIDDLE EAR FUNCTION USING MULTI-FREQUENCY, MULTI-COMPONENT TYMPANOMETRY FROM BIRTH TO 6 MONTHS OF AGE  by ANIKA CAI B.A., The University of British Columbia, 2007  A THESIS SUBMITTED IN PARTIAL FUFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in The Faculty of Graduate Studies (Audiology and Speech Sciences)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) January 2010  © Anika Cai 2010  Abstract Objectives: The specific goals of this study were: 1) To understand the mechanoacoustical properties of the normal ear canal and middle ear and its maturation as a function of age using conventional and high frequency tympanometry 2) to establish tympanometric guidelines and normative data of the normal ear canal and middle ear in infants birth to 6 months of age. Design: Thirty-one normal hearing newborns were tested longitudinally in 1-month intervals up to 6 months of age for a total of 6 visits. Tympanograms were recorded and the distributions of patterns were analyzed using the Vanhuyse model at 226 Hz, 678 Hz, and 1000 Hz. Additionally, tympanometric recordings of admittance (Ya), susceptance (Ba), and conductance (Ga) were analyzed at 226 Hz and 1000 Hz probe tones. Lastly, the variation of compensated susceptance (∆B) and conductance (∆G) were recorded at extended frequencies from 250-2000 Hz in 50 Hz intervals for 16 infants. Results: Results showed that 1000 Hz tympanograms were the simplest to quantify as most recordings were single-peaked. 226 Hz and 678 Hz recordings were often multipeaked. Both positive and negative admittance and susceptance tail values increased with age for 226 Hz and 1000 Hz. However, tail values at 1000 Hz increased faster than for 226 Hz. Negative tail values were smaller compared to positive tail values which resulted in smaller compensated admittance values for the positive tails compared to negative tails across all 6 visits. Admittance magnitude decreased with age at 226 Hz as susceptance increased and conductance decreased. However, at 1000 Hz, admittance magnitude increased as susceptance remained relatively constant and conductance increase. Conclusion: Results suggest that the infant middle ear and ear canal develop towards compliance with age although is not yet a purely acoustically compliant system by 6 months of age, particularly at high frequencies. An increase in volume in the middle ear cavity, reduction of middle ear debris, and overall decrease in resistive elements may be contributing to these changes. Significant differences were observed between each visit and warrant the use of age-specific norms when applying tympanometric data to infants below 6 months of age. ii  Table of Contents Abstract .............................................................................................................................. ii Table of Contents ............................................................................................................. iii List of Tables .................................................................................................................... vi List of Figures .................................................................................................................. xii Acknowledgements ...................................................................................................... xviii Dedication ....................................................................................................................... xix  1 Introduction .................................................................................................................... 1 1.1  External and Middle Ear Structure and Function in Adults ................................ 5  1.2  Maturation of the External and Middle Ear ........................................................ 8  1.2.1  Maturation of the External Ear .................................................................... 9  1.2.2  Maturation of the Middle Ear .................................................................... 11  1.3  Functional Implications of External and Middle Ear Structural Changes on Auditory Sensitivity........................................................................ 12  1.4  Pathology of the Middle Ear in Infants .............................................................. 13  1.5  Clinical Implications .......................................................................................... 16  1.5.1  Impact of Maturation on Transducers ......................................................... 16  1.5.2  Impact of Maturation on Amplification Measures...................................... 17  1.6  Measures of External and Middle Ear: Tympanometry ..................................... 18  1.6.1  Fundamentals and Principles of Acoustic Immittance Measurements ....... 21  1.6.2  Clinical Application of Tympanometry ...................................................... 27  1.6.2.1  Conventional 226 Hz Tympanometry ............................................ 28  1.6.2.2  Multi-frequency, Multi-component Tympanometry ...................... 32  1.6.2.3  Classification of Tympanometric Shapes ....................................... 34  1.6.2.4  Resonant Frequency and Frequency Corresponding to Admittance Phase Angle of 45 degrees ............................................................. 36  1.6.2.5  Diagnostic Utility of Multi-frequency, Multi-component Tympanometry..................................................................................... 37 iii  1.7  1.7.1  Peak Compensated Static Admittance ........................................................ 41  1.7.2  Compensation for Ear Canal Volume ......................................................... 43  1.7.3  Consideration of Probe Tone Frequency .................................................... 45  1.7.4  Classification of Tympanometric Shapes ................................................... 48  1.7.5  Tympanometric Peak Pressure .................................................................... 50  1.8  2  Immittance Measurements: Principles and Application in Infants .................... 39  Purpose of Study ................................................................................................ 51  Longitudinal Investigation of Middle Ear Function Using Multi-frequency, Multi-component Tympanometry in Infants Birth to 6 months of age .............. 54 2.1  Methods .............................................................................................................. 54  2.1.1  Recruitment ................................................................................................. 54  2.1.2  Subjects ....................................................................................................... 55  2.1.3  Instrumentation and Procedure ................................................................... 56  2.1.3.1  Transient Evoked Otoacoustic Emission Test .................................... 57  2.1.3.2  Tympanometry.................................................................................... 57  2.1.3.3  Real-ear to Coupler Difference Measurement .................................... 59  2.1.3.4  Wide Band Reflectance ...................................................................... 59  2.1.4  Challenges in Testing Young Infants .......................................................... 59  2.1.5  Treatment of the Data ................................................................................. 62  2.2  Results ................................................................................................................ 63  2.2.1  Analysis of Tympanometric Shapes ........................................................... 64  2.2.2  Variation of Admittance, Susceptance, and Conductance Tails with Age . 68  2.2.3  Variation of Peak Compensated Static Admittance Calculated from Admittance Tympanograms and Rectangular Components with Age ........ 75  2.2.4  Variation of Peak Compensated Static Susceptance and Compensated Conductance with Age ................................................................................ 81  2.2.5  Variation of Tympanometric Pressure with Age ........................................ 88  2.2.6  Analysis of Change in Compensated Susceptance (ΔB) and Conductance (ΔG) Across Age ......................................................................................... 89 iv  2.2.7  Data Comparison of Infants at Visit 6 to School-aged Children and Adults ................................................................................................... 92  2.2.8  2.3  Case Studies ................................................................................................ 99  2.2.8.1  Case 1: (Sensorineural Hearing Loss) ................................................ 99  2.2.8.2  Case 2: (Conductive Hearing Loss-Transient) .................................. 102  2.2.8.3  Case 3: (Conductive Hearing Loss-Transient) .................................. 105  Discussion ........................................................................................................ 110  2.3.1  Comparison of Results .............................................................................. 111  2.3.1.1  Tympanometric Shapes ..................................................................... 112  2.3.1.2  Equivalent Ear Canal Volume ........................................................... 116  2.3.1.3  Peak Compensated Static Admittance ............................................... 119  2.3.1.4  Peak Compensated Static Susceptance and Conductance ................. 123  2.3.1.5  Individual Longitudinal Data From 5 Subjects ................................. 123  2.3.1.6  Tympanometric Differences Observed Between Infants at Visit 6 to School-aged Children and Adults ...................................................... 124  2.3.2  2.3.2.1  Tympanometric Shape ....................................................................... 127  2.3.2.2  Equivalent Ear Canal Volume ........................................................... 128  2.3.2.3  Peak Compensated Static Admittance ............................................... 129  2.3.2.4  Peak Compensated Static Susceptance and Conductance ................. 124  2.3.3  3  Clinical Implications ................................................................................. 126  Directions For Future Research ................................................................ 134  References............................................................................................................... 136  Appendices ..................................................................................................................... 160 Appendix I ................................................................................................................... 160 Appendix II ................................................................................................................. 168 Appendix III ................................................................................................................ 172 Appendix IV ................................................................................................................ 174  v  List of Tables  Table 1.1:  Terminology List ........................................................................................ 18  Table 1.2: Rectangular Notation and Polar Notation Conversion Formulas (Adapted from Shahnaz, 2007 with permission) .............................................................. 25 Table 1.3: 1000 Hz Normative Data from Margolis et al. (2003) (65 infants -mean age of 4 weeks) .................................................................................... 47 Table 1.4: 1000 Hz Normative data from Kei et al. (2003) (106 infants- mean age of 3.26 days) ................................................................................ 47 Table 2.1: Subject Age Range and Number of Ears Tested............................................. 56 Table 2.2: Distribution of Vanhuyse et al. (1975) Patterns For Infants Across 6 Visits .................................................................................................................. 67 Table 2.3: The 5th, 50th, and 95th Percentiles of Admittance at Positive and Negative Tails .............................................................................................. 69 Table 2.4: The 5th, 50th, and 95th Percentiles of Susceptance at Positive and Negative Tails .............................................................................................. 71 Table 2.5: The 5th, 50th, and 95th Percentiles of Conductance at Positive and Negative Tails .............................................................................................. 74 Table 2.6: The 5th, 50th, and 95th Percentiles of Peak Compensated Static Admittance (Positive and Negative) ..................................................................................................... 76 Table 2.7: The 5th, 50th, and 95th Percentiles of Peak Compensated Static Susceptance (positive & negative)......................................................................................................... 83  vi  Table 2.8: The 5th, 50th, and 95th Percentiles of Peak Compensated Static Conductance (Positive & Negative) ....................................................................................................... 83 Table 2.9: The 5th, 50th, and 95th Percentiles of Tympanometric Peak Pressure ............. 88 Table 2.10: The Mean, 50th, and 95th Percentiles of Admittance at Positive Tail For Infants at Visit 6 (Current Study), School-aged Children (Bosaghzadeh, in progress) and Adults (Shahnaz & Bork, 2008) ................................................................................. 94 Table 2.11: The Mean, 50th, and 95th Percentiles of Positive Peak Compensated Static Admittance from Ya Tympanogram For Infants at Visit 6 (Current Study), School-aged Children (Bosaghzadeh, in progress) and Adults (Shahnaz & Bork, 2008) ..................... 95 Table 2.12: The Mean, 50th, and 95th Percentiles of Positive Peak Compensated Static Susceptance and Conductance For Infants at Visit 6 (Current Study), School-aged Children (Bosaghzadeh, in progress) and Adults (Shahnaz & Bork, 2008) ..................... 97 Table 2.13: Summary Comparison of Data to Previous Published Works .................... 129 Table 2.14: Recommended Clinical Cut-off Values For Equivalent Ear Canal Volume For Infants Between Birth and 6 Months of Age ............................................................ 129 Table 2.15: Recommended Clinical Cut-off Values For 226 Hz Positive Peak Compensated Static Admittance For Infants Between 3-6 months of Age .................... 131 Table 2.16: Recommended Clinical Cut-off Values For 226 Hz Negative Peak Compensated Static Admittance For Infants Between 3-6 months of Age .................... 131 Table 2.17: Recommended Clinical Cut-off Values For 1000 Hz Positive Peak Compensated Static Admittance For Infants Between Birth and 6 Months of Age ....... 132 Table 2.18: Recommended Clinical Cut-off Values For 1000 Hz Negative Peak Compensated Static Admittance For Infants Between Birth and 6 Months of Age ....... 132 vii  Table 2.19: Recommended Clinical Cut-off Values For Peak Compensated Static Susceptance For Infants Between Birth and 6 Months of Age ....................................... 133 Table 2.20: Recommended Clinical Cut-off Values For Peak Compensated Conductance Conductance For Infants Between Birth and 6 Months of Age ...................................... 134 Table A1.1: Summary of ANOVA For Infants Admittance at Tails at 226 Hz With Two Within-subject Factors (6 Levels: Number of Visits) and (2 Levels: Positive versus Negative tail). ....................................................................... 160 Table A1.2: Summary of ANOVA For Infants Admittance at Tails at 1000 Hz With  Two Within-subject Factors (6 Levels: Number of Visits) and  (2 Levels: Positive Versus Negative tail)........................................................................ 160 Table A1.3: Summary of ANOVA For Infants Susceptance and Conductance at  Positive Tails at 226 Hz With Two Within-subject Factors  (6 Levels: Number of Visits) and (2 Levels: Rectangular Component). ........................ 161 Table A1.4: Summary of ANOVA For Infants Susceptance and Conductance at Negative Tails at 226 Hz With Two Within-subject Factors (6 Levels: Number of Visits) and (2 Levels: Rectangular Component). ..................... 161 Table A1.5: Summary of ANOVA For Infants Susceptance and Conductance at Positive Tails at 1000 Hz With Two Within-subject Factors (6 Levels: Number of Visits) and (2 Levels: Rectangular Component). ...................... 162 Table A1.6: Summary of ANOVA For Infants Susceptance and Conductance at Negative Tails at 1000 Hz With Two Within-subject Factors (6 Levels: Number of Visits) and (2 Levels: Rectangular Component). ....................... 162  viii  Table A1.7: Summary of ANOVA For Infants Ytm from Ya Tympanograms at 226 Hz With Two Within-subject Factors (6 Levels: Number of Visits) and (2 Levels: Positive Versus Negative Tails). .................................................................... 163 Table A1.8: Summary of ANOVA For Infants Ytm from Ya Tympanograms at 1000 Hz With Two Within-subject Factors (6 Levels: Number of Visits) and (2 Levels: Positive Versus Negative Tail). ..................................................................... 163 Table A1.9: Summary of ANOVA For Infants Ytm from B/G Tympanograms at 226 Hz With Two Within-subject Factors (6 Levels: Number of Visits) and (2 Levels: Positive Versus Negative Tail). ..................................................................... 164 Table A1.10: Summary of ANOVA For Infants Ytm from B/G Tympanograms at 1000 Hz With Two Within-subject Factors (6 Levels: Number of Visits) and (2 Levels: Positive Versus Negative Tail). ..................................................................... 164 Table A1.11: Summary of ANOVA For Infants Positive Compensated Susceptance and Conductance at 226 Hz With Two Within-subject Factors (6 Levels: Number of Visits) and (2 Levels: Rectangular Component) ......................... 165 Table A1.12: Summary of ANOVA For Infants Negative Compensated Susceptance and Conductance at 226 Hz With Two Within-subject Factors (6 Levels: Number of Visits) and (2 Levels: Rectangular Component) ......................... 165 Table A1.13: Summary of ANOVA For Infants Positive Compensated Susceptance and Conductance at 1000 Hz With Two Within-subject Factors (6 Levels: Number of Visits) and (2 Levels: Rectangular Component) ......................... 166  ix  Table A1.14: Summary of ANOVA For Infants Negative Compensated Susceptance and Conductance at 1000 Hz With Two Within-subject Factors (6 Levels: Number of Visits) and (2 Levels: Rectangular Component) ......................... 166 Table A1.15:Summary of ANOVA For Infants Tympanometric Peak Pressure With Two Within-subject Factors (6 Levels:Number of Visits) and (2 Levels:Probe Tone) .. 167 Table A2.1: The 5th, 50th, and 95th Percentiles of Change in Susceptance (ΔB) Between 260-900 Hz Across Age ................................................................................... 168 Table A2.2: The 5th, 50th, and 95th Percentiles of Change in Susceptance (ΔB) Between 1000- 2000 Hz Across Age .............................................................................. 169 Table A2.3: The 5th, 50th, and 95th Percentiles of Change in Conductance (ΔG) Between 260- 900 Hz Across Age .................................................................................. 170 Table A2.4: The 5th, 50th, and 95th Percentiles of Change in Conductance (ΔG) Between 1000-2000 Hz Across Age ............................................................................... 171 Table A3.1: Summary of ANOVA For Infants at Visit 6/School-aged Children/Adults Positive Peak Compensated Ytm from Ya tympanograms at 226 Hz With Two Within-subject Factors (3 Levels: Age Groups) and (1 Level: Ytm) ............................. 172 Table A3.2: Summary of ANOVA For Infants at Visit 6/School-aged Children/Adults Admittance at Positive Tail From Ya Tympanograms at 226 Hz With Two Within-subject Factors (3 Levels: Age Groups) and (1 Level: Ytm) ..................... 172 Table A3.3: Summary of ANOVA For Infants at Visit 6/School-aged Children/Adults Positive Peak Compensated Static Admittance/Susceptance/Conductance at 226 Hz With Two Within-subject Factors (3 Levels: Age Groups) and (3 Levels: Rectangular Components and Ytm)............................................................... 173 x  Table A3.4: Summary of ANOVA For Infants at Visit 6/School-aged Children/Adults Positive Peak Compensated Static Admittance/Susceptance/Conductance at 1000 Hz With Two Within-subject Factors (3 Levels: Age Groups) and (3 Levels: Rectangular Componentsand Ytm)................................................................ 173  xi  List of Figures  Figure 1.1: Gross Anatomy of the External, Middle, and Inner Ear (adapted from Gelfand, 2004) ............................................................................................. 5 Figure 1.2: Middle and External Ear of a Newborn Versus Adult (adapted from Ballachanda, 1995) .................................................................................... 11 Figure 1.3: Graphic Illustration of Impedance (Za) Versus Admittance (Ya) .................. 22 Figure 1.4: Admittance Terminology (Adapted from Shahnaz, 2007 with permission) 24 Figure 1.5: Acoustic Admittance (Ya) As a Function of Probe Tone Frequency (adapted from Margolis & Shanks, 1991)......................................................................... 26 Figure 1.6: Typical Ya-226 Hz Tympanogram Recorded From a Normal Adult Ear Showing Positive Compensated Static Admittance (Ytm) and Tympanometric Peak Pressure (TPP) (Adapted from Shahnaz, 2007 with permission) ..................................... 29 Figure 1.7: GSI-Tympstar Sweep Frequency Recording of Variation of Susceptance (ΔB) and Conductance (ΔG) (in mmho) Using Positive Compensation. (Adapted from Shahnaz, 2007) ......................................................................................... 33 Figure 1.8: Vanhuyse Classification Model (Vanhuyse et al., 1975) to Account For Four Normal Patterns of Acoustic Susceptance (Ba) and Conductance (Ga) Tympanograms Recorded Using a 678 Hz Probe Tone. (Adapted from Fowler & Shanks, 2002) ........... 35 Figure 2.1: TEOAE Testing On a Subject ....................................................................... 60 Figure 2.2: Tympanometric Testing On a Subject ........................................................... 60 Figure 2.3: Proportion of Single versus Multiple-peak Ya Tympanograms Across 6 Visits .................................................................................................................. 65 xii  Figure 2.4: Distribution of Vanhuyse et al.(1975) Patterns for Infants Across 6 Visits .. 67 Figure 2.5: Median Admittance Tail Magnitudes (Positive and Negative Tails) For 226 Hz Probe Tone Across 6 Visits .................................................................................. 70 Figure 2.6: Median Admittance Tail Magnitudes (Positive and Negative Tails) For 1000 Hz Probe Tone Across 6 Visits ................................................................................ 70 Figure 2.7: Median Susceptance (Ba) and Conductance (Ga) Negative Tail Magnitude For 226 Hz Probe Tone Across 6 Visits ........................................................................... 72 Figure 2.8: Median Susceptance (Ba) and Conductance (Ga) Negative Tail Magnitude For 1000 Hz Probe Tone Across 6 Visits ......................................................................... 72 Figure 2.9: Median Susceptance (Ba) and Conductance (Ga) Positive Tail Magnitude For 226 Hz Probe Tone Across 6 Visits ........................................................................... 73 Figure 2.10: Median Susceptance (Ba) and Conductance (Ga) Positive Tail Magnitude For 1000 Hz Probe Tone Across 6 Visits ......................................................................... 73 Figure 2.11: Median Peak Compensated Static Admittance (Ytm) Calculated from Ya Tympanograms Using Positive and Negative Compensation For 226 Hz Across 6 Visits ............................................................................................... 77 Figure 2.12: Median Peak Compensated Static Admittance (Ytm) Calculated from Btm/Gtm Tympanograms Using Positive and Negative Compensation For 226 Hz Across 6 Visits ............................................................................................... 78 Figure 2.13: Median Peak Compensated Static Admittance (Ytm) Calculated from Ya tympanograms Using Positive and Negative Compensation For 1000 Hz Across 6 Visits ............................................................................................. 80  xiii  Figure 2.14: Median Peak Compensated Static Admittance(Ytm) Calculated from Btm/Gtm tympanograms Using Positive and Negative Compensation at 1000Hz Across 6 Visits .................................................................................................................. 81 Figure 2.15: Median Positive Compensated Susceptance (Btm) and Conductance (Gtm) For 226 Hz Across 6 Visits................................................................ 84 Figure 2.16: Median Negative Compensated Susceptance (Btm) and Conductance (Gtm) For 226 Hz Across 6 Visit ................................................................. 84 Figure 2.17: Median Positive Compensated Susceptance (Btm) and Conductance (Gtm) For 1000 Hz Across 6 Visits.............................................................. 87 Figure 2.18: Median Negative Compensated Susceptance (Btm) and Conductance (Gtm) For 1000 Hz Across 6 Visits.............................................................. 87 Figure 2.19: Median Tympanometric Peak Pressure (TPP) For 226 Hz and 1000 Hz Across 6 Visits ................................................................................................... 89 Figure 2.20: Variation of Susceptance (ΔB) as a Function of Probe Tone Frequency For Infants Across 6 Visits (Current study) and Compared to Adult Data (Shahnaz & Bork, 2008)................................................................................. 91 Figure 2.21: Variation of Conductance (ΔG) as a Function of Probe Tone Frequency For Infants Across 6 Visits (Current study) and Compared to  Adult  Data (Shahnaz & Bork, 2008) ........................................................................................... 92 Figure 2.22: Mean Static Admittance at Positive Tail (+200) from Ya Tympanogram For infants at VISIT 6 (current study) compared to School-aged Children (Bosaghzadeh, in progress) and Adults (Shahnaz & Bork, 2008) .................................... 94  xiv  Figure 2.23: Mean Positive Peak Compensated Static Admittance (Ytm) from Ya Tympanogram  For infants at Visit 6 (current study) compared to School-aged Children  (Bosaghzadeh, in progress) and Adults (Shahnaz & Bork, 2008) .................................... 96 Figure 2.24: 226 Hz Mean Positive Peak Compensated Static Admittance (Ytm) from Btm/Gtm Tympanograms and Positive Compensated Susceptance (Btm) and Conductance (Gtm) For infants at Visit 6 (current study) compared to School-aged Children (Bosaghzadeh, in progress) and Adults (Shahnaz & Bork, 2008) ..................... 98 Figure 2.25: 1000 Hz Mean Positive Peak Compensated Static Admittance (Ytm) from Btm/Gtm Tympanograms and Positive Compensated Susceptance (Btm) and Conductance (Gtm) For infants at Visit 6 (current study) compared to School-aged Children (Bosaghzadeh, in progress) and Adults (Shahnaz & Bork, 2008) ..................... 98 Figure 2.26: Case 1 Visit 1 Right Ear Ya-Tympanogram Recording 1000 Hz.............. 100 Figure 2.27: Case 1 Visit 1 Left Ear Ya-Tympanogram Recording 1000 Hz ................ 100 Figure 2.28: Case 1 Visit 1 Right Ear B/G-Tympanogram Recording 1000 Hz ........... 100 Figure 2.29: Case 1 Visit 1 Left Ear B/G-Tympanogram Recording 1000 Hz ............. 100 Figure 2.30: Case 1 Visit 1 Right Ear Ya- Tympanogram Recording 226 Hz............... 101 Figure 2.31: Case 1 Visit 1 Left Ear Ya- Tympanogram Recording 226 Hz ................. 101 Figure 2.32: Case 1 Visit 1 Right Ear B/G-Tympanogram Recording 226 Hz ............ 101 Figure 2.33: Case 1 Visit 1 Left Ear B/G-Tympanogram Recording 226 Hz ............... 101 Figure 2.34: Case 2 Visit 1 Right Ear Ya-Tympanogram Recording 1000 Hz ............. 103 Figure 2.35: Case 2 Visit 1 Left Ear Ya-Tympanogram Recording 1000 Hz ............... 103 Figure 2.36: Case 2 Visit 1 Right Ear B/G-Tympanogram Recording 1000 Hz ........... 103 Figure 2.37: Case 2 Visit 1 Left Ear B/G-Tympanogram Recording 1000 Hz ............. 103 xv  Figure 2.38: Case 2 Visit 1 Right Ear Ya- Tympanogram Recording 226 Hz............... 104 Figure 2.39: Case 2 Visit 1 Left Ear Ya-Tympanogram Recording 226 Hz .................. 104 Figure 2.40: Case 2 Visit 1 Right Ear B/G-Tympanogram Recording 226 Hz ............. 104 Figure 2.41: Case 2 Visit 1 Left Ear B/G-Tympanogram Recording 226 Hz ............... 104 Figure 2.42: Case 3 Visit 4 Right Ear Ya-Tympanogram Recording 1000 Hz ............ 106 Figure 2.43: Case 3 Visit 4 Left Ear Ya-Tympanogram Recording 1000 Hz ................ 106 Figure 2.44: Case 3 Visit 4 Right Ear B/G-Tympanogram Recording 1000 Hz ........... 106 Figure 2.45: Case 3 Visit 4 Left Ear B/G-Tympanogram Recording 1000 Hz ............. 106 Figure 2.46: Case 3 Visit 4 Right Ear Ya-Tympanogram Recording 226 Hz ............... 107 Figure 2.47: Case 3 Visit 4 Left Ear Ya-Tympanogram Recording 226 Hz .................. 107 Figure 2.48: Case 3 Visit 4 Right Ear B/G-Tympanogram Recording 226 Hz ............. 107 Figure 2.49: Case 3 Visit 4 Left Ear B/G-Tympanogram Recording 226 Hz ............... 107 Figure 2.50: Case 3 Follow Up Right Ear Ya-Tympanogram Recording 1000 Hz ....... 108 Figure 2.51: Case 3 Follow Up Left Ear Ya-Tympanogram Recording 1000 Hz ......... 108 Figure 2.52: Case 3 Follow Up Right Ear B/G-Tympanogram Recording 1000 Hz..... 108 Figure 2.53: Case 3 Follow Up Left Ear B/G-Tympanogram Recording 1000 Hz ....... 108 Figure 2.54: Case 3 Follow Up Right Ear Ya-Tympanogram Recording 226 Hz ......... 109 Figure 2.55: Case 3 Follow Up Left Ear Ya-Tympanogram Recording 226 Hz ........... 109 Figure 2.56: Case 3 Follow Up Right Ear B/G-Tympanogram Recording 226 Hz ..... 109 Figure 2.57: Case 3 Follow Up Left Ear B/G-Tympanogram Recording 226 Hz ......... 109 Figure A1.1: Overview of Positive Tail and Positive Peak Compensated Admittance and Susceptance Longitudinal Data for Subject A ............................................................... 174  xvi  Figure A1.2: Variation of Susceptance (ΔB) and Conductance (ΔG) as a Function of Probe Tone Frequency for Subject A …... ..................................................................... 175 Figure A1.3: Overview of Positive Tail and Positive Peak Compensated Admittance and Susceptance Longitudinal Data for Subject B ............................................................... 176 Figure A1.4: Variation of Susceptance (ΔB) and Conductance (ΔG) as a Function of Probe Tone Frequency for Subject B…… ..................................................................... 177 Figure A1.5: Overview of Positive Tail and Positive Peak Compensated Admittance and Susceptance Longitudinal Data for Subject C ............................................................... 178 Figure A1.6: Variation of Susceptance (ΔB) and Conductance (ΔG) as a Function of Probe Tone Frequency for Subject C………. ................................................................ 179 Figure A1.7: Overview of Positive Tail and Positive Peak Compensated Admittance and Susceptance Longitudinal Data for Subject D ............................................................... 180 Figure A1.8: Variation of Susceptance (ΔB) and Conductance (ΔG) as a Function of Probe Tone Frequency for Subject D……… ................................................................. 181 Figure A1.9: Overview of Positive Tail and Positive Peak Compensated Admittance and Susceptance Longitudinal Data for Subject E ............................................................... 182 Figure A1.10: Variation of Susceptance (ΔB) and Conductance (ΔG) as a Function of Probe Tone Frequency for Subject E……. .................................................................... 183  xvii  Acknowledgements  Many people have been a part of my graduate education, as friends, teachers, and colleagues. Dr. Navid Shahnaz, first and foremost, has been all of these. The best advisor and teacher I could have wished for, he is actively involved in the work of all his students, and clearly always has their best interest in mind.  Thank you for your  unwavering support through patience, motivation, enthusiasm, and immense knowledge throughout the entire thesis process. I am grateful for your many words of encouragement as a thesis supervisor, but more importantly, as a friend.  I would also like to show my gratitude to my thesis committee, Sharon Adelman and Dr. Anna Van Maanen, for their contributions of time and full hearted dedication to enriching the learning experience and empowering future audiologists.  Thank you to Dr. Lorienne Jenstad for her contributions and support for this project and Kristina Bingham and Dr. Li Qi for their help in data collection.  I also want to  acknowledge The British Columbia Early Hearing Program and the University of British Columbia Faculty Of Graduate Studies, for providing funding for this research.  Finally, I would like to thank my parents, Michael Crickmer and Katie Dunn who share my happiness and make me happy.  xviii  Dedication  To my mother, Dr. Jiang-Hong Gong, who has dedicated her entire life to the betterment of humanity through her research. Her constant oasis of ideas and passions in science has exceptionally inspired and enriched my growth as a learner, teacher, and person. Thank you for your love.  xix  1  Introduction Hearing loss in early childhood and infancy often goes undetected because it has  no obvious indications and symptoms. Often, it may be mistaken for developmental delay or attention deficit disorder (Hogan, Stratford, & Moore, 1997). However, since 80% of a child‟s ability to develop speech, language and related cognitive skills is established by the time the child is 6 months of age, permanent hearing loss in infants results in communication problems that can ultimately interfere with learning and social development (Yoshinaga-Itano, 2000). The process of developing language competency is very difficult for infants with hearing loss, particularly for those with hearing loss prior to language acquisition. Infants with hearing loss lack sufficient auditory input necessary for phonological awareness. Additionally, impaired language development results in poor literacy skills which greatly affect academic success (Yoshinaga-Itano, 2000). Hearing loss in infants may be sensorineural or conductive in nature and may be caused by various factors including but not limited to genetics, ototoxic drugs, complications during prenatal development, and middle ear pathology. Yoshinaga-Itano (2001) found that infants, who are identified with hearing loss, and given appropriate intervention before 6 months of age, maintain language development consistent with their cognitive abilities. In a study of 120 children with bilateral hearing loss, those with hearing loss confirmed by 9 months of age scored significantly higher in both expressive and receptive language skills at a mean age of 7.9 years of age compared to those who were confirmed after 9 months of age (Kennedy et al., 2006). In response to the positive outcomes associated with early detection and management of hearing loss in infants,  1  newborn hearing screening programs have become a public health initiative across many parts of the world, including the province of British Columbia. One of the main goals of the British Columbia Early Hearing Program (BCEHP) is to distinguish infants with a permanent sensorineural or conductive hearing loss from those with transient conductive losses or normal hearing (BCEHP, 2006). Measures of otoacoustic emissions (OAEs) and auditory brainstem response (ABR) are currently used for hearing screening in infants. OAEs are low intensity sounds emitted by the ear in response to auditory stimuli that can be detected by a microphone placed in the ear canal (Roush, 2001). ABR reflects electrical activity of the VIII cranial (auditory) nerve and brainstem, in response to auditory stimuli (Roush, 2001).  However, successful  recordings of OAEs and ABR not only require a healthy inner ear, but also necessitate normal or near normal middle ear functioning (Fowler & Shanks, 2002; Roush, 2001). While ABR depends only on the forward transmission of the signal through the middle ear, OAEs requires the signal to pass via both forward and backward transmission. Consequently, OAEs is more likely to be affected by conductive components than ABR (Allen et al 2005). The structural and functional immaturity of the middle ear in infants, being different than older children and adults, may in part, contribute to false positive (Clemens, Davis & Bailey, 2000; Marchant, McMillan & Shurin 1986). In addition, pathologies of the external and middle ear may also lead to false-positive test results. An ear with underlying normal hearing may not pass OAEs or ABR screening due to transient conductive dysfunction. These false positives arise because current screening tests, such as ABR and OAEs cannot distinguish between transient middle ear problems and permanent sensorineural hearing loss. This is especially problematic in the infant 2  population as the prevalence of middle ear effusion is very high. Several studies have shown that otitis media with effusion (OME), an inflammation of the middle ear accompanied by middle ear fluid with no signs of active infection, occurs commonly in healthy neonates. The prevalence of OME in infants has been shown to be 20 per 1000 births (Maxon White, Vohr, & Behrens, 1993). About 9% of all infants will have an episode of OME by 3 months of age (Klein, 1986). The incidence of OME within the first 2 months of life has been shown to be 33% (Marchant, Shurin, Turczyk, Wasikowski, Tutihasi, & Kinney, 1984).  More recently, Engel, Anteunis, Volvics,  Hendriks, and Marres (1999) reported the prevalence of OME in healthy newborns to be around 19%. Moreover, OME has been documented in up to 30% of infants in Neonatal Intensive Care Units (NICU) probably due to the use of nasotracheal tubes for ventilation in the NICU (Berman, Balkany, & Simmons 1978; Petalozza, 1984; Salamy, Eldredge, & Tooly, 1989). Therefore, there is evidence that the incidence of OME is reasonably high both in normal newborns and newborns in the NICU. In contrast, sensorineural hearing loss occurs far less frequently with an incidence of approximately 3 per 1000 births (Northern & Downs, 2002). Proper diagnosis and treatment may be delayed, which may have a negative impact on the child‟s speech and language development (Kennedy et al., 2006; Luotonen et al., 1996; Yoshinaga-Itano, 2003). Acoustic immittance measures, specifically tympanometry, have proven over the years to be a very good audiologic procedure for assessing middle ear functioning (Hall & Mueller, 1997).  The high  sensitivity of acoustic immittance measures in screening for middle ear disorders forms the basic rationale for the use of such measures in screening protocols. Tympanometry has been widely used in adults and school-aged children for many years and more 3  recently, studies have shown its diagnostic utility in newborn infants (Alaerts, Lutz, & Woulters, 2006; Kei et al 2003; Margolis, Bass-Ringdahl, Hanks, Holte & Zapala, 2003; Shahnaz, Miranda & Polka, 2008). However, during the development of the infant ear, several anatomical changes take place, which influence the mechanical properties of the ear canal and middle ear. Consequently, the diagnostic utility of tympanometry in young infants may require age-specific norms.  To date, we do not have a clear picture of how  fast the middle ear changes and at what age it becomes adult-like.  As middle ear  dysfunction is more prevalent in infant ears than sensorineural hearing loss, there is a critical need for a better understanding of middle ear development and function in infants (Keefe, Folsom, Gorga, Vorh, Bulen & Norton, 2000). Knowledge of the development of the middle ear system contributes to meaningful interpretation of audiologic measures in infants, including universal newborn hearing screening outcomes.  There is an  immense need for research that will facilitate the development of guidelines and diagnostic protocols to provide age-specific norms for better sensitivity and specificity for the detection of middle ear disease in young infants. The external and middle ear are important parts of the conductive mechanism that can seriously affect the integrity of the signal delivered to the cochlea and higher auditory processing centers (Hall, 2000; Saunders, Doant, & Cohen, 1993). Investigating the maturation of the external and middle ear will also help to understand the development of hearing sensitivity. Operationally, peripheral auditory structures function as serial processing elements (Saunders et al., 1993), and therefore, any restriction imposed by the external and middle ear system will affect the function of more proximal structures such as the inner ear and central auditory system. It is also believed that the frequency 4  response of the auditory system is largely determined by external and middle ear function (Relkin, 1988; Rosowski, 1994). The subsequent section will provide a brief overview of the anatomy, physiology, and development of the external and middle ear which is essential to understand the mechano-acoustic mechanisms involved in sound transmission. A brief overview of acoustic immittance principles and a review of current research and literature on external and middle ear function in young infants will also be provided.  1.1  External and Middle Ear Structure and Function in Adults The peripheral structures of the fully developed, adult auditory system act in a  serial processing manner in which the acoustic signal must pass through the outer and middle ear prior to reaching the more proximal structures such as the inner ear and central auditory system (Saunders et al., 1993). The gross anatomy of the external, middle, and inner ear is shown in figure 1.1.  Figure 1.1: Gross anatomy of the external, middle, and inner ear (adapted from Gelfand, 2004) 5  The outer ear consists of the pinna and the auditory canal. The pinna is comprised primarily of skin-covered elastic cartilage and functionally serves as the principal means of sound source localization (Blauert, 1997). The auditory canal, commonly known as the ear canal, is a long tunnel-like s-shaped structure that leads from the pinna to the tympanic membrane with the outer one-third being cartilaginous and the remaining twothirds bony (Gelfand, 2004).  The ear canal acts similar to a closed ended tube,  effectively boosting sounds within its characteristic resonance, which is approximately between 2000 Hz and 5000 Hz in adults (Shaw, 1974). However, unlike a simple tube, the ear canal is more complex in shape, and the tympanic membrane and canal walls are absorptive rather than rigid, which introduces damping effects (Gelfand, 2004). Consequently, the length, diameter, and damping characteristics of the ear canal result in varying peaks of resonance which depend on the shape of individual adult ears. The middle ear is a small air-filled, mucosa-lined cavity within the petreous portion of the temporal bone.  Mechanical structures of the middle ear include the  tympanic membrane, ossicular chain and intratympanic muscles and ligaments. Together, these mechanisms act to transfer airborne sound efficiently into the fluid-filled inner ear. This is important because fluid offers more opposition, or impedance, to the flow of sound energy than air. As a result, only 0.1% of the airborne sound energy would be transmitted into the cochlea without the middle ear (Bennett, 1984). The middle ear is able to compensate for the impedance mismatch between the air and cochlear fluids by acting as an impedance matching transformer utilizing the principles of area ratio, curved membrane mechanism, and ossicular lever action (Wever & Lawrence, 1954). It is  6  necessary then to understand how each of the middle ear structures contributes to this process. Separating the ear canal from the middle ear is a thin, translucent membrane called the tympanic membrane, which contributes to the area ratio and curved membrane mechanisms of the middle ear.  In adults, the tympanic membrane lies relatively  perpendicular to the ear canal and tilts at the top at an angle of approximately 55 degrees (Wever & Lawrence, 1954). With an average area of 64.3mm2, the tympanic membrane is approximately twenty times larger in area than the oval window, which is a mere 3.2mm2 (Wever & Lawrence, 1954). Given that pressure (P) is equal to force (F) per unit area (A), sound pressure energy at the level of the tympanic membrane would be effectively amplified in the direction of the oval window.  The curved membrane  mechanism first proposed by Helmholtz (1873) allows the system to exert more effective force upon the ossicular chain allowing the sound energy to become more localized into the middle ear cavity. This is accomplished as three interconnected bones called the malleus, incus, and stapes suspended within the middle ear cavity link the tympanic membrane to the oval window. The manubrium or „arm‟ of the malleus is attached roughly to the centre of the tympanic membrane making that section stiffer than either the anterior or posterior quadrants of the membrane. As the products of force and amplitude of displacement on the tympanic membrane are equal, the smaller displacement at the manubrium is accompanied by much greater force.  As a result, the action of the  tympanic membrane will cause an amplification of force to the ossicles. Finally, the three bones of the middle ear constitute the ossicular lever mechanism. The malleus and incus, fixed at the malleoincudal joint, generally move together as one unit (Barany, 7  1938). Together, these structures create a lever-like system where the malleus constitutes the longer leg and the incus the shorter leg. As the amplitude of displacement of the incus is smaller than that of the malleus, it causes a greater force against the stapes, which is attached to the oval window. As the last process of the middle ear, the footplate of the stapes rocks in a piston-like motion in the oval window with maximum force. Together these mechanical processes efficiently transfer airborne sound energy from the ear canal into the fluid-filled inner ear so they may be further processed for audition. In this fashion, the outer, middle, and inner ear work in a serial processing manner in which the acoustic signal must pass through the outer and middle ear prior to reaching the more proximal structures and before sound can become meaningful (Saunders et al., 1993). Consequently, any restriction imposed by the external and middle ear systems can decrease hearing sensitivity by affecting the integrity of the signal delivered to the cochlea and higher auditory processing centers (Hall, 2000; Sauders et al., 1993). The anatomy of the external and middle ear of the human neonate is immature compared to that of adults and accordingly, may process sound differently. It is important then, to understand the development of this system and its contribution to auditory sensitivity as a function of age.  1.2  Maturation of the External and Middle Ear In infants, the middle ear and external auditory canal are structurally immature  compared to those of adults. These structures undergo significant changes over the course of postnatal development which influences the mechanical and acoustical properties of the ear. The differences in the ear canal and middle ear transmission 8  systems due to anatomical differences between infants, children, and adults may be partially regarded as contributory factors to test outcome differentials in adult and infant populations (Keefe & Levi, 1996; Holte, Margolis, & Cavanaugh, 1991)  1.2.1 Maturation of the External Ear The auditory canal does not reach full maturity until the age of 9 (Northern & Downs, 2002), up to which time changes occur in the diameter, length, orientation and in the structure of the canal wall. The anatomical differences between an adult and infant external and middle ear are depicted in figure 1.2. More specifically, in newborns, the canal is oval in diameter, narrower, shorter, and straighter in orientation compared to an adult‟s ear canal (See figure 1.2). At birth, the infant‟s canal is only about 22.5 mm in length (McLellan & Webb, 1957) and 4.4 mm in diameter (Keefe & Levi, 1996) compared to an adult length of 25-27 mm (Shaw, 1978) and a diameter of 8mm (Keefe & Levi, 1996). In adults, the inner two thirds of the ear canal wall are bony while the outer third is composed of soft tissue (McLellan & Webb, 1957). In contrast, the canal wall in the newborn is composed exclusively of thin cartilage and soft tissue, making it flexible and compliant (Anson & Donaldson, 1981). As the infant matures, the cartilage and canal wall thicken and bone is formed, causing the structure to become stiffer, more rigid, and consequently less compliant (Anson & Donaldson, 1981). These changes of ear canal greatly determine the characteristic impedance of the ear canal (Keefe, Bulen, Arehart & Burns, 1993), as canal volume increases, impedance decreases. Since the characteristic impedance of a 1 month old infant‟s ear canal is approximately 6 times  9  higher than that of an adult‟s, transfer of sound energy to the middle ear will be reduced in infants (Keefe et al., 1993). Developmental variations in ear canal length will produce changes in the resonant properties of the unoccluded ear canal. In adults, resonance peaks around 3 kHz (Djupesland & Zwislocki, 1973). In neonates, the ear canal is shorter; therefore, the canal has a higher resonant frequency. Assuming the ear canal acts as a tube with one open end and an infant‟s average ear canal length is approximately 2.5 cm, the resonant frequency would peak at 3430 Hz calculated using the quarter wavelength rule. Furthermore, the gain of the resonance in the adult ear canal is about 10-12 dB. This gain is due to the diameter of the canal, the input impedance of the middle ear, and the compliance of the ear canal wall (Killion & Dallos, 1979). In neonates, where the diameter of the ear canal is small and the wall is compliant, the resonance gain should be less than that seen in the adult. Thus, in neonates the resonance of the ear canal occurs at higher frequencies and exhibits a lower gain than that seen in the adult (Saunders et al., 1993). This may have an impact on the sensitivity of the auditory system especially at higher frequencies.  10  Figure 1.2: Middle and External Ear of a newborn versus adult (adapted from Ballachanda, 1995)  1.2.2 Maturation of the Middle ear The tympanic cavity in an adult ear is approximately one and a half times larger than that of an infant under 1 year of age (Ikui, Sando, Haginomori and Sudo, 2000) The tympanic cavity volume is affected by the expansion of the mastoid air sinuses, which increases the size of the temporal bone in infants (Anson & Donaldson, 1981) and results in pneumatization of mastoid air cells (Ikui et al., 2000). This volume of air acts as the major stiffness component of the middle ear. Therefore, as cavity size increases with maturation, there is an overall decrease in stiffness (Meyer, Jardine & Deverson, 1997). As the infant matures, the ossicular joints and stapes footplate attachment to the oval window will tighten and the tympanic ring will fuse resulting in overall decrease in resistance (Anson & Donaldson, 1981). The tympanic membrane lies nearly parallel to the canal (horizontal) at birth and slowly becomes more vertical in axis until it reaches an adult-like position of 50-60 11  degrees between the ages of 3-4 years (Eby & Nadol, 1986). Furthermore, portions of the membrane itself are thicker and more vascular in infants compared to adults making it more massive and less compliant (Roush, 2001). As the infant matures, bone erosion of the stapes causes a decrease in bone density and therefore also a decrease in overall mass (Meyer et al., 1997). Mesenchyme (unabsorbed fetal tissue), residual amniotic fluid, and other cellular debris have been observed within the middle ear cavities of infant temporal bones (Eavey, 1993). Temporal bone studies by De Sa (1973) revealed amniotic fluid or mucoid effusion in 60% of 130 temporal bones of neonates. Paparella et al. (1980) also found a significant retention of mesenchymal tissue within the middle ear cavity of 111 temporal bones of infants from birth to 2 years of age. These substances may reside within the tympanic cavity up to 5 months after birth and may contribute mass to the middle ear system (Paparella, Shea, Meyerhooff, & Goycoolea, 1980). However, a study on newborn chinchillas suggests that the middle ear cavity is free of debris and seem mature in structure (Hsu, Margolis and Schacher, 2000). Combined, the gradual decrease in tympanic membrane thickness, bone density, and possible residual debris, result in an overall decrease in mass and resistance as the infant develops.  1.3  Functional Implications of External and Middle ear Structural Changes on Auditory Sensitivity The mechano-acoustic system of the middle ear consists of spring (stiffness),  mass, and resistive elements.  Together, these elements interact and determine the  impedance or total opposition to the flow of the energy of the middle ear system (Wiley & Stoppenbach, 2002).  The volume of air within the middle ear cavity is mainly 12  responsible for the stiffness component and affects the conductance of low frequencies. Mass elements control the conduction of the high frequency response of the middle ear and are mainly affected by the ossicles. The shape of the frequency response is driven by the resistive elements, which consist of the ossicular joints, the annular ligament around the footplate of the stapes, and cochlear fluids (Moller, 1975). For an infant, the overall mass and resistive elements of the middle ear are high at birth (Holte et al., 1991; Shahnaz et al., 2008) and then decrease as the infant matures (Holte et al., 1991). Mass elements may also be attributed to the presence of amniotic fluid and mesenchyme within the middle ear cavity of newborns (Paparella et al., 1980). A mass dominated system at birth can affect the conductance of high frequencies into the cochlea.  Partially, this may explain why young infants are shown to have higher  thresholds than adults when measured using ABR, particularly in the higher frequencies (Sininger, 2003). Since the cochlea is believed to be mature at the time of birth, the middle ear in neonates may be responsible for loss of energy transfer into the cochlea during the forward transmission of sound (Abdala & Keefe, 2006).  1.4  Pathology of the Middle Ear in Infants Otitis media (OM), inflammation of the middle ear, is the second most prevalent  childhood disorder and the leading cause of conductive hearing loss in children. Between 84% and 93% of all children will experience at least one episode of OM (Northern & Downs, 2002). Moreover, the incidence of OM has been shown to be highest between 4 and 12 months of age (Saes, Goldberg, & Montovani, 2005). OM can compromise the air conduction sound pathway and may cause mild to moderate degrees of hearing loss. 13  Eustachian tube dysfunction is almost always the underlying cause of this disease (Northern & Downs, 2002). The eustachian tube, a connector between the middle ear cavity and the nasopharynx, has three functions: protection of the middle ear from microbes, drainage of middle ear fluid, and equalization of pressure (Northern & Downs, 2002). In cases where the eustachian tube malfunctions, bacteria (most commonly due to upper respiratory infections) can travel up into the middle ear space and cause inflammation. Furthermore, if effusion builds up, the fluid cannot be cleared resulting in significant pressure build up (Northern & Downs, 2002). The prevalence of OM in infants is likely due to anatomical differences of the eustachian tube between newborns and adults. In newborns, the eustachian tube is shorter, more horizontal, and composed of relatively flaccid cartilage compared to that of adults (Northern & Downs, 2002). These structural characteristics more easily permit retrograde reflex of bacteria from the nasopharynx into the middle ear cavity (Northern & Downs, 2002). Although there are several different types of OM, the variations of the disease are simply expressions of a single underlying problem that manifests in a way that can be viewed in a continuum. Acute otitis media (AOM) is characterized by middle ear fluid with acute inflammation.  Symptoms may include otalgia, otorrhea, irritability,  restlessness, poor feeding or fever (Dowell, Marcy, Phillips, Gerber, and Schwartz, 1998). In contrast, the diagnosis of OME is distinguished from AOM by the presence of fluid with a lack of signs or symptoms of fever, pain, and infection (Kempthorne and Giebink, 1991). In approximately 40% to 50% of cases of OME, neither affected infants nor their caregivers describe significant complaints (American Academy of Pediatrics,  14  2004). The lack of infection symptoms makes OME more difficult to diagnose than AOM (Hendley, 2002). There are several motivating factors for which a safe and cost effective procedure in diagnosing OME and AOM should be considered and implemented. The most obvious reason for seeking correct diagnosis is that transient conductive hearing loss is the most common consequence of both types of OM. In infants, normal hearing sensitivity is crucial for the development of language, speech, and cognitive skills (Kennedy et al., 2006; Luotonen, Uhari, Aitola, et al., 1996; Yoshinaga-Itano, 2001). Transient hearing loss associated with the disease may result in an incomplete or an inconsistent auditory signal leading to weak encoding of auditory based phonemic distinctions (Gravel & Nozza, 1997) resulting in poorer language, speech, and literacy skills (Kennedy et al., 2006; Luotonen et al., 1996; Yoshinaga-Itano, 2001). In addition to being harmful to the individual and their family, OM has resulted in tremendous national health expenditures. According to Coyte, Asche, and Elden (1999), in Canada, approximately 611 million dollars a year is spent in direct and indirect costs associated with OME and AOM. Another major consequence of over-diagnosis of OME and AOM is the negative impact associated with over-prescription of antibiotics. Although antibiotics are more commonly prescribed for cases of AOM, it has been recommended that those with persistent OME are given a course of antibiotics prior to referral for ventilation tubes (British Columbia Medical Association, 2004).  AOM  accounts for approximately 30% of all pediatric antimicrobial prescriptions (Garbutt, Jeffe, & Shackelford, 2003). Antibiotics can be harmful and can result in antibiotic  15  resistance when taken unnecessarily; therefore, should only be prescribed when necessary and disease is confirmed (Hendley, 2002). Without symptoms of systemic infection, a firm diagnosis of OME is difficult. Myringotomy, while being the gold standard of diagnosis, is not usually indicated in the diagnosis or treatment of OME except for the relief of severe symptoms. Obviously, the risks and costs involved in surgical procedures and the use of anesthetics motivate the need to implement a noninvasive diagnostic protocol with the highest sensitivity and specificity for correct diagnosis of OME in infants. Furthermore, detection of OME in infants may prevent recurrent episodes.  In fact, when medical intervention was  implemented in early infancy, results indicated a significant reduction in future attacks of OME (Biedel, 1978; Gray, 1982; Perrin, Charney, MacWhinney, McInery, Miller, & Nazarian, 1974).  1.5  Clinical Implications The developmental changes in the external and middle ear may also affect many  common clinical measures used for audiologic assessment and amplification. These may include tests of hearing sensitivity performed under insert phones and real ear verification measures.  1.5.1 Impact of Maturation on Transducers The output SPL of any transducer used to measure hearing thresholds is calibrated to a normative reference value measured in a standardized coupler, which represents the residual volume in the average adult ear canal. However, the SPL measured in the ear 16  canal varies between individuals and differs from the calibrated SPL in the standard coupler. These differences may be partially due to structural differences of the ear canal and middle ear, including ear canal length, diameter and impedance of the tympanic membrane (Voss & Herrmann, 2005). Since the ear canal and middle ear transmission properties in infant ears differ significantly from adults, the SPL levels measured in infant ears are even more variable and significantly higher than the SPL levels present in average adult ears (Bagatto, Scollie, Seewald, Moodie, and Hoover, 2002; Bingham, Jenstad, & Shahnaz, 2009) . Higher SPL levels measured in infant ear canals may have significant clinical implications. In tests of hearing sensitivity, including OAE and ABR, an output SPL is converted into averaged hearing level (HL) values, where 0 dB HL is equal to the average adult hearing threshold. However, if the SPL measured in the infant‟s ear canal is unknown, HLs may not be accurate. Therefore, the differences observed between adult and infant middle ears, due to developmental immaturities, may directly affect tests of hearing sensitivity. Inaccurate results may affect proper diagnosis and treatment.  1.5.2 Impact of Maturation on Amplification Measures Another clinical implication involves real ear measures during hearing aid fitting. In order to compensate for the differences in SPL measured in the infant ear canal and standard coupler, a real ear to coupler difference (RECD) measurement is taken. An RECD measure is a difference in decibels, as a function of frequency, between the SPL at a specified measurement point in the ear canal and the SPL in a 2cc coupler, for a specified input signal. However, since many clinicians may find this procedure to be 17  time consuming, an averaged RECD factor is commonly used. This method may result in over-or under-amplification as individual RECD values may vary significantly between infants. As RECD measures may be reflected and affected by changes in the properties of the external and middle ear (Bingham, Jenstad, and Shahnaz, 2009), it is important to understand how these structures change as the infant matures. The degree of impact of middle ear transmission properties on RECD values in young infants is largely unknown. By investigating the changes that occur in the ear canal and middle ear as a function of age, we will have a better understanding of how sound is affected when it is transmitted through the middle ear. This in turn can provide critical information regarding whether correction factors need to be implemented for hearing testing and when and how often RECD values need to be measured in young infants.  1.6  Measures of External and Middle ear: Tympanometry Definitions of the most important terms relevant to the context of this study are  provided in Table 1.1. Table 1.1: Immittance  Terminology list  Measures of acoustic impedance (opposition to the flow of energy) or acoustic admittance (ease with which energy flows) within the middle ear system. Measures of a) opposition to energy flow or b) ease of energy flow in the ear canal as a function of changes in air pressure. Performed  Tympanometry  by introduction of an acoustic signal and measurement of sound pressure level of the signal in the ear canal as pressure is varied above (+) and below (-) atmospheric pressure.  18  A continuous tone delivered into the ear by the probe speaker. Probe tone  Acoustic immittance in analyzed by monitoring of probe tone sound pressure level in the ear canal by means of the probe microphone. Low (226 Hz) or high (678/1000 Hz) tones can be used to measure middle ear mobility.  Peak Compensated Static Admittance  The point of maximum compliance on a tympanogram which indicates the degree of energy flow within the middle ear system minus the effects of the ear canal.  Tympanometric  The pressure value where maximum compliance occurs (can be  Peak Pressure  peak or notch in cases of multi-peaked tympanograms) and which approximates pressure within the middle ear space.  Acoustic Susceptance (Ba)  Interaction between compliance elements and mass elements of a system.  Susceptance is positive at lower frequencies when a  system is stiffness controlled and negative when it is controlled by mass at higher frequencies.  Acoustic Conductance (Ga)  Relates to the impact of friction elements on susceptance and refers to the resistance to energy flow.  Acoustic  Represent ease of flow of acoustic energy and is determined by  Admittance (Ya)  susceptance (B) and conductance (G) components of the middle ear system.  Tympanometry is a safe and quick routine clinical procedure used to assess the integrity of the middle ear. It is a measurement of acoustic immittance, which is the ease with which, (acoustic admittance) or opposition to (acoustic impedance) sound energy flow through the ear at varying points above (+) and below (-) atmospheric pressure (Wiley & Fowler, 1997). A primary rationale for the clinical use of acoustic immittance measures is that they are sensitive to middle ear pathologies even in the absence of 19  recordable hearing loss and require no behavioral response on the part of the patient. Due to the high prevalence of middle ear effusion and difficulty associated with obtaining behavioral responses in young infants, tympanometry is an essential test procedure in the audiometric test battery. All tympanometric instruments are equipped with three primary subsystems including a miniature loudspeaker (sound pressure source), a microphone (to monitor and analyze the SPL of the probe signal), and a manometer (to vary and monitor air pressure changes in the ear canal) (Wiley & Fowler, 1997). Together, these components attach to one soft tipped probe which is inserted into the opening of the ear canal to create a hermetically sealed (airtight) space. Acoustic immittance is assessed by introducing a probe tone to the ear while measuring SPL of the signal in the ear canal as air pressure changes (Wiley & Fowler, 1997). The changes in probe tone intensity in response to changes in the air pressure are measured in SPL which serves as an indirect index of acoustic admittance or acoustic impedance (Wiley & Fowler, 1997). Specifically, the SPL measured at the probe tip is directly proportional to the acoustic impedance of the system. Consequently, a higher SPL measurement equates to higher acoustic impedance and lower acoustic admittance of the ear under measurement.  However, as the  measurement plane is at the level of the probe tip and not at the plane of the tympanic membrane, the recorded acoustic immittance represents the effect of the ear canal, tympanic membrane, and all the components of the middle ear (Wiley & Fowler, 1997). While the measurement plane is clinically useful, particularly in providing acoustic estimates of the equivalent ear canal volume, it cannot directly obtain a measurement of acoustic immittance provided by the middle ear in isolation (Wiley & Fowler, 1997). 20  Since placing the probe tip directly at the tympanic membrane is not feasible using commercially available instruments, the effect of the ear canal should be subtracted from the total impedance value at the probe tip in order to obtain a measurement of the middle ear alone (Wiley & Fowler, 1997). Measures that are used to extract the effects of the ear canal are termed compensated acoustic measures (Wiley & Fowler, 1997). Acoustic immittance measures can either be positively compensated (subtracting the positive tail of the tympanogram from the peak/notch value) or negatively compensated (subtracting the negative tail of the tympanogram from the peak/notch value). In order to better understand tympanometric measures, a discussion of the underlying physical and acoustic principles of acoustic immittance is provided in the subsequent section.  1.6.1 Fundamentals and Principles of Acoustic Immittance measurements Immittance is a generic term that encompasses impedance, admittance, and their components. Acoustic Impedance (Za), measured in acoustic ohms, is the opposition to the flow of energy, and acoustic admittance (Ya) measured in acoustic millimhos (mmhos), is the ease with which energy flows into a system. The subscript “a” is used to denote that the measures are based on acoustic measurement principles. The terms acoustic impedance and acoustic admittance are reciprocal terms and can be described by the equations: Za=1/Ya Ya=1/Za Therefore, the human ear, an acoustic transmission system, which offers high acoustic admittance to the flow of sound, has low acoustic impedance (Wiley & Fowler, 1997). 21  As noted previously, since impedance and admittance are reciprocal terms, the tympanograms recorded in figure 1.3 are identical in magnitude but opposite in phase angle, creating a mirror image (Fowler & Shanks, 2002).  Figure 1.3: Graphic illustration of impedance (Za) versus admittance (Ya) tympanograms (Adapted from Wiley & Fowler, 1997)  Although both terms can be used to describe acoustic measurements of middle ear function, most current commercially available acoustic immittance instruments typically provide measures of admittance (Shahnaz, 2007). Advantages of admittance measures over impedance measures include fewer tympanogram shape variance due to ear canal volume (Shanks, 1984) and higher sensitivity to middle ear conditions by changes in 22  tympanometric shape (Fowler & Shanks, 2002). For these reasons, admittance terminology will be used throughout this paper unless otherwise specified. Acoustic admittance is determined by in phase (real) components and out of phase (imaginary) components: total acoustic susceptance (Ba) and total acoustic conductance (Ga) respectively. Ba may be of two types: compliant (the inverse of stiffness) acoustic susceptance denoted by Bs and mass acoustic reactance denoted by Bm.  Ba is the  algebraic sum of mass and compliance elements which are in the same plane with the applied force: sound pressure. Ba is plotted along the Y-axis in the Cartesian plot of acoustic admittance in figure 1.4. That is, Ba = Bm + Bs Bs lies on the positive axis of Y, therefore, if total susceptance is positive, a system is stiffness controlled. Conversely, Bm lies on the negative axis of Y and a system is mass controlled if the value of Ba is negative. Acoustic conductance (Ga) which determines the dissipation of acoustic energy is primarily driven by the force of friction and is plotted along the X-axis (figure 1.4) (Shahnaz, 2007). The value of Ga is always positive and out of phase with the applied force: sound pressure.  When Ba is zero, also known as resonant frequency, Ga is the  only element that contributes to total admittance. Admittance (Ya) is a vector sum of the components Ba and Ga. This vector sum is the total magnitude plus a direction (or phase angle) that results from combining the two forces.  23  Figure 1.4: Admittance terminology [Bm: mass susceptance; Bs: Compliant susceptance; |Y|: absolute admittance magnitude; y: admittance phase angle]. (Adapted from Shahnaz, 2007 with permission)  This relationship can be expressed mathematically in either rectangular notation or in polar notation, described by either vectors or phasors, respectively. In rectangular notion, a complex number is represented by both Ba and Ga components that comprises acoustic admittance. Thus, acoustic admittance in rectangular notation can be expressed as: Ya = G + jBa Where j is an imaginary number equal to √-1 in complex number notation and the subscript  a  stands for total susceptance (Shahnaz, 2007). As Ga and Ba are vectors that  operate in different directions, they cannot be simply combined using addition. In polar notation, admittance is expressed by a single number with an associated phase angle. The 24  angle formed by admittance vector and the horizontal axis is denoted by the phase angle, y. Polar notation can be therefore expressed as, |Y|  y  Mathematically, rectangular and polar notions are related to one another. Conversion formulas to express this relationship are shown in Table 1.2.  Table 1.2: Rectangular notation and Polar notation conversion formulas  (Adapted from Shahnaz, 2007 with permission)  Acoustic admittance varies both in magnitude and phase angle depending on the probe tone frequency. Bm and Bs are both frequency dependant. Specifically, Bm is directly proportional to frequency and Bs is inversely proportional to frequency. Ga alone is not an independent of frequency as its interaction with reactive elements causes frequency dependant changes in a complex manner as expressed in the following equation, Ga = - __Ra____ Ra2+ Xa2 25  Where Ra is acoustic resistance and Xa is acoustic reactance. Figure 1.5 illustrates the effect of probe tone frequency on the resultant acoustic admittance (Ya) in a normal adult ear.  It can be observed that when the admittance vector lies between 0º and 90º  (frequencies below resonance), the system is stiffness dominated and when the admittance vector lies between 0º and -90º (frequencies above resonance), the system is mass dominated (Shahnaz, 2007). Between the angles 0º and +⁄- 45º Ga is greater than Ba between the angles of 45º and 90º, and -45º and -90º Ba is greater than Ga (Shahnaz, 2007). At resonance (RF), Bm and Bs are equal and Ba equals zero (0 mmhos) (Shahnaz, 2007).  Ga is the only component contributing to the admittance of the system at  resonance (RF) (Shahnaz, 2007).  Figure 1.5: Acoustic admittance (Ya) as a function of probe tone frequency (adapted from Margolis & Shanks, 1991).  26  1.6.2 Clinical Application of tympanometry Tympanometry has been widely used as part of routine clinical evaluation of the ear in North America since the 1970s (Fowler & Shanks, 2002).  By definition,  tympanometry is the dynamic measure of acoustic immittance in the external ear canal as a function of changes in air pressure in the ear canal (ANSI, 3.39-1987). The recorded points of immittance along various pressure points are then graphed to form a tympanogram. The middle ear transmission system is made up of physical components of membranes, ligaments, muscles, bones, and air cavities which contribute to susceptance and conductance characteristics (Fowler & Shanks, 2002). Together, they make up the acoustic admittance of the middle ear that is measured with tympanometry (Lilly, 1973). Pathological conditions of the middle ear alter the mechanoacoustic properties of the system and consequently result in changes of admittance. The shape of tympanograms can also be altered by pathologic conditions.  The goal of the diagnostic use of  tympanometry is to separate the changes that are caused by pathologic conditions from the changes that are associated with normal variability (Wiley & Fowler, 1997). Current commercially available instruments are capable of recording various tympanometric procedures including conventional 226 Hz tympanometry and multicomponent tympanometry at various frequencies. One commercially available system is capable of measuring immittance at a wide range of frequencies. Although conventional 226 Hz tympanometry has shown to provide the least amount of diagnostic information it is still the most common tympanometric measure used in the clinic. Accordingly, a  27  review of clinical procedures will be provided beginning with conventional 226 Hz tympanometry followed by multi-frequency, multi-component tympanometry.  1.6.2.1  Conventional 226 Hz Tympanometry The most commonly used tympanometric measure, termed conventional  tympanometry, involves the measurement of one component, usually admittance magnitude (Ya) as a function of air pressure at 226 Hz (Wiley & Fowler, 1997). The reasons for using this particular probe tone as opposed to higher frequencies has been due to issues with calibration and limitations of instrumentation dating back to the 1970‟s (Fowler and Shanks, 2002). Also, it was chosen, in part, „by random‟ rather than upon scientific evidence showing that it was superior to other probe tone frequencies (Terkilden & Scott Nielson, 1960). Although modern instruments are capable of reliably recording tympanometric measures using higher probe tones frequencies, the 226 Hz probe tone acoustic admittance tympanogram (Ya-226 Hz) continues to be the most commonly used assessment today (Shahnaz, 2007). The Ya-226 Hz tympanogram as shown in figure 1.6 is obtained by plotting acoustic admittance as a function of changes in ear canal pressure usually between +200 daPa and -400 daPa. The tympanogram typically peaks near atmospheric pressure (0 daPa) in normal adult ears and denotes the pressure corresponding to maximum admittance. The admittance at the peak represents the admittance of the ear canal and middle ear (Fowler & Shanks, 2002). At pressures above and below this point, the system stiffens, causing admittance to decrease. Thus, at both pressure extremes, the ear canal effectively becomes a hard walled cavity and the admittance of the middle ear is 28  minimal. As a result, the admittance measured at the extreme pressures is largely from the volume of air in the ear canal. In order to obtain a value representing the acoustic admittance of the middle ear alone, the admittance due to the volume of air in the ear canal is subtracted from the admittance at the peak. Acoustic admittance of the middle ear can be calculated from a Ya-226 Hz tympanogram by subtracting the acoustic admittance (in mmho) of the ear canal either from the positive tail or negative tail from the admittance of the middle ear at the peak of the tympanogram. The resulting acoustic admittance value is termed peak compensated static acoustic admittance (Ytm) and can be compensated either through positive or negative tails. The term static means that the measure is taken only at one pressure, not as a function of varying pressures.  Figure 1.6: Typical Ya-226 Hz tympanogram recorded from a normal adult ear showing positive peak compensated static admittance (Ytm) and tympanometric peak pressure (TPP) (Adapted from Shahnaz, 2007 with permission)  29  Ytm calculated from Ya-226 tympanograms have been well documented in adults and children and provide some clinical value in the evaluation of middle ear pathologies (Liden, 1969; Feldman, 1976; Nozza, Bluestone, Kardatzke, & Bachman, 1992). Pathological conditions may result in reduced Ytm, increased Ytm, as well as notching of the tympanogram (multi-peak tympanogram) (Wiley & Fowler, 1997). A low probe tone of 226 Hz forces the adult middle ear system to behave as a stiffness dominated system and can be used to assess pathologies that increases the stiffness of the middle ear (Fowler & Shanks, 2002). However, one of the major diagnostic limitations in using Ytm calculated from Y-226 Hz tympanograms is substantial overlap in the range of values recorded from normal and diseased ears (Shanks & Shelton, 1991). Many authors have found that despite initial stiffening of the ossicular chain in cases of otosclerosis, Ytm from Y-226 Hz is often indistinguishable from those obtained from normal ears (Liden, 1969; Shahnaz & Polka, 1997; Shahnaz & Polka, 2002; Shahnaz et al. 2009; Zhao, Wada, Koike, Ohyama, Kawase, & Stephens, 2002). Nozza et al. (1992) investigated children with histories of chronic OME whom were scheduled for surgery. At the time of surgery, those with effusion had shown a 90% range for 226 Hz-Ytm between 0.10 and 0.60 mmho while those with dry ears had a 90% 226 Hz-Ytm range of 0.01 to 1.95 mmho. The ranges for the two groups demonstrated significant overlap, making it difficult to diagnostically separate cases with effusion from cases without effusion. Furthermore, the data from this study of children with abnormal middle ear function also overlapped with data obtained from another study of children with normal middle ear function (Margolis & Heller, 1987). Based on the findings by Nozza, Bluestone, Kardatzke, & Bachman (1994), the current clinical cut-off value for Ytm calculated from Y-226 Hz tympanogram 30  is 0.2 mmhos for separating ears with effusion from those without (ASHA, 1997). However, using Ytm of 0.2 mmhos alone yields a sensitivity of only 46% with a specificity of 92% resulting in a high level of false negatives (miss). Another measure commonly taken from the Y-226 Hz tympanogram is tympanic peak pressure (TPP) which is the pressure at which the peak of the tympanogram occurs (Wiley & Fowler, 1997). If the eustachian tube is blocked or malfunctioning, negative middle ear pressure may result prior to the development of effusion, and a negative TPP value may be recorded. However, in 25% of children with normal middle ear systems, values as low as -250 daPa can be observed (Lindholdt, 1980). Due to the significant variation within normal ears, TPP is not a good screening predictor of effusion and may result in over referral rates in screening programs (Roush & Tait, 1985). Tympanometric width (TW) is another common measure taken from the Y-226 Hz tympanogram. By definition, it is the width of the tympanogram (in daPa) measured at half of the height from the peak to the tail (Wiley & Fowler, 1997). ASHA (1990, 1997) guidelines specify that the tail value is to be estimated from the tympanogram at +200 daPa. The general application of TW has been in identifying pathologies that increase the width, for example, in some cases of OME. Since the prevalence of OME in adults is low, ASHA (1997) does not provide TW limits for adults (Wiley & Fowler, 1997). Nozza et al. (1994) reported a sensitivity of 81% and a specificity of 82% using a criterion of >275 daPa in separating children with and without middle ear effusion. The authors identified this variable as the single best diagnostic variable for separating these two groups of children. There is not, however, consensus regarding which tympanometric parameter offers the best assessment of middle ear status. A study by 31  Margolis, Schachern, and Fulton (1998), for example, found that TW was not an effective test for detecting significant middle ear pathology in simulated middle ear lesions in chinchillas.  1.6.2.2  Multi-frequency, Multi-component Tympanometry Although conventional 226 Hz tympanometry can provide useful information of  middle ear status in adults and children, the development of modern immittance equipment allows for tympanometric procedures that can simultaneously measure Ba and Ga while varying pressure, known as multi-component tympanometry (B/G tympanometry) (Wiley & Fowler, 1997: 56). B/G tympanograms can be recorded at various probe tones frequencies (typically at 226, 678, and 1000 Hz). Sweeping pressure in a descending direction (+ to -) is more common, as sweeping pressure in ascending direction (- to +) has been shown to elicit more multiple notching of tympanograms (Wilson, Shanks, & Kaplan, 1984; Margolis et al., 1985).  Another method for  conducting multi-frequency tympanometry is sweep frequency. In this method, pressure is held constant while frequency is swept in steps of 50 Hz intervals between 250-2000 Hz. In some commercially available systems, the frequency sweep is conducted at two distinct pressure points corresponding to the tail and the peak 226 Hz Ya tympanogram. Measurements of peak compensated susceptance (Bpeak- Btail or Btm) or peak compensated conductance (Gpeak – Gtail or Gtm) are taken at each frequency point. The plotted values are called delta B (ΔB) and delta G (ΔG), respectively. A recording of ΔB and ΔG in a normal adult ear plotted as a function of frequency (in Hz) from one such system, the GSI Tympstar demonstrated in figure 1.7. 32  Figure 1.7: GSI-Tympstar sweep frequency recording of ΔB and ΔG (in mmho) using positive compensation. If ΔB is positive, the system is stiffness dominated; if it is negative the system is mass dominated. The crossing of ΔB with ΔG corresponds to phase angle of 45 degrees. If ΔB is zero, the system is at resonance. (Adapted from Shahnaz, 2007)  The evaluation of Ba and Ga at higher frequencies is especially important because it offers information on the relative contribution of mass and stiffness to the admittance tympanogram indicated by the various shapes of the B/G tympanograms. Many of the shapes are classified as normal versus abnormal in adults and can help the clinician to identify the probable cause of the middle ear disorder (Wiley & Fowler, 1997). Other useful parameters that can be derived from MFT include measurements of resonant frequency (RF) and frequency corresponding to admittance phase angle of 45 º (F45º) where the value of Ba is equal to Ga (Shahnaz, 2007). The greatest impact of middle ear pathologies is at probe tones frequencies close to the RF (Liden, Harford, & Hallen, 1974; Margolis & Shanks, 1991; Shanks, 1984) or admittance phase angle of 45º 33  (Shahnaz & Polka, 1997).  Note that when examining recordings using the sweep  frequency method as in figure 1.5, the frequency at which ΔB crosses 0 mmho corresponds to the resonant frequency of the middle ear while the frequency at which ΔB and ΔG intersect corresponds to admittance phase angle of 45º (Shahnaz, 2007). Furthermore, as previously discussed, complex components of admittance must be measured in order to accurately calculate compensated static admittance (Ytm) magnitude. Given that the two values, uncompensated peak admittance and admittance at ear canal volume (admittance at either the positive or the negative end), have different phase angles, simple subtraction of tail value from peak value in conventional tympanometry results in mathematical error (Shahnaz, 2007).  1.6.2.3  Classification of Tympanometric Shapes Component tympanogram recordings are often complex in shape which may  include notching and multiple peaks.  Therefore, descriptors used to measure  conventional tympanograms cannot be used to describe B/G tympanograms. Vanhuyse, Creten, and Van Camp (1975) proposed a classification system to be used with B/G tympanograms recorded with high frequency probe tones.  The Vanhuyse model  classifies the tympanograms based on the number of extrema on the Ba and Ga tympanograms. This model predicts 4 tympanometric patterns at 678 Hz probe tone which are associated with specific Ya vector locations depicted in figure 1.8 (Vanhuyse et al., 1975). Later, this model was extended to higher probe tones frequencies (Margolis & Goycoolea, 1993).  34  Figure 1.8: Vanhuyse classification model (Vanhuyse et al., 1975) to account for 4 normal patterns of acoustic susceptance (Ba) and conductance (Ga) tympanograms recorded using a 678 Hz probe tone. (Adapted from Fowler & Shanks, 2002)  The 4 tympanometric patterns observed in adults with normal ears include single peaked B/G tympanograms denoted by 1B1G; when notched, it can have 2 maxima (peaks) and 1 minima denoted by 3B1G; 3 maxima and 2 minima denoted by 3B3G; and a 5 maxima 3 minima denoted by 5B3G. Measurements of Ytm should be calculated from the centre of the tympanogram whether it be at a peak or a notch (Vanhuyse et al., 1975). Note how an examination of tympanometric shape can indicate the approximate location of the admittance vector within the Cartesian plot. Between 90º and 45º, both Ba and Ga are single peaked. Ba does not begin to notch until the admittance vector rotates below 45º. Once Ba notches, if the notch lies above the tail value than total susceptance remains positive, and the middle ear is still stiffness dominated. If the Ba notch is equal to the tail value then the middle ear is in resonance and the admittance vector is at 0º. Finally, if the 35  Ba notch lies below the tail value, Ga also begins to notch, the admittance vector will be below 0º and the middle ear is then mass dominated. In relation to probe tone, a normal adult ear progress through the 4 tympanometric shapes as probe tone frequency increases (Wiley & Fowler, 1997). At a low probe tone of 226 Hz, almost all adult ears will demonstrate the 1B1G pattern (Wilson et al., 1984). Consequently, any pattern other than 1B1G elicited by an adult ear using a 226 Hz probe tone would be considered abnormal (Wiley & Fowler, 1997). Abnormal B/G tympanograms using 678 Hz probe tone have also been described by Van de Heyning, Van Camp, Creten, and Vanpeperstraete (1982) and deviations from normal recordings are based on width and height of Ba and Ga components in relation to each other.  1.6.2.4  Resonant Frequency and frequency corresponding to admittance phase angle of 45 degree (F45) By utilizing either the shape of B/G tympanograms or recordings of ΔB using the  sweep frequency method, resonant frequency (RF) can be determined.  In B/G  tympanograms, RF can be determined when the notch of the Ba portion of a 3B1G tympanogram is equal to the value of the tail(s). Using a sweep frequency method, RF is represented by the frequency in which Btm crosses zero. A normal adult middle ear typically has a RF at around 900 – 1000 Hz (Ferekidis, 2003). Changes in RF are used to assess the pathology of the middle ear system. Specifically, pathologies that reduce stiffness such as ossicular discontinuity can lower the RF while those that are stiffening such as otosclerosis can increase the RF (Wiley & Fowler, 1997).  36  1.6.2.5  Diagnostic Utility of Multi-frequency, Multi-component Tympanometry The diagnostic value of using multi-frequency multi-component tympanometry  over conventional tympanometry to detect pathologies has been documented in adults (Funasaka & Kumakawa, 1988; Shahnaz & Polka, 1997; Shahnaz & Polka, 2002; Shahnaz et al., 2009; Zhao et al., 2002) and school-aged children (Harris, Hutchinson, and Moravec, 2005).  Multi-frequency tympanometry (MFT) utilizes more than one  probe tone frequency in assessing the middle ear. Using a MFT procedure, Funasaka and Kumakawa (1988) correctly diagnosed 61% of adult ears with ossicular fixation and 83% of ears with ossicular discontinuity, whereas only 32% and 42% were correctly diagnosed using conventional 220 Hz tympanometry, respectively. The frequency in which phase angle is at 45º (F45º where Bt and Ga are equal) has been shown to be the best predictor of otosclerosis in adult ears (Shanks, 1984; Shahnaz & Polka, 1997; Shahnaz et al., 2009). Accordingly, Shahnaz and Polka (2002) found that Ytm obtained at 630 and 710 Hz are more sensitive in distinguishing normal from otosclerotic ears when compared to 226 Hz. Although several studies have shown promising results in detection of OME using conventional 226 Hz tympanometry (Nozza et al., 1994; Palmu et al., 1998), sequelae and subtle changes in middle ear mechanics following OME are not detectable using low frequency tympanometry (Margoli, Hunter, & Giebnik, 1994; Vlachou, Ferekidis, Tsakanikos, Apostolopoulos, and Adamopoulos, 1999). Vlachou et al. (1999) compared the results of conventional 226 Hz tympanometry to MFT in 86 children affected by acute OME. Tympanometry measures were taken the day of initial evaluation for middle 37  ear disease and again 3 months later. Vlachou et al. (1999) categorized conventional tympanograms as normal with either Type A or C for determination of OME resolution. The comparison revealed that many children who experienced recurrent OME or persistence of middle ear effusion during the follow-up appointment had normal compliance (Type A or C), indicating the resolution of middle ear effusion. MFT revealed abnormal or borderline tympanograms, indicating that the disease had not completely resolved. These findings suggested that MFT is able to detect subtle changes in the middle ear system following OME that conventional tympanometry is unable to detect. The authors suggest that abnormal resonant frequency values and recordings by MFT right after an episode of acute OME indicate persistence of changes in the mass and stiffness balance of the middle ear, not demonstrated by conventional tympanometry that could be responsible for a higher probability of sequelae. Middle ear mechanics are changed in some children who have recovered from chronic OME, and the alteration in mechanics may not be reflected in the conventional 226 Hz tympanogram (Hanks & Robinette, 1993). Moreover, a recent study of 21 children aged 1 to 10 years of age, conducted by Harris, Hutchinson, and Moravec (2005) reported improved sensitivity at 678 Hz and 1000 Hz over conventional 226 Hz tympanometry for the detection of effusion. For all subjects scheduled for myringotomy, cases identified as abnormal by conventional tympanometry were also detected by 678 Hz and 1000 Hz findings. However, lowfrequency tympanometry identified some cases as normal that were classified abnormal by high-frequency tympanometry. The study revealed that although the use of 226 Hz tympanometry was found to be a successful predictor of presence or absence of fluid in 38  the middle ear (with a sensitivity rating of 80% and a specificity rating of 100%), 226 Hz tympanometry incorrectly identified 3 of 10 cases as normal that were identified as abnormal by 678 and 1000 Hz tympanometry. The authors suggest that it is possible that these abnormal recordings indicate a delayed recovery of the middle ear system or some permanent changes not demonstrated by low probe tone tympanometry. The use of higher probe tone tympanometry has shown to be more sensitive for the detection of fluid presence compared low-frequency tympanometry with a sensitivity rating of 100% and 95% for 1000 Hz and 678 Hz, respectively. These studies demonstrate that although conventional low frequency tympanometry has shown promising results in detection of OME, there are limitations and drawbacks compared to the use of higher probe tone tympanometry.  1.7  Immittance Measurements: Principles and Application in Infants As newborn hearing screening programs are rapidly expanding, a need for a  simple and objective method to evaluate and interpret middle ear status in infants is becoming increasingly important. Although the utility of tympanometry in the diagnosis of middle ear pathology has been well documented in adults and children, the interpretation of tympanometry is controversial in the infant population, especially for infants under 7 months of age (Paradise et al., 1976). The universal normative data classification system that exists for conventional 226 Hz tympanometry for adults and older children cannot be applied to infants. This is because tympanometric patterns observed in newborn infants do not conform to the classic patterns found in older infants, children, and adults.  Major problems demonstrated by conventional 226 Hz 39  tympanometry include high false positive and high false negative rates; both abnormal and normal tympanograms coexist in both normal ears and those with confirmed middle ear effusion (Meyer et al., 1997; Paradise et al., 1976; Petrak, 2002; Purdy & Williams, 2002).  The reason for false positive and false negative results is unclear although  physiological differences between infant and adult middle ear transmission properties may be a contributing factor (Keefe & Levi, 1996; Paradise et al., 1976; Petrak, 2002). A number of studies have shown that the use of higher probe tone frequencies yield recordings that are more informative (Alaerts et al., 2007; Baldwin, 2006; Hirsch, Margolis, & Rykken, 1992; Hunter & Margolis, 1992; Kei et al. (2003); Kei, Mazlan, Hickson, Gavranich, & Linning, 2007; Marchant et al., 1986; Margolis et al., 2003; Rhodes, Margolis, Napp, & Hirsch, 1999; Shahnaz et al., 2008). Additionally, the use of component tympanometry (B/G tympanometry) to calculate Ytm is more important in the infant population than for adults (Calandruccio, Fitzgerald, & Prieve, 2006; Holte, Margolis, & Cavanaugh, 1991; Kei et al., 2007; Shahnaz et al., 2008). Although limited, research has shown that use of higher probe tone frequencies (678 Hz and 1000 Hz) have better sensitivity and specificity than conventional 226 Hz tympanometry in detection of OME in newborns (Hirsch et al., 1992; Meyer et al., 1997; Rhodes et al., 1999; Sutton Gleadle, & Rowe, 1996). Although many early hearing screening programs have adopted the 1000 Hz probe tone for middle ear assessment in infants, there is still insufficient age-specific normative data available. Little is known about the functional development of the middle ear between 1 and 6 months of life.  Before the clinical utility of  tympanometry can be established, a better understanding of the characteristics of normal tympanograms in the first 6 months of life is needed. Although limited, a review of the 40  existing literature on the mechano-acoustic properties of the infant external and middle ear as measured by tympanometry will follow.  1.7.1 Peak Compensated Static Admittance (Ytm) Most studies examining infant tympanograms have investigated Ytm either by computing from positive or negative tails of Ya tympanograms or from rectangular components. Holte et al. (1991) investigated 23 subjects from 1-7 days old over a period of 4 months longitudinally.  Although they found no significant change in mean  admittance magnitude with age at 226 Hz, mean admittance magnitude for higher probe tones (450, 710, and 900 Hz), increased with age during the first 4 months of life and remained lower than adult values even at 4 months of age. Similar results from 33 infants were reported by Calandruccio et al. (2006) in a longitudinal study, where mean admittance magnitude for 226 Hz probe tone remained essentially stable from 4-10 weeks up to 2 years of age while 630 Hz and 1000 Hz showed increase in admittance magnitude as a function of age. Moreover, the authors reported that even at 2 years of age; mean Ytm was significantly lower compared to that of adults across all frequencies. Shahnaz et al. (2008) compared admittance magnitude using a 1000 Hz probe tone between 3 week old infants and adults and also found that Ytm was significantly higher for adults than for infants. Developmental expansion of the middle ear cavity has been proposed to be a contributing factor to the increase in admittance magnitude with age (Holte et al., 1991). In a study of 87 neonates by Margolis et al. (2003), Ytm calculated using negative tail compensation method from admittance (Ya)-1000 Hz tympanograms indicated that Ytm was substantially higher for those who passed DPOAE screening. Specifically, with 41  a fifth percentile cut-off for negative compensated Ytm of 0.6 mmho, 91% of tested ears that passed DPOAE screen had Ytm that exceeded the pass-fail criterion. The authors propose that the strong correlation between DPOAE pass-fail status and Ytm magnitude may be indicative that many screening failures may result from middle ear rather than inner ear factors. Shahnaz et al 2008 also found strong correlation between normal tympanogram at 1000 Hz and present TEOAEs. Ytm calculated by subtracting the admittance at the tympanometric tail from the magnitude at the peak, is commonly used in the clinic. The use of this method of calculation assumes that the external and middle ear act as a completely acoustic compliant system and that the admittance phase angle does not change with variation in ear canal pressure. These two conditions are approximate in the normal adult ear at 226 Hz leading to negligible mathematical error (Shahnaz, 2007). However, these conditions are not met in infants; therefore, using the peak-minus-tail method results in false estimates of true admittance at the middle ear (Holte et al., 1991; Shahnaz, 2007). For this reason, calculation of static admittance magnitude using the rectangular component of admittance in this population yields a more mathematically accurate measure (Holte et al., 1991). Kei et al. (2007) reported results on 1000 Hz probe tone Ytm recorded using the peak-minus-tail method and from rectangular components taken from 36 healthy neonates. The authors found that positive compensation admittance calculated from the peak-minus-tail method was significantly lower (mean of 0.65 mmho) than from rectangular components (mean of 1.00 mmho). Furthermore, the accuracy of the middle ear admittance estimates relies on obtaining an accurate estimate of the effects of the ear canal. 42  1.7.2 Compensation for Ear Canal Volume The volume of air between the probe tip and the tympanic membrane is estimated using a 226 Hz probe tone either at high positive (commonly +200 daPa) or negative (commonly -400 daPa) static pressures, by driving the admittance of the middle ear toward zero (Shanks & Lilly, 1981). However, probe tone frequencies can still be heard at these extreme pressures. Therefore, admittance does not reach zero, but an equivalent ear canal volume (Vea) can be obtained which represents an estimation of admittance attributed to the ear canal alone.  This measure of the ear canal is critical for the  estimation of middle ear admittance as the ear canal effects are subtracted from the peak admittance in order to derive admittance of the middle ear alone. In adults, the ear canal is bony and rigid therefore Vea is a close estimate of true ear canal volume. Between the two tails, the negative tail (-400 daPa) has been shown to be a more accurate measure of ear canal volume than the positive tail (+400 daPa) (Shanks & Lilly, 1981). Despite this finding, Vea is most commonly estimated in the clinic from the admittance positive tail due to better test-retest reliability (Margolis & Goycoolea, 1993). Moreover, Shanks and Lilly (1981) also demonstrated that the use of susceptance tail rather than admittance tail may be a more accurate estimate of Vea. Due to the anatomical immaturity of the infant ear canal, caution needs to be taken when measuring Vea within this population. The size of the ear canal both in length and diameter is significantly smaller, and the soft tissue along the walls makes it much more compliant when compared to the adult‟s (Holte et al., 1991; Qi, 2008). Holte et al. (1991) have shown that the greater mobility of the ear canal in young infants results in distention and deformation in response to extreme static pressures used in tympanometry. 43  This effect is most prominent on the tails of tympanograms (rather than the peak), and more so for negative pressure than for positive pressures. There is controversy as to which tail is more appropriate for estimating Vea and middle ear admittance in the infant population. Kei et al. (2007) reported results on 1000 Hz probe tone Ytm recorded using the peak-minus-tail method and from rectangular components taken from 36 healthy neonates. They found that although admittance derived from rectangular components using negative compensation (-400 mmho) attained higher mean values than from positive compensation (+200 mmho), negative compensation demonstrated lower testretest reliability. Therefore, the authors recommend the use of the positive tail for calculating Ytm values. Shahnaz et al. (2008) have also examined the interaction effects of compensation and admittance calculated from Ya tympanograms and rectangular components. They found that the mean and 90% range for static admittance calculated from the positive tail (+250 daPa) of Ya tympanograms is lower and narrower than static admittance computed from positively compensated rectangular components; however, when negative compensation was used (-300 daPa), the mean and 90% range between the two calculation methods was less apparent. The authors proposed that this finding may indicate that static admittance derived from the negative tail (-300 daPa) may not result in a significant mathematic error. However, to date, there have been no studies comparing the test performance of these two measures in confirmed cases of middle ear pathology. Furthermore, little is known about the maturational effects of positive versus negative compensation on static admittance. 44  1.7.3 Consideration of Probe Tone Frequency Keith (1973) first noted that recorded tympanograms using a low probe tone frequency were multi-peaked in 7 out of 40 babies aged 36 to 151 hours. This was significantly different than the single-peaked tympanograms found in normal children and adults. Since then, others have also confirmed that tympanograms of young infants elicited by low probe tone frequencies result in irregular multi-peaked recordings, which were inconsistent with the Vanhuyse et al. (1975) model (Calandruccio et al., 2006; Himelfarb, Popelka, & Shanon, 1979; McKinley, Grose, & Roush, 1997; Shahnaz et al., 2008; Sprague, Wiley, & Goldstein, 1985). Studies have shown that 50-95% of neonates have notched tympanograms for low-frequency probe tones (Himelfarb et al., 1979; McKinley et al., 1997; Sprague et al., 1985). Holte et al. (1991) reported that B/G tympanogram shapes at 226 Hz did not become adult-like until the age of 4 months. Although valuable information can be gathered from multi-peaked tympanograms, the complexities of the shapes makes them difficult to interpret for diagnostic purposes and are susceptible to greater subjective interpretation. Therefore, procedures that elicit tympanograms that are simple and single peaked are more desirable for clinical use. Mass and resistive elements are more predominate at low frequencies (226 Hz) in the newborn (Himelfarb et al., 1979; Holte et al., 1991) whereas in contrast, the adult ear is stiffness controlled at these frequencies. Consequently, many studies have shown that the use of a higher probe tone frequency, such as 1000 Hz, may be more informative in newborns (Alaerts et al., 2007; Baldwin, 2006; Calandruccio et al., 2006; Kei et al., 2003; McKinley et al., 1997; Rhodes, et al., 1999; Shahnaz et al., 2008). As probe tone frequency increases, the tympanograms of newborns become less complex, whereas in 45  adults, as probe tone frequency increases tympanograms become more complex (Shahnaz et al., 2008). Alaerts et al. (2007) investigated choice of probe tone frequency in infants with passing TEOAEs between the ages of birth to 32 months. They reported that for infants younger than 3 months, 1000 Hz tympanometry performed significantly better in evaluating the middle ear system with 91% pass rate versus only 35% using 226 Hz. Furthermore, the use of 226 Hz tympanometry resulted in 58% false positive results. In contrast, the authors reported that for children older than 9 months, 226 Hz tympanometry was more reliable showing a correct passing rate of 75%, whereas 1000 Hz tympanometry resulted in up to 63% false positive results. For infants between the ages of 3 to 9 months, there was no significant difference in reliability between 226 and 1000 Hz tympanometry (70% pass results using 1000 Hz versus 57% using 226 Hz). However, when a two-stage evaluation was used, pass results increased to 87%. The two-stage evaluation process was done in cases of a failed 1000 Hz tympanogram in which case a second stage 226 Hz tympanogram was performed. Margolis et al. (2003) reported that Ya tympanograms recorded in their study of 65 infants (mean age of 4 weeks) using 1000 Hz probe tone were almost always single peaked and free of irregular patterns. Similar patterns were observed in the Kei et al. (2003) study of 122 neonates where 92.2% of Ya tympanograms were single peaked at 1000 Hz. Normative values from 1000 Hz Ya tympanograms reported by Margolis et al. (2003) and Kei et al. (2003) are presented in tables 1.3 and 1.4. Although limited in sample size, these two studies have provided preliminary guidelines for clinical practice in high frequency tympanometry. Nonetheless, more normative data from large scale studies are imperative for the development of a standardized classification system. 46  Table 1.3: 1000 Hz Normative Data from Margolis et al. (2003) (65 infants -mean age of 4 weeks)  Tympanometric Peak Pressure (daPa)  Admittance at +200 daPa (mmho)  Admittance at -400 daPa (mmho)  Positive Peak Compensated Static Admittance (mmho)  Negative Compensated Static Admittance (mmho)  Mean  -10  1.4  0.8  1.3  1.9  SD  68  0.4  0.4  1.0  1.3  5th percentile  -133  0.8  0.3  0.1  0.6  95th percentile  113  2.2  1.4  3.5  4.3  Table 1.4: 1000 Hz Normative data from Kei et al. (2003) (106 infants-mean age of 3.26 days) Tympanometric Peak Pressure (daPa)  Tympanometric Width (daPa)  Ears combined  Left Ear  Right Ear  Admittance at +200 daPa (mmho) Left Right Ear Ear  Mean  18.3  97.7  107.6  3.20  SD  41.6  30.1  28.0  5th percentile  -58.0  46.1  95th percentile  86.6  144.2  Positive Peak Compensated Static Admittance (mmho) Left Ear  Right Ear  3.06  1.04  1.16  1.11  1.07  0.51  0.58  56.6  1.54  1.40  0.39  0.39  154.0  5.09  5.01  1.95  2.28  47  1.7.4 Classification of Tympanometric Shapes As an alternative to using numerical cut-offs for admittance values, Baldwin (2006) proposed a trace classification system based on a method adapted from Marchant et al. (1986). The author reported results obtained from 211 infants between the ages of 2-19 weeks including both normal hearing and those with temporary conductive hearing loss. The goal was to propose a simple classification system in which complex shaped recordings could be easily evaluated to reduce subjectivity of the decision process. In this method, a baseline is drawn between +200 and -400 daPa. Normal Ya tympanograms were characterized by a positive peak while abnormal ones were of a negative or trough configuration. The authors found that the use of 1000 Hz probe tone Ya tympanograms evaluated using this method yielded 99% sensitivity and 89% specificity for the detection of eustachian tube dysfunction. Very few studies have analyzed tympanometric shapes observed in infants using the Vanhuyse model.  Existing studies have consistently demonstrated that  tympanometric shapes observed in infants do not conform to classic patterns observed in adults. In a study of 44 neonates, Sprague et al. (1985) reported that 99% of their infants showed a 1B1G pattern in their 660 Hz tympanograms, whereas in contrast, 1B1G was the least common pattern observed from their 220 Hz tympanograms. Holte et al. (1991) found that the majority of tympanometric patterns observed at a 900 Hz probe tone in their 11 to 22 day old infants were complex in shape and did not conform to Vanhuyse patterns. McKinley et al. (1997) observed that at 678 Hz 62% of newborns displayed flat tympanograms, and at 1000 Hz, most tympanograms could not be classified according to the Vanhuyse model. 48  Shahnaz et al. (2008) investigated the B/G tympanometric patterns obtained from 31 ears of 16 3 week old infants and compared them to the results obtained from 32 ears of 16 adults. They found that the distribution of the Vanhuyse patterns at 226 and 1000 Hz were very different between the infants and adults. All adults showed a 1B1G Vanhuyse pattern at the standard 226 Hz probe tone, which is consistent with stiffness dominated system.  In contrast, only 13% of the 3 week old infants had 1B1G  tympanograms at this frequency and complex multi-peak tympanograms accounted for the remaining 85%, which is consistent with a mass dominated system. However, 1000 Hz probe tone adults showed mainly 3B1G (69%) and 3B3G (31%) patterns, while infants showed predominantly 3B1G (50%) and 1B1G (38%) patterns. This suggests that for a 1000 Hz probe tone, the middle ear is mass dominated in adults while it is stiffness dominated in infants. Calandruccio et al. (2006) reported similar findings for 1000 Hz tympanograms; infants and toddlers (age 4 weeks to 2 years) were equally likely to show either a 1B1G or a 3B1G pattern, whereas most adults (80%) demonstrated a 3B1G pattern. In a cross sectional study by Alaerts et al. (2007), the authors also found an equally dominant distribution of 1B1G and 3B1G (91% combined) in their <3 month old age group. As age increased, the distribution of 3B1G increased to approximately 55% in their 3-9 month old age group and over 65% in their 9-32 month old age group. Some researchers believe that the unusual tympanometric shapes found in neonates may be, in part, due to anatomical differences of the external ear canal (Paradise et al., 1976; Zarnoch & Balkany, 1978). The osseous portion of the ear canal is not completely formed until about 1 year of age (Anson and Donaldson, 1981). Therefore, the canal portion of the infant ear, comprised primarily of soft tissue is much more 49  compliant than an adult‟s.  The consequences of pressurizing the ear canal during  tympanometry may result not only in the movement of the tympanic membrane, but also in the distention of the ear canal walls. However, data collected by Holte et al. (1991) comparing tympanograms to ear canal mobility as shown by pneumatic otoscopy revealed that ear canal characteristics alone cannot account for the differences found in tympanometric shapes. Thus, the middle ear must be a large contributing factor in the differences observed between adult and infant tympanometric shapes. Little is known about maturational changes in tympanogram shapes.  1.7.5  Tympanometric Peak Pressure (TPP) The diagnostic value of measuring tympanometric peak pressure (TPP) is that it  can detect the presence of negative pressure in the middle ear. If the eustachian tube malfunctions as a result of disease, negative pressure may develop as a consequence which is commonly observed during the initial stages of OME.  However, TPP is  considered to have little clinical value for screening purposes due to the amount of normal pressure variation, which may lead to over-referral rates in screening programs (Wiley & fowler, 1997). Mazlan et al. (2007) reported 1000 Hz TPP results on 42 healthy neonates with recordings taken at birth and again at 6-7 weeks of age. The authors reported large variability within this measure with a mean value of 12.46 daPa (44.76 daPa SD) documented at birth and -2.08 daPa (67.99 daPa SD) at 6-7 weeks of age. No significant changes were observed over time. Similar results were obtained by Kei et al. (2007) of 106 neonates showing a mean 1000 Hz TPP of 18.3 daPa (41.6 daPa SD). Margolis et al. (2003) reported 1000 Hz TPP data on 30 2-4 week old infants 50  showing a mean 1000 Hz TPP of -10 daPa (68 daPa SD). The authors also provided a 90th percentile range of -188 to 145 daPa but cautioned that it is not clear how the data is able to contribute to a pass or fail criterion of a screening algorithm. To date, whether an abnormal TPP is significant in the infant population is not known. Furthermore, due to the large ranges that exist in TPP within the normal hearing infant population, it is unclear whether maturational effects exist. However, more data needs to be obtained in order to investigate if there is an effect on TPP with age.  1.8  Purpose of Study As shown by tympanometric measures, developmental changes that occur in the  middle ear and the external auditory canal can affect the mechano-acoustic properties of the middle ear system during the first 2 years of life (Calandruccio et al., 2006; Holte et al., 1991; Prieve, Chasmawala, & Jackson, 2005). Furthermore, several studies have shown that the middle ear function in young infants is significantly different from children and adults (Alaerts et al., 2007; Calandruccio et al., 2006; Shahnaz et al., 2008). However, these observed differences and the specific course of maturation have not been fully investigated.  Available data suggest that age-specific norms for middle ear  measurements may be necessary for infants and toddlers. Normative data are available for infants under 1 month of age (Kei et al., 2003; Margolis et al., 2003; Swanepoel et al., 2007; Kei et al., 2007; Shahnaz et al., 2008) and above 6 months of age (Roush et al., 1992; De Chicchis, Todd & Nozza, 2000). However, very few studies have investigated the mechano-acoustic properties of the middle ear using tympanometric measures on normal, healthy infants between the ages of 1-6 months. As functional development is 51  the focus of interest, two concepts are central to its definition. The first is the concept of change in function. The second is the concept of time. Longitudinal research design provides the best information about the continuity of changes over time and allows for the individual tracking of patterns of change, as well as trends of development, within a similar group. The reason that cross sectional design is not as effective is that many healthy babies may develop OME, which can effectively mask the effect of middle ear maturation.  Therefore, young infants should be tested at regular intervals to avoid  detection bias. Longitudinal data on infants less than 6 months were obtained by Holte et al. (1991) and Calandruccio et al. (2006). Holte et al. (1991) reported data from 23 infants up to 4 months of age. Calandruccio et al. (2006) provided data of 33 infants over a 24 month observation period; however, age ranges within each visit were large, ranging from 6-9 weeks for visits below 6 months.  Consequently, very little normative data is  available for infants in this age group although audiological diagnosis and intervention are most likely to occur during this period. If significant differences exist between young infants and older infants then each age group may require narrower age-specific norms when hearing is being assessed within a clinical setting. There is a significant need for data to document the rapid mechanical changes that occur within the middle ear during the first 2 years of life. Before the clinical utility of tympanometry can be established, the characteristics of normal tympanograms in the first few months of life need to be understood. In order to meet this demand, the current study analyzed longitudinal data in 1month intervals in order to determine the rate of change of the mechanical properties of 52  the middle ear and whether additional sets of norms are warranted. This study also served to replicate and add to the findings of previous research that have assessed the relative contribution of stiffness, mass and resistive elements across a wide range of frequencies.  By doing so, this may enable researchers to better understand the  contribution of the middle ear to the development of auditory sensitivity, to gather narrow age-specific norms, and to provide information necessary to create more specific and sensitive assessment protocols for diagnosing middle ear disease in young infants. The objective of the current study was to define the time course during which functional maturation of the middle ear occurs. The specific goals of this project were: 1) To understand the mechano-acoustical properties of the normal ear canal and middle ear and their maturation as a function of age and 2) to establish guidelines and normative data to characterize the acoustical properties of the normal ear canal and middle ear in newborn infants. These goals were addressed by investigating the variations of the peak compensated static admittance (Ytm); peak compensated static susceptance and conductance (Gtm and Btm); changes in resonant frequency (F0); tympanometric peak pressure (TPP) and tympanometric patterns at multiple probe tone frequencies as a function of age.  53  2  Longitudinal Investigation of Middle ear Function Using Multi-frequency, Multi-component Tympanometry in Infants Birth to 6 months of Age  2.1  Methods This project was approved by the University of British Columbia‟s Clinical  Research Ethics Board, as well as the Children's and Women's Health Centre of British Columbia Research Review Committee.  2.1.1  Recruitment Subjects were recruited through pre-natal classes offered by St. Paul‟s Hospital,  Douglas College Pre-natal Program, and the Lower Mainland Childbearing Society. The researchers provided a brief talk regarding the importance of hearing for speech and language development as well as the benefits of early intervention if hearing loss is detected.  In addition, the study was explained to parents and opportunities to ask  questions were given. Interested parents were then given a copy of an invitational letter and parental consent form and asked to contact the researchers, 1-2 weeks after their child was born. Subjects were also recruited through the Children's and Women's Health Centre of British Columbia. Researchers explained the study to hospital nurses, who then discussed the study with new parents and distributed invitational letters. Additional recruitment was conducted through word of mouth.  54  2.1.2  Subjects Subjects consisted of 31 infants totaling 62 ears (11 females, 20 males) ranging in  age from 7-30 days with a mean age of 16 days at the time of the initial visit. For statistical purposes, chronological age was calculated in days. All infants were full term gestation age (>38 weeks) with uneventful birth histories and normal birth weight (>2,500 grams). Subjects had normal hearing sensitivity determined by presence of transient evoked otoacoustic emissions (TEOAEs).  A total of 2 subjects (4 ears) were  excluded from the study: 1 subject did not demonstrate normal hearing sensitivity (did not pass TEOAE test) and the other exhibited middle ear effusion bilaterally. Due to the nature of longitudinal studies, not all subjects completed all 6 visits. Reasons for attrition included development of middle ear effusion (2 ears), non-passing TEOAE screening (4 ears), moved out of town (2 ears), and lack of interest (8 ears). In some cases, one or more visits were missed although subsequent visits were completed. Detailed description of number of ears with mean age and range of groups per visit is shown in Table 2.1. For those subjects who did not pass TEOAE screening, an Automated Auditory Brainstem Response (AABR) test in accordance with the British Columbia Early Hearing Program (BCEHP) protocol was conducted and referral into the program was initiated if a pass was not obtainable. Subjects were tested regardless of sleep state; however, parents were advised to feed babies 30 minutes prior to testing in order to facilitate sleep.  55  Table 2.1: Subject age range and number of ears tested Mean age (days)  Age range (days)  No. Ears  Visit 1  16  9-30  57  Visit 2  53  41-74  54  Visit 3  85  77-101  44  Visit 4  114  96-130  47  Visit 5  146  129-166  36  Visit 6  176  161-193  41  2.1.3  Instrumentation and Procedure In this study, maturation of middle ear function was assessed using conventional,  multi-component, and multi-frequency tympanometry. The use of cross-sectional design is subject to lower validity as many healthy babies may develop OME, which can change the mechano-acoustic properties of the middle ear and may mask the effect of middle ear maturation. For this reason, a longitudinal design was implemented. A total of 31 infants were tested in 1-month intervals over a period of 6 months with the first measurements performed while the infants were within 4 weeks of age. Frequent testing with short time intervals was implemented to avoid detection bias due to OME and also because the middle ear and outer ear is expected to undergo rapid mechano-acoustic changes during the first year of life. In order to reduce attrition as a result of follow-up, tests were carried out at the subjects‟ own residences. Data from this study was collected as part of a larger longitudinal study of maturation effects of the external and middle ear. Therefore, 4 tests, although not all 56  reported as part of this study, were completed at each of the 6 visits: TEOAEs, tympanometry, RECD measurements and wide band reflectance (WBR).  2.1.3.1  Transient evoked otoacoustic emission (TEOAE) test TEOAE is a screening test that is sensitive to cochlear hearing loss of above 30  dB HL or more and can verify cochlear and middle ear function (Bonfils & Uziel, 1989). As a requirement for inclusion in the study all subjects needed to demonstrate normal middle and inner ear function at each of the 6 visits as measured by repeated TEOAE test using Otodynamics ILO-292 (Otodynamics Ltd., Hatfield, England). Calibrations were performed based on the operations manual of the manufacturer. Stimuli consisted of 84 dB SPL clicks. A 3 dB signal-to-noise ratio (SNR) was required at 1 and 1.5 kHz and a 6dB SNR was required at 2, 3 and 4 kHz. In accordance with the protocol adopted from the work of Norton et al. (2000), to pass, a response at 4 out of the 5 frequency bands was required, along with 70% reproducibility and a minimum of 50 sweeps.  2.1.3.2  Tympanometry Tympanograms were recorded using a GSI Tympstar version 2 immittance  system. The system was calibrated in accordance to the manufacturer‟s operation manual in three different hard-wall cavities and results were within acceptable tolerance values. Admittance (Ya), susceptance (Ba), and conductance (Ga) tympanograms were recorded using a positive to negative sweep pressure method from +200 to -400 daPa with a pump speed of 200 daPa/sec. Decreasing pressure (positive to negative) was used for all recordings because this approach results in fewer irregular tympanograms (Wilson, 57  Shanks & Kaplan, 1984) and a reduced chance of collapsing canals (Holte, Margolis & Cavanaugh, 1991), when compared with an increasing direction of pressure change. The positive and negative tail immittance values were taken at the static pressures of +200 and -400 daPa, respectively. All measures were recorded at each of two probe tone frequencies (226 and 1000 Hz). Additionally, Ba and Ga tympanograms were also recorded at a probe tone of 678 Hz for analysis of tympanogram shape. Ytm was calculated using two different methods. In the first method, the peak (notch in cases of multi-peak tympanograms) was subtracted from the positive (+200 daPa) and negative (-400) tail on the admittance tympanograms. In the second method, Ytm was derived from the rectangular components of susceptance and conductance using the following equation:  Y = (B ± )2 + (G ± )2 The rectangular components Ba and Ga were corrected for ear canal admittance at +200 daPa (positive compensation) and -400 daPa (negative compensation). The use of the positive tail has been shown to have better test-retest reliability in adults (Margolis & Goycoolea,1993) as well as newborns (Kei et al., 2007) while the negative tail is a more accurate measure of equivalent ear canal volume (Shanks and Lilly,1981). Tympanometric peak pressure (TPP) was recorded from all Ya-tympanograms by measuring the point in which maximum admittance occurs (mid-peak in single-peak and mid-trough in multi-peak tympanograms).  58  Finally, multi-frequency peak compensated susceptance and conductance (ΔB and ΔG) were recorded using sweep frequency method at extended frequencies between 2502000 Hz in 50 Hz intervals during each visit for 16 infants.  2.1.3.3  Real-ear to coupler difference (RECD) measurement RECD measurements were obtained on visits 1 and 2 as part of the larger  longitudinal study that has been published in another paper (Bingham, Jenstad, and Shahnaz, 2009). Results are not reported here.  2.1.3.4  Wide band reflectance (WBR) WBR was also measured as part of a larger longitudinal study, but the results are  not reported here.  2.1.4  Challenges in Testing Young Infants Testing was conducted in the subject‟s home in a quiet room. In most instances  the infant was held by a parent and the exposed ear was tested first. Figure 2.1 shows a subject with an inserted TEOAE probe-tip. Figure 2.2 shows a subject with an inserted immittance probe-tip. At times, middle ear testing within this population posed specific challenges. Difficulties were encountered during recruitment, scheduling testing times, travel, and test procedures.  59  Figure 2.1: TEOAE Testing On a Subject  Figure 2.2: Tympanometric Testing On a Subject  60  Longitudinal research on newborn infants requires recruitment during the prenatal period. It was a challenge to arrange talks during pre-natal classes in order to recruit subjects as many pregnant women already had much to prepare for. Talks that focused on the importance of hearing screening yielded the highest success rate for recruitment. Once subjects were recruited, it was a challenge to schedule testing times that were convenient for two testers and the parents. Furthermore, testing times had to coincide with times during the day in which the infant was most calm and not agitated. In order to follow as close as possible within 1-month testing intervals, infants were recruited in 2 separate occasions. Over a period of 6-months, 15 infants were tested until they reached 6 months of age. Testing of an additional 17 infants commenced after the first group was complete. This was done to reduce the active subject load and to make scheduling testing times more manageable. Over the span of 6 months, winter often fell within the testing period. Harsh weather conditions not only made travel to families‟ homes difficult but many infants developed colds and resulted in cancelled appointments. Moreover, families often travelled and had other commitments during the Christmas season which resulted in frequent missed appointments and rescheduling. Challenges that were encountered during testing included retention of probe-tips within the ear and the infant‟s physical state. Due to the small size of the ear canal opening, a hermetic seal was often difficult to attain. Probe-tip insertion into a very small compliant ear canal occasionally resulted in retention issues, wax blockage, and collapse of the ear canal wall. Moreover, the infant‟s physical state affected testing times and recording quality. Although parents were told to feed the infant 30 minutes prior to testing to promote calmness and sleep, infants would occasionally wake up, become 61  agitated, and engage in significant movement. These factors made it difficult to insert and retain probe-tips. Recorded measures under sufficient physical movement such as head turning, crying, sucking, and/or swallowing resulted in poor recordings and in most cases resulted in re-testing and increased testing times.  2.1.5 Treatment of the data Statistical analysis was performed with Statistica (version 6.1, Statsoft Inc.). A repeated-measure analysis of variance (ANOVA) was conducted to investigate whether the variations observed in susceptance, conductance and admittance across the 6 visits at 226 and 1000 Hz for both tails were statistically different. All significant findings were subject to Greenhouse-Geisser (GG) correction for repeated-measures sphericity and inflated type-I error.  Within-subject factors included number of visits (6 levels),  component (3 levels: Btm vs. Gtm vs. Ytm), tail pressure (2 levels: positive vs. negative, and probe tone (2 levels: 226 Hz vs 1000 Hz). A 95% confidence interval along with a significance level of α= 0.05 was used for statistical tests. Significant findings were further analyzed using a post-hoc Tukey Honestly Significantly Different (HSD) test in order to determine the interaction between visits and other within-subject factors. In order to investigate whether tympanometric measures observed in infants during the sixth visit (6 months old) are comparable to those obtained from school-aged children (5-7 years old) (Bosaghzadeh, in progress) and young adults (18-36 years old) (Shahnaz & Bork, 2008), either a univariate test of significance or a repeated-measure ANOVA was conducted.  In univariate tests of significance, age groups (2 levels),  component (2 levels: Btm vs. Gtm), tail pressure (2 levels: positive vs. negative) and 62  frequency (2 levels: 226 Hz vs. 1000 Hz) served as within-subject factors. For repeatedmeasure ANOVA tests, within-subject factors included age groups (3 levels) and component (3 levels: Ytm vs. Btm vs. Gtm).  2.2  Results The main objective of this study was to observe the changes that occur during  development of the middle ear from 1-6 months of age in 1-month intervals as measured using tympanometry. First, proportions of single versus multi-peak 226 and 1000 Hz Ya tympanograms will be reported as a function of age followed by proportions of B/G tympanogram shapes. B/G tympanograms were classified as one of the 4 Vanhuyse et al. patterns (1975) at 226, 678, and 1000 Hz probe tone frequencies. Those that could not be categorized as one of the classic Vanhuyse patterns were labeled as „other‟. Secondly, admittance, susceptance, and conductance tails are examined at both positive and negative pressures as a function of age 1. Third, significant findings for Ytm from Ya tympanograms and from the rectangular components Ba and Ga using both positive and negative compensation as a function of age were reported. In addition, results on peak compensated static susceptance (Btm) and conductance (Gtm) using both positive and negative compensation across age were described. Tympanic peak pressure ranges (TPP) are analyzed across visits. Furthermore, multi-frequency variation in peak compensated  1  Findings on admittance, susceptance and conductance tails on current data have been described in a  graduate paper. Detailed results can be found in Qi (2009)  63  susceptance ( B) and compensated conductance ( G) will be reported including changes in resonant frequency and relative contribution of mass and stiffness.  Lastly,  tympanometric results obtained for infants at visit 6 are compared to data of school-aged children (Bosaghzadeh, in progress) and adults (Shahnaz & Bork, 2008). Tympanometric results are reported with median and 5th–to 95th-percentile values for each measure from the first to the sixth visit for both 226 and 1000 Hz. Statistical findings from repeated-measures ANOVA and Univariate Test of Significance are described followed by Tukey HSD post-hoc analysis for significant findings. Detailed ANOVA tables are provided in Appendices I, II, and III.  2.2.1 Analysis of Tympanometric Shapes The proportion of single-peak Ya tympanograms at 226 and 1000 Hz probe tone frequencies is shown in figure 2.3 for all 6 visits. At 226 Hz, Ya tympanograms become simpler in shape with age, thus, the proportion of single-peak Ya tympanograms increases. Specifically, at visit 1 (V1) 226 Hz Ya tympanograms were predominantly multi-peak and become predominantly single-peak by visit 2 (V2) and thereafter. In contrast, infants across all visits almost exclusively showed single-peak Ya tympanograms at 1000 Hz probe tone with a slight decrease with age.  64  Figure 2.3: Proportion of Single versus Multiple Peak Ya Tympanograms Across 6 Visits (V=visit)  Figure 2.4 shows the proportion of ears displaying various Vanhuyse patterns at 226, 678, and 1000 Hz probe tone frequencies across all visits. The proportions are also provided in numerical form in Table 2.2. As figure 2.4 shows, there was a wide variety of tympanometric patterns observed. Recordings using 678 Hz probe tone yielded the greatest variety of patterns. At 226 Hz, infants at V1 and V2 showed predominately 3B1G and 3B3G patterns with no occurrences of simple 1B1G tympanograms at V1. However, by visit 3 (V3), 1B1G became the dominant pattern observed in 59% of infants ears followed by 3B1G observed in 39% of ears. By visit 6 (V6), almost all (98%) tympanograms showed a 1B1G pattern at 226 Hz.  65  At 678 Hz probe tone, infants yielded a greater variety of tympanometric patterns. Recorded tympanometric shapes were equally scattered across patterns with the majority not confined to the patterns observed under the Vanhuyse model in adults. Specifically, at V1, V2, and V3, a significant number of recordings were labeled as „other‟ at 32%, 52%, and 37% respectively. Despite the variance, the proportion of simpler 1B1G and 3B1G tympanograms progressively increased with age. By V6, 678 Hz tympanograms were, for the most part, 1B1G at 71% followed by 3B1G at 17%. The occurrences of complex shapes also decreased with age with combined 3B3G, 5B3G, and „other‟ patterns recorded at only 12% at V6. In contrast, different patterns were observed at 1000 Hz probe tone. 1B1G and 3B1G patterns occurred most often and the distribution across visits remained constant. More specifically, 1B1G was recorded 49% of the time and 3B1G was recorded 47% of the time at V1. These proportions remained relatively consistent up to visit 5 (V5). By V6, 3B1G pattern become predominant pattern (51%) followed by 1B1G pattern (38%). There were very few recordings of complex patterns, with combined 3B3G, 5B3G, and „other‟ patterns consisting of less than 15% across all visits.  66  Figure 2.4: Distribution of Vanhuyse et al. (1975) Patterns for Infants across 6 Visits (V=Visits)  Table 2.2: Distribution of Vanhuyse et al. (1975) Patterns for Infants Across 6 Visits Proportion of different tympanometric configurations in % 1B1G Visit  3B1G  3B3G  5B3G  OTHER  Frequency (Hz) 226  678  1000  226  678  1000  226  678  1000  226  678  1000  226  678  1000  1  0  16  49  44  16  47  56  21  2  0  16  0  0  32  2  2  30  22  50  54  7  43  17  15  6  0  4  2  0  52  0  3  59  18  56  39  18  37  2  14  2  0  14  5  0  37  0  4  89  25  49  9  21  38  2  21  6  0  11  2  0  21  4  5  97  57  50  3  19  44  0  19  3  0  0  0  0  5  3  6  98  71  38  0  17  51  2  6  5  0  3  3  0  3  3  67  2.2.2 Variation of Admittance, Susceptance, and Conductance Tails with Age Tables 2.3, 2.4, and 2.5 provide summary descriptive statistics for admittance, susceptance, and conductance tails for 226 Hz and 1000 Hz. Appendix I (Table A1.1A1.6) provides the ANOVA summary tables. As part of another study, comprehensive analysis of tail data values have been reported and discussed in a paper by Qi (2009). As a result, detailed statistical analysis will not be reported here. Equivalent ear canal volume (Vea) is typically taken as a measurement of admittance at either positive or negative tails for 226 Hz tympanograms. Descriptive statistics for admittance (Ya) at positive (Ya+) and negative (Ya-) tails for 226 Hz and 1000 Hz for each of the 6 visits are shown in Table 2.3. Results indicate that admittance at both tails increased as age increased.  This was observed for both tails at both  frequencies. Median Ya+ at 226 Hz was 0.50 mmho at V1 and increased to 0.70 mmho at V6. Median Ya- at 226 Hz also increased from 0.25 mmho to 0.5 mmho between V1 to V6. For 1000 Hz probe tone, mean Ya+ was 1.67 mmho at V1 and increased to 2.73 mmho by V6. Similarly, Ya- at 1000 Hz increased from 0.69mmho at V1 to 1.85 mmhos at V6.  68  Table 2.3: The 5th, 50th, and 95th percentiles of the admittance at positive and negative tails Admittance positive tail (Ya+) value  Admittance negative tail (Ya-) value  (mmho)  (mmho)  226 Hz  1000 Hz  226 Hz  1000 Hz  Visit Median  5th-to-95th  Median  5th-to-95th  Median  5th-to-95th  Median  5th-to-95th  1  0.50  0.40-0.72  1.67  1.22-2.32  0.25  0.20-0.40  0.69  0.44-0.99  2  0.60  0.39-0.93  1.86  1.31-2.65  0.40  0.25-0.50  1.07  0.65-1.45  3  0.70  0.50-0.80  2.25  1.67-3.00  0.45  0.35-0.50  1.38  0.89-1.76  4  0.60  0.50-0.87  2.47  1.89-3.19  0.45  0.35-0.55  1.65  1.17-2.21  5  0.60  0.50-0.90  2.59  1.93-3.45  0.45  0.35-0.60  1.66  1.35-2.10  6  0.70  0.50-0.90  2.73  2.13-4.33  0.50  0.40-0.65  1.85  1.46-2.58  Median Ya+ and Ya- values are plotted across the 6 visits for 226 Hz (figure 2.5) and 1000 Hz (figure 2.6). Vertical bars denote 95% confidence intervals. Figures indicate that with age, admittance at 1000 Hz increases faster than at 226 Hz for both tails. For positive tails, the median admittance at 1000 Hz increases by a factor of about 0.63 from V1 to V6, while the median admittance at 226 Hz increases by a factor of only about 0.40 for the same period. Similarly, for negative tails, the median susceptance at 1000 Hz increases by a factor of about 1.68 while the median susceptance at 226 Hz only increases by a factor of about 1.  69  Figure 2.5: Median Admittance Tail Magnitudes (Positive and Negative Tails) for 226 Hz Probe Tone Across 6 Visits  Figure 2.6: Median Admittance Tail Magnitudes (Positive and Negative Tails) for 1000 Hz Probe Tone Across 6 Visits  70  Table 2.4 provides a summary of descriptive statistics for susceptance at positive (Ba+) and negative (Ba-) tails at both 226 and 1000 Hz across the 6 visits. Generally, susceptance at both tails increased as age increased. For Ba+, median value was 0.52 mmho at V1 and increased to 0.61mmho at V6 for 226 Hz. Similarly, for the Ba-, median value was 0.19 mmho at V1 and increased to 0.46 mmho at V6. Similar trends were observed for 1000 Hz probe tone. Table 2.4: The 5th, 50th, and 95th percentiles of the Susceptance at Positive and Negative Tails Susceptance positive tail (Ba+) value  Susceptance negative tail (Ba-) value  (mmho)  (mmho)  226 Hz Visit  1000 Hz  226 Hz  1000 Hz  Median  5th-to-95th  Median  5th-to-95th  Median  5th-to-95th  Median  5th-to-95th  1  0.52  0.37-0.78  1.26  0.79-1.91  0.19  0.05-0.41  0.25  0.00-0.54  2  0.55  0.37-0.83  1.51  1.02-2.38  0.30  0.17-0.46  0.52  0.12-1.04  3  0.60  0.47-0.79  1.88  1.28-2.69  0.40  0.30-0.48  0.95  0.22-1.40  4  0.62  0.48-0.83  2.11  1.34-2.90  0.41  0.30-0.54  1.15  0.27-1.89  5  0.61  0.47-0.83  2.31  1.62-3.17  0.42  0.32-0.54  1.26  0.79-1.76  6  0.61  0.44-0.92  2.45  1.55-3.91  0.46  0.32-0.69  1.58  0.96-2.21  Median Ba- and Ga- are plotted across the 6 visits at 226 Hz (Figure 2.7) and 1000 Hz (figure 2.8). Similar graphs are plotted for positive tails at 226 Hz (Figure 2.9) and 1000 Hz (Figure 2.10). Vertical bars denote 95% confidence intervals. Figures indicate that Ba at 1000 Hz increased faster than at 226 Hz for both tails across visits. For negative tails, median Ba increased by a factor of 5.32 at 1000 Hz but only by 1.42 at 226 Hz for the same period. Similarly, for positive tails, the median Ba at 1000 Hz increased by a factor of about 0.94 compared to only 0.17 for 226 Hz. 71  Figure 2.7: Median Susceptance (Ba) and Conductance (Ga) Negative Tail Magnitude for 226 Hz Probe Tone Across 6 Visits  Figure 2.8: Median Susceptance (Ba) and Conductance (Ga) Negative Tail Magnitude for 1000 Hz Probe Tone Across 6 Visits  72  Figure 2.9: Median Susceptance (Ba) and Conductance (Ga) Positive Tail Magnitude for 226 Hz Probe Tone Across 6 Visits  Figure 2.10: Median Susceptance (Ba) and Conductance (Ga) Positive Tail Magnitude for 1000 Hz Probe Tone Across 6 Visits  73  Table 2.5 provides a summary of descriptive statistics for conductance at positive (Ga+) and negative (Ga-) tails at both 226 and 1000 Hz across the 6 visits. As shown in figures 2.6 and 2.8, Ga at both tails remained constant between V1 and V6 at 226 Hz at an approximate median value of 0.17 mmho. Ga at 1000 Hz shown in figures 2.7 and 2.8 was overall higher than at 226 Hz for both tails and also increased in range between V1 and V6. For Ga+, median values at V1 were 0.19 mmho and 1.06 mmho for 226 Hz and 1000 Hz, respectively. At V6, Ga+ remained relatively stable with a median value of 0.15 mmho at 226 Hz and 1.18 mmhos at 1000 Hz. Similar trends were observed for Ga-.  Table 2.5: The 5th, 50th, and 95th percentiles of the Conductance at Positive and Negative Tails  Visit  Conductance positive tail (Ga+) value (mmho)  Conductance negative tail (Ga-) value (mmho)  226 Hz  226 Hz  1000 Hz th  5th-to-95th  0.06-0.62  0.63  0.36-0.95  0.17  0.10-0.46  0.88  0.60-1.19  0.89-1.52  0.17  0.11-0.25  0.96  0.73-1.29  1.17  0.91-1.88  0.16  0.11-0.23  1.02  0.81-1.60  0.12-0.30  1.26  0.85-1.72  0.15  0.12-0.20  1.01  0.78-1.34  0.08-0.43  1.18  0.42-1.79  0.15  0.08-0.29  0.96  0.25-1.58  5 -to-95  1  0.19  2  th  Median  5 -to-95  0.12-0.41  1.06  0.23  0.13-0.49  3  0.20  4  th  1000 Hz Median  Median  th  th  Median  5 -to-95  0.76-1.43  0.13  1.16  0.73-1.57  0.11-0.40  1.23  0.19  0.11-0.41  5  0.18  6  0.15  74  th  2.2.3 Variation of Peak Compensated Static Admittance (Ytm) Calculated from Admittance Tympanograms and Rectangular Components with Age Table 2.6 provides a summary of descriptive statistics for Ytm using positive and negative compensation at both 226 Hz and 1000 Hz across the 6 visits. Overall, Ytm from negative compensation was higher than from positive compensation for both frequencies across the 6 visits. Ytm calculated from B/G tympanograms were also higher than Ytm calculated from Ya tympanograms for both frequencies across the 6 visits. For the 226 Hz probe tone, admittance decreased across visits. This was observed for admittance derived from both Ya and B/G tympanograms using both positive or negative compensation methods. As shown in Table 2.6, median positive Ytm derived from Ya tympanograms at V1 was 0.70 mmho and decreased to 0.40 mmho by V6. Corresponding values calculated from B/G tympanograms were 1.21 mmho at V1 which decreased to 0.47 mmho by V6.  Similar trends were observed from negative  compensation.  75  Table 2.6: The 5th, 50th, and 95th percentiles of the Admittance with Positive and Negative Compensation Visit  Computed  Peak Compensated Static Admittance (Ytm)  Peak Compensated Static Admittance (Ytm) from  from positive tail (mmho)  negative tail (mmho)  226 Hz  1000 Hz th  226 Hz th  th  1000 Hz  Median  5 -to-95  Median  5 -to-95  Median  5 -to-95  Median  5th-to-95th  B/G  1.21  0.76-1.67  1.21  0.23-2.42  1.17  0.64-1.70  1.66  0.92-3.83  Ya  0.70  0.12-1.20  0.85  0.11-2.05  1.05  0.47-1.51  1.75  0.77-3.51  B/G  1.14  0.56-1.89  1.28  0.23-2.60  1.19  0.67-1.95  1.52  0.88-3.08  Ya  0.80  0.23-1.33  0.87  0.00-2.21  0.95  0.44-1.63  1.67  0.83-3.17  B/G  0.80  0.39-1.41  1.18  0.41-2.93  0.98  0.51-1.61  1.83  0.99-3.54  Ya  0.60  0.40-1.10  0.82  -0.07-1.97  0.85  0.50-1.35  1.62  0.83-3.71  B/G  0.65  0.25-1.79  1.66  0.31-3.42  0.81  0.38-1.64  1.85  0.63-4.28  Ya  0.50  0.25-1.42  1.06  0.03-2.51  0.65  0.34-1.40  1.71  0.74-4.18  B/G  0.58  0.25-0.87  1.72  0.57-3.44  0.78  0.37-1.06  2.09  1.03-4.25  Ya  0.50  0.20-0.72  1.04  0.24-2.54  0.65  0.34-1.05  2.13  0.92-3.97  B/G  0.47  0.29-1.03  1.92  0.53-3.60  0.62  0.37-1.22  2.20  0.68-4.78  Ya  0.40  0.20-0.70  1.19  0.36-2.32  0.60  0.35-1.00  2.17  1.07-4.54  From  th  th  th  1  2  3  4  5  6  Figure 2.11 shows median Ytm derived from Ya at 226 Hz across all 6 visits. Vertical bars denote 95% confidence intervals. A repeated-measure ANOVA was conducted with age (6 levels) and tail pressures (2 levels) as within-subject factors. Appendix I (Table A1.7) provides the statistical summary tables.  Repeated-measure ANOVA for admittance  derived from Ya tympanograms at 226 Hz were statistically significant across visits [F (5,145)= 12.34; p<0.05] and tail pressures [F(1,29)= 308.28; p<0.05] indicating that Ytm obtained from Ya varies across visits and between tail pressures. Interaction between visits and tail pressures was also significant [F(5,145)=10.15; p<0.05] indicating that the 76  effect of tail pressures on Ytm obtained from Ya varied differently between each visit . To probe the interaction, simple effects of tail pressure were examined at each visit. Further investigation using Tukey HSD post-hoc analysis revealed that admittance from positive tails was significantly lower for V1 and V3 relative to V2. From V3 onwards, admittance became progressively lower until V5. However, V6 showed no significant difference from V5. Admittance from negative tails became progressively lower from V2 to V6. However, V2 and V1 were not significantly different from each other.  Figure 2.11: Median Peak Compensated Static Admittance (Ytm) Calculated from Ya tympanograms using Positive and Negative Compensation for 226 Hz Across 6 Visits  Figure 2.12 shows Ytm derived from B/G tympanograms at 226 Hz across all 6 visits. Vertical bars denote 95% confidence intervals. A repeated-measure ANOVA was conducted with age (6 levels) and tail pressures (2 levels) as within-subject factors. Appendix I (Table A1.9) provides the statistical summary tables Repeated-measure ANOVA for admittance derived from B/G tympanograms at 226 Hz were statistically 77  significant across visits [F(5,145)= 25.75; p<0.05] and tail pressures [F(1,29)= 90.20; p<0.05] indicating that Ytm obtained from B/G varies across visits and between tail pressures. Interaction between visits and tail pressures was also significant [F(5,145)= 2.93;p<0.05] indicating that the affect of tail pressures on Ytm obtained from B/G varied differently between visits . To probe the interaction, simple effects of tail pressure were examined at each visit.  Further investigation using Tukey HSD post-hoc analysis  revealed that admittance for positive tails became progressively lower between V1 and V6. However, between V2 and V1, and between V6 and V5 no significant differences were observed. Post-hoc analysis for admittance from negative tails revealed that V2 was significantly higher than visit 1. However, admittance became progressively lower from V2 to V5 with no significant difference between V5 and V6.  Figure 2.12: Median Peak Compensated Static Admittance (Ytm) Calculated from Btm/Gtm tympanograms using Positive and Negative Compensation for 226 Hz Across 6 Visits  78  In contrast, at 1000 Hz probe tone, on average, Ytm increased with age, particularly after the third visit. This was observed for admittance derived from both Ya and B/G tympanograms using both positive or negative compensation. As shown in Table 2.6, median positive Ytm at 1000 Hz derived from Ya tympanograms at V1 was 0.85 mmho and increased to 1.19 mmho by V6. Corresponding values calculated from B/G tympanograms were 1.21 mmho at V1 which increased to 1.92 mmho by V6. Similar trends were observed from negative compensation. Admittance increased faster when derived from B/G tympanograms than from Ya tympanograms. Specifically, for positive tails, the median admittance from B/G tympanograms increases by a factor of 0.71 from the first visit to the sixth visit, while the median admittance from Ya tympanograms increases by a factor of only 0.4 for the same period. Figure 2.13 shows Ytm derived from Ya tympanograms at 1000 Hz across all 6 visits. Vertical bars denote 95% confidence intervals. A repeated-measure ANOVA was conducted with age (6 levels) and tail pressures (2 levels) as within-subject factors. Appendix I (Table A1.8) provides the statistical summary tables. Repeated-measure ANOVA for admittance derived from Ya tympanograms at 1000 Hz were statistically significant across visits [F(5,145)= 9.17; p<0.05] and tail pressures [F(1,29)= 372.15; p<0.05] indicating that Ytm obtained from Ya varies across visits and between tail pressures. Interaction between visits and tail pressures were not found to be significant. To further probe the changes of Ytm across visits, a post-hoc Tukey HSD analysis was applied. Results revealed that Ytm for 1000 Hz probe tone did not show any changes between V1 to V5. However, Ytm at V6 was significantly higher (p<0.05) compared to all other visits except V5 and admittance at V5 was significantly higher than at V2. 79  Figure 2.13: Median Peak Compensated Static Admittance (Ytm) Calculated from Ya tympanograms using Positive and Negative Compensation for 1000 Hz Across 6 Visits  Figure 2.14 shows Ytm derived from B/G tympanograms at 1000 Hz across all 6 visits. Vertical bars denote 95% confidence intervals. A repeated-measure ANOVA was conducted with age (6 levels) and tail pressures (2 levels) as within-subject factors. Appendix I (Table A1.10) provides the statistical summary tables. Repeated-measure ANOVA for admittance derived from B/G tympanograms at 1000 Hz were statistically significant across visits [F(5,145)= 12.17; p<0.05] and the two tails [F(1,29)= 68.59; p<0.05] indicating that Ytm obtained from B/G varies across visits and between tail pressures. Interaction between visits and tail pressures was also significant [F(5,145)= 2.92;p<0.05] indicating that the affect of tail pressures on Ytm obtained from B/G varied differently between each visit . To probe the interaction, simple effects of tail pressure were examined at each visit. Further investigation using Tukey HSD post-hoc analysis revealed that Ytm from both positive and negative tails were statistically lower for V1, V2, and V3 (p<0.05) than all other visits but not significantly different from each other. V4 and V5 were significantly higher than V3, V2, and V1 but not different from each 80  other. For positive compensation only, V6 was significantly higher than all other visits. For negative compensation only, V6 was higher than all other visits except V5.  Figure 2.14: Median Peak Compensated Static Admittance (Ytm) Calculated from Btm/Gtm tympanograms using Positive and Negative Compensation for 1000 Hz Across 6 Visits  2.2.4 Variation of Peak Compensated Static Susceptance (Btm) and Conductance (Gtm) with Age Table 2.7 provides a summary of descriptive statistics for compensated susceptance (Btm) and compensated conductance (Gtm) from both positive and negative tails at 226 and 1000 Hz probe tone frequencies across all 6 visits. At 226 Hz probe tone, Btm obtained using both positive and negative compensations, increased with age, particularly within the first 3 visits, and Gtm decreased with age. Btm values were overall lower for positive compensation than for negative compensation. Median values for positively compensated Btm during V1 were -0.50 mmho and increased to 0.37 mmho by 81  V6. Corresponding values for negatively compensated Btm was -0.17 mmho at V1 and increased to 0.55mmho by V6. Median negatively compensated Btm values increased rapidly within the first 3 visits by a factor of 4.6 but did not increase from the third to the sixth visit.  Both negative and positively compensated Gtm decreased with age.  Specifically, at V1, median value was 1.03 mmho for positively compensated Gtm and decreased to 0.48 mmho by V6.  Similar results were obtained for negatively  compensated Gtm for the same time period. Figure 2.15 shows positively compensated Btm and Gtm values at 226 Hz probe tone across all 6 visits. Figure 2.16 shows the corresponding graph using negative compensation.  Vertical bars denote 95% confidence intervals.  A repeated-measure  ANOVA was conducted with age (6 levels) and rectangular component (2 levels) as within-subject factors. Appendix I (Table A1.11and Table A1.12) provides the statistical summary tables.  Significant main effects of visits [positive compensation F(5,145)=  7.08; p<0.05; negative compensation F(5,145)= 8.68; p<0.05] and rectangular components [positive compensation F(1,29)= 78.38; p<0.05; negative compensation F(1,29)= 38.97; p<0.05] were found indicating that Btm and Gtm at 226 Hz varies across visits and between components. Interaction between visits and rectangular components were also significant [positive compensation F(5,145)= 67.20; p<0.05; negative compensation F(5, 145)= 64.45; p<0.05] indicating that compensated Btm and Gtm at 226 Hz varies differently between components and visits. To probe the interaction, effects of rectangular components were examined at each visit. Further investigation using Tukey HSD post-hoc analysis revealed that for 226 Hz probe tone, both positive and negative compensated Btm values progressively became higher from V1 to V3. V4, V5, and V6 82  were significantly higher than V1 to V3; however, they were not significantly different from each other. Table 2.7: The 5th, 50th, and 95th Percentiles of Compensated Susceptance (positive & negative) Compensated Susceptance (Btm) from  Compensated Susceptance (Btm) from  positive tail (mmho)  negative tail (mmho)  226 Hz Visit  1000 Hz th  226 Hz  5th-to-95th  -0.56-0.42  0.80  -0.10-2.12  0.29  -0.48-0.77  0.92  -0.40-181  -0.88-1.00  0.62  0.04-0.74  1.01  0.21-2.32  -0.09  -1.32-0.81  0.58  0.20-0.92  0.84  -0.07-1.99  0.18-0.60  -0.11  -1.18-1.14  0.60  0.34-0.84  0.88  0.36-2.49  0.22-0.58  0.09  -1.60-0.92  0.55  0.32-0.85  1.01  -0.24-2.25  5 -to-95  1  -0.50  2  th  Median  5 -to-95  -1.00-0.00  -0.05  0.13  -1.00-0.52  3  0.36  4  th  1000 Hz Median  Median  th  th  Median  5 -to-95  -1.07-0.72  -0.17  -0.05  -1.33-0.88  -0.20-0.54  0.14  0.40  -0.06-0.65  5  0.38  6  0.37  th  Table 2.8: The 5th, 50th, and 95th Percentiles of Compensated Conductance (positive & negative) Compensated Conductance (Gtm) from  Compensated Conductance (Gtm) from  positive tail (mmho)  negative tail (mmho)  226 Hz Visit  1000 Hz  226 Hz  1000 Hz  Median  5th-to-95th  Median  5th-to-95th  Median  5th-to-95th  Median  5th-to-95th  1  1.03  0.56-1.51  0.97  0.16-2.23  1.11  0.59-1.69  1.43  0.52-2.64  2  1.06  0.40-1.74  0.99  0.11-2.46  1.09  0.44-1.92  1.18  0.44-2.98  3  0.60  0.23-1.39  1.09  0.30-2.77  0.68  0.21-1.55  1.36  0.53-3.05  4  0.48  0.16-1.26  1.47  0.22-3.12  0.51  0.14-1.36  1.73  0.30-4.11  5  0.36  0.14-0.73  1.64  0.56-3.35  0.39  0.14-0.79  1.71  0.75-3.64  6  0.28  0.09-0.81  1.68  0.37-3.30  0.33  0.12-0.99  1.78  0.51-4.00  83  Figure 2.15: Median Positive Compensated Susceptance (Btm) and Conductance (Gtm) for 226 Hz Across 6 Visits  Figure 2.16: Median Negative Compensated Susceptance (Btm) and Conductance (Gtm) for 226 Hz Across 6 Visit  84  For 1000 Hz probe tone, Btm and Gtm revealed a different pattern. As seen in Table 2.7, Btm remained relatively constant across visits while Gtm increased with age for both tail pressures at 1000 Hz. Mean Btm from negative tail was lower across visits when compared to mean Gtm from positive tail. At V1, median value for positive compensated Btm was -0.05 mmho which increased minimally to 0.09 mmho by V6. Corresponding values for negative compensated Btm was 0.80 mmho at V1 and 1.01 mmho at V6. Positive compensated Gtm at V1 was 0.97 mmho and increased to 1.68 mmho by V6. Similar trends were observed using negative compensation. Mean Gtm increased faster at a factor of 1.7 from positive compensation between V1 and V6 when compared to negative compensation which increased by a factor of 1.2. Figure 2.17 shows positive compensated Btm and Gtm values at 1000 Hz probe tone across all 6 visits. Figure 2.18 shows the corresponding graph using negative compensation.  Vertical bars denote 95% confidence intervals.  A repeated-measure  ANOVA was conducted with age (6 levels) and rectangular component (2 levels) as within-subject factors. Appendix I (Table A1.13 and A1.14) provides the statistical summary tables. For positive compensation, significant effects were observed between visits [F(5,145)= 10.05; p<0.05] and between rectangular components [F(1,29)= 86.77; p<0.05] indicating that positively compensated Btm and Gtm at 1000 Hz varies across visits and between components. Interaction between visits and rectangular components were also significant [F(5,145)= 3.17;p<0.05] indicating that Btm and Gtm at 1000 Hz varies differently between components and visits.  Interactions between visits and  rectangular components were also significant indicating that at 1000 Hz using positive compensation, Btm and Gtm values vary differently across the visits between the two 85  components. When the same measures were observed with negative compensation, similar results were obtained. Again, Btm and Gtm values were statistically significant across visits [F(5,145)= 5.17;p<0.05] and between rectangular components [F(1,29)= 24.13;p<0.05] indicating that negatively compensated Btm and Gtm at 1000 Hz varies across visits and between components. Similarly, interaction effects between visits and rectangular components were also statistically significant [F(5,145)= 2.64; p<0.05] indicating that at 1000 Hz using negative compensation, Btm and Gtm values vary differently across the visits between the two components.  In order to investigate the  interaction effects, Tukey HSD post-hoc analysis tests were applied. Post-hoc results indicated that there was no significant change in Btm values across all 6 visits for either positive or negative compensation methods. For Gtm however differences between visits were observed. Gtm from positive compensation at 1000 Hz probe tone showed that V1, V2, and V3 were not significantly different from each other. However, Gtm at V4 was significantly higher than V2 but not V3 or V1. V5 and V6 were significantly higher than V1 to V3; but were not different from each other. Gtm with negative compensation showed that V5 and V6 were higher than V2. V6 was also higher than V3. Gtm was not statistically different between any other visits.  86  Figure 2.17: Median Positive Compensated Susceptance (Btm) and Conductance (Gtm) for 1000 Hz Across 6 Visits  Figure 2.18: Median Negative Compensated Susceptance (Btm) and Conductance (Gtm) for 1000 Hz Across 6 Visits  87  2.2.5 Variation of Tympanometric Pressure with Age Table 2.9 provides a summary of descriptive statistics for tympanometric peak pressure (TPP) at both 226 Hz and 1000 Hz across the 6 visits. The 5th to 95th percentile ranges for TPP was large at both frequencies and across all 6 visits showing no specific pattern.  Table 2.9: The 5th, 50th, and 95th percentiles of Tympanometric Peak Pressure Tympanometric Peak Pressure (daPa) 226 Hz Visit  1000 Hz th  Median  5th-to-95th  -46.00-17.00  -10.00  -79.00-57.00  -10.00  -51.50-21.50  0.00  -69.00-50.00  3  0.00  -78.00-9.50  -7.50  89.25-39.25  4  -25.00  -73.50-24.00  -15.00  -99.00-53.50  5  -15.00  -64.00-40.00  -10.00  -98.75-86.25  6  -20.00  -100.00-55.00  -15.00  -100.00-55.00  Median  5 -to-95  1  -5.00  2  th  Figure 2.19 shows variation of TPP at 226 Hz and 1000 Hz probe tone across all 6 visits. Vertical bars denote 95% confidence intervals. A repeated-measure ANOVA was conducted with age (6 levels) and frequency (2 levels) as within-subject factors. Appendix I (Table A1.15) provides the statistical summary tables. No statistically significant differences were obtained between TPP values across visits or frequency indicating that TPP does not significantly vary with age or probe tone frequency.  88  Figure 2.19: Median Tympanometric Peak Pressure (TPP) for 226 Hz and 1000 Hz Across 6 Visits  2.2.6 Analysis of Change in Compensated Susceptance (ΔB) and Conductance (ΔG) Across Age ΔB and ΔG were recorded using the sweep frequency method from 250 to 2000 Hz in 50 Hz intervals in 16 infants. Figure 2.20 shows ΔB plotted as a function of probe tone frequency for infants across visits and compared to adult data (Shahnaz & Bork, 2008). Detailed descriptive statistics for each test frequency across 6 visits for ΔB and ΔG are provided in appendix II. Btm above 0 mmho represents a stiffness-dominated middle ear system, Btm close to 0 mmho represent the resonant frequency, and Btm below 0 mmho represent a mass-dominated middle ear system. Overall pattern indicates that Btm in young infants lie largely below 0 mmho suggesting a mass-dominated middle ear system even at low frequencies.  As age increases, Btm gradually rises in the low 89  frequencies becoming more stiffness-dominated at later visits. Resonant frequency also shifts upwards with age, reaching approximately to 600 Hz by V6. Although the middle ear system becomes more stiffness-dominated at lower frequencies with age, it is not yet adult-like.  ΔB decreases in adults in an orderly fashion as probe tone frequency  increases, where resonant frequency lies approximately at 900 Hz and is stiffnessdominated below this point and mass-dominated above. In contrast, infants even at V6 demonstrate lower resonant frequency with lower Btm at low frequencies and higher Btm at higher frequencies compared to adults. A repeated-measure ANOVA was applied to Δ B with age (6 levels: 6 visits) and probe tone frequencies (20 levels) as within-subject variables. These factors were beyond the scope of the current study and were not controlled for explicitly. Appendix II (Table A2.1and A2.2) provides the statistical summary tables. Repeated-measure ANOVA for ΔB was significant across visits [F(5,40)= 6.60; p<0.05] and frequencies [F(19,152)= 3.90; p<0.05].  The interaction between visit and  frequencies was also significant [F(95,760)=2.84; p<0.05] indicating that Btm varies differently across the visits at various frequencies.  90  2.00  Delta B 1.00 0.00  Mag nitude - mmho  260 300 350 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 -1.00 -2.00 -3.00 -4.00 -5.00 -6.00 -7.00  F requenc y - Hz V1  V2  V3  V4  V5  V6  Adult  Figure 2.20: Δ B (ΔB) as a Function of Probe Tone Frequency for Infants Across 6 Visits(Current study) and Compared to Adult Data (Shahnaz & Bork, 2008) (V=Visit) Figure 2.21 shows ΔG plotted as a function of probe tone frequency for infants across visits and compared to adult data (Shahnaz & Bork, 2008). At low frequencies (below approximately 400 Hz), positively compensated Gtm decreases with age. Between approximately 400 Hz and 900 Hz, Gtm for V1 to V4, decreases then rises again above 900 Hz. However, at V6, Gtm does not decrease but progressively rises in an adult-like pattern, although not yet at the same magnitude. Overall Gtm increases with age above approximately 400 Hz although adult-like values are not yet reached by V6. A repeatedmeasure ANOVA was applied with age (6 levels: 6 visits) and probe tone frequencies (20 levels) as within-subject variables. Appendix II (Table A2.3 and A2.4) provides the statistical summary tables. Repeated-measure ANOVA for ΔG was significant across 91  visits [F(5,40)= 2.65; p<0.05] and frequencies [F(19,152)= 21.42; p<0.05].  The  interaction between visit and frequencies was also significant [F(95,760)=1.61; p<0.05] indicating that Gtm varies differently across the visits at various frequencies. Delta G  3.00  2.50  Mag nitude - mmho  2.00  1.50  1.00  0.50  0.00 0  500  1000  1500  2000  2500  -0.50  -1.00  F requenc y - Hz V1  V2  V3  V4  V5  V6  Adult  Figure 2.21: Variation of Conductance (ΔG) as a Function of Probe Tone Frequency for Infants across 6 Visits (Current study) and Compared to Adult Data (Shahnaz & Bork, 2008) (V=Visit)  2.2.7 Data Comparison of Infants at Visit 6 to School-aged Children and Adults Various tympanometric results obtained from infants at visit 6 in the current study were compared to data of school-aged children (Bosaghzadeh, in progress) and adults (Shahnaz & Bork, 2008). Measurements were made by (Bosaghzadeh, in progress) in 104 children (5-7 years, 154 ears) and by Shahnaz and Bork (2008) in 53 adults (18-36 years, 92  94 ears). Although unpublished, Bosaghzadeh (in progress) has used identical procedure and instrumentation used in the current study which renders itself nicely for data comparison. It is important to ascertain that all confounding variables (including procedural issues and instrumentation) are accounted for in maturational studies as differences observed could be easily attributed to these confounding variables. The comparison measures included admittance at positive tail (Y+) at 226 Hz, Ytm from Ya tympanograms at 226 Hz, and Ytm, Btm and Gtm from B/G tympanograms at 226 Hz and 1000 Hz. Only positive compensation methods were compared as the positive tail has been shown to have better test-retest reliability when compared to the negative tail values (Kei et al., 2007; Margolis & Goycoolea, 1993) and is the most commonly used method in current clinical practice today. For comparison purposes, descriptive statistics for admittance at positive tails at 226 Hz also commonly referred to as equivalent ear canal volume (Vea) for infants at V6 in the current study are reported next to those obtained from school-aged children and adults in Table 2.10. Figure 2.22 shows mean Vea at positive tail for each of the 3 age groups. Vertical bars denote 95% confidence intervals. Results indicate that admittance at the positive tail increases as age increases. In order to investigate whether Vea at positive tail in infants during visit 6 (6 months old) is comparable to Vea at positive tail in school-aged children and adults, a univariate test of significance was conducted. In this model the age (3 levels: infants at V6, school-aged children, and adults) served as a between-subject factor. Appendix III (Table 3.1) provides the statistical summary table. The effect of age was significant [F(2,304)=18.59;p<0.05] indicating that Vea at positive tail varies between different age groups. Post-hoc test revealed that Vea at positive tail 93  for infants at V6 and school-aged children were significantly lower than adults. Vea at positive tail in infants at V6 were also significantly lower than for school-aged children  Table 2.10: The mean, 50th, and 95th percentiles of Admittance at Positive Tail for Infants at V6 (current study), School-aged Children (Bosaghzadeh, in progress) and Adults (Shahnaz & Bork, 2008) Admittance (Ya) positive tail value (mmho) 226 Hz Group  Mean  5th-to-95th  Infants at V6 (current Study)  0.67  0.50-0.90  0.81  0.60-1.10  1.26  0.80-1.70  School-aged Children (Bosaghzadeh, in progress) Adult (Shahnaz & Bork, 2008)  Figure 2.22: Mean Static Admittance at Positive Tail (+200) from Ya Tympanogram for infants at V6 (current study) compared to School-aged Children (Bosaghzadeh, in progress) and Adults (Shahnaz & Bork, 2008)  94  Descriptive statistics for positive compensated static admittance (Ytm) derived from Ya tympanograms at 226 Hz and 1000 Hz for infants at V6 in the current study are shown next to those obtained from school-aged children and adults in Table 2.11. Figure 2.23 shows mean Ytm at 226 Hz for each of the 3 age groups. Vertical bars denote 95% confidence intervals. Results indicate that Ytm increases as age increases. In order to investigate whether Ytm in infants during V6 (6 months old) is comparable to Ytm in school-aged children and adults, a univariate test of significance was conducted. In this model the age (3 levels: infants at V6, school-aged children, and adults) served as a between-subject factor. Appendix III (Table 3.2) provides the statistical summary table. The effect of age was significant [F(2,307)=167.03;p<0.05] indicating that Ytm varies differently between different age groups. Post hoc revealed that while Ytm for infants at V6 and for children were significantly lower than for adults; however, they were not significantly different from each other.  Table 2.11: The mean, 50th, and 95th Percentiles of Positive Peak Compensated Static Admittance from Ya Tympanogram for Infants at V6 (current study), School-aged Children (Bosaghzadeh, in progress) and Adults (Shahnaz & Bork, 2008) Peak Compensated Static Admittance (Ytm) from Ya Tympanogram from Positive tail (mmho) 226 Hz Group Infants at V6 (current Study) School-aged Children  1000 Hz th  Mean  5th-to-95th  0.29-1.03  2.00  0.56-3.60  0.53  0.25-0.96  2.87  1.01-6.58  0.70  0.22-1.31  2.71  0.64-5.81  Mean  5 -to-95  0.55  th  (Bosaghzadeh, in progress) Adult (Shahnaz & Bork, 2008)  95  Figure 2.23: Mean Positive Peak Compensated Static Admittance (Ytm) from Ya Tympanogram for infants at V6 (current study) compared to School-aged Children (Bosaghzadeh, in progress) and Adults (Shahnaz & Bork, 2008)  Descriptive statistics for infants at V6 in the current study are shown next to those obtained from school-aged children and adults for positive Btm and Gtm in Table 2.12. Mean Ytm (derived from B/G tympanograms), Btm, and Gtm values for each of the 3 age groups are shown in figure 2.24 for 226 Hz and figure 2.25 for 1000 Hz. Vertical bars denote 95% confidence intervals. In order to investigate whether Ytm, Btm, and Gtm in infants during visit 6 (6 months old) is comparable to those of school-aged children and adults, a repeatedmeasure ANOVA was conducted. In this model the age (3 levels: infants at V6, schoolaged children, and adults) served as a between-subject factor and the component (3 levels: Ytm, Btm, and Gtm) served as a within-subject factor. Appendix III (Tables 3.3-3.4) provides the ANOVA summary tables. For 226 Hz, significant effects were observed between age groups [F(2,620)= 10.61; p<0.05] and between components [F(2,620)= 397.10; p<0.05]. Interactions between age groups and components were also significant [F(2,620)= 23.30;p<0.05] indicating that at 226 Hz Ytm, Btm and Gtm values vary 96  differently between each other and across age groups. Post hoc revealed that Ytm and Btm for both infants at V6 and school-aged children were significantly lower than adults but were not significantly different from each other. There were no significant differences observed for Gtm across age groups. For 1000 Hz, significant effects were observed between age groups [F(2,620)= 3.25; p<0.05] and between components [F(2,620)= 302.73; p<0.05].  Interactions  between age groups and components were also significant [F(2,620)= 4.50;p<0.05] indicating that at 1000 Hz Ytm, Btm and Gtm values vary differently between each other and across age groups. Post hoc analysis revealed that Ytm and Gtm of infants at V6 were significantly lower than adults and children.  There were no significant differences  observed for Btm across age groups.  Table 2.12: The mean, 50th, and 95th percentiles of Positive Compensated Susceptance (Btm) and Conductance (Gtm) for Infants at V6 (current study), School-aged Children (Bosaghzadeh, in progress) and Adults (Shahnaz & Bork, 2008) Compensated Susceptance (Btm)  Compensated  from Positive tail (mmho)  from  226 Hz Group Infants  at  V6  (current Study) School-Aged Children (Bosaghzadeh, in progress) Adult (Shahnaz & Bork, 2008)  1000 Hz th  Mean  5-to-95  Mean  0.37  0.22-0.58  -0.07  0.46  0.21-0.84  -0.44  0.59  0.19-1.13  -0.54  Conductance  Positive tail (mmho)  226 Hz 5-to-95 -1.600.92 -3.180.97 -2.881.24  97  th  (Gtm)  1000 Hz Mean  5-to-95th  0.09-0.81  1.81  0.37-3.30  0.27  0.12-0.52  2.54  0.84-5.80  0.36  0.11-0.66  2.48  0.56-5.61  Mean  5-to-95  0.39  th  Figure 2.24: 226 Hz Mean Positive Peak Compensated Static Admittance (Ytm) from B/G Tympanograms and Positive Compensated Susceptance (Btm) and Conductance (Gtm) for infants at V6 (current study) compared to School-aged Children (Bosaghzadeh, in progress) and Adults (Shahnaz & Bork, 2008)  Figure 2.25: 1000 Hz Mean Positive Peak Compensated Static Admittance (Ytm) from B/G Tympanograms and Positive Compensated Susceptance (Btm) and Conductance (Gtm) for infants at V6 (current study) compared to School-aged Children (Bosaghzadeh, in progress) and Adults (Shahnaz & Bork, 2008 98  2.2.8 Case Studies Among the infants tested as part of this study, 5 ears did not pass otoacoustic emission screening at one or more visits. The results from these infants will be reviewed in the following cases and illustrate the value of 1000 Hz probe tone tympanometry along with tympanometric shape in the assessment of the middle ear in infants.  2.2.8.1 Case 1: (Sensorineural Hearing Loss) Case 1, male, with no family history of hearing loss was initially tested at 22 days of age using otoacoustic emission screen and tympanometry. He failed to meet the passing criterion for OAE screen in both ears using both TEOAE and DPOAEs. As seen in figures 2.26 (right ear) and 2.27 (left ear), normal, single-peaked 1000 Hz Ya tympanograms were recorded bilaterally with 1B1G patterns observed for B/G tympanograms shown in figure 2.28 (right ear) and figure 2.29 (left ear). Positive Ytm was within normal range at 0.53 mmho and 0.74 mmho for right and left ears, respectively. However, 226 Hz Ya tympanogram recordings shown in figures 2.30 (right ear) and figure 2.31 (left ear) and B/G tympanogram recordings shown in figures 2.32 (right ear) and figure 2.33 (left ear) were more complex and difficult to interpret. Ipsilateral middle ear muscle reflex using a broadband noise signal and 1000 Hz probe tone was recorded at 90 dB HL in both ears. The outcome of tympanometric results at 1000 Hz and OAEs suggests sensorineural hearing loss bilaterally. He was referred for diagnostic air-conduction (AC) and bone-conduction (BC) tone-evoked ABR at BC Children‟s Hospital and was seen at 4 weeks of age. Results revealed a bilateral mild to moderately-severe sensorineural hearing loss. 99  Figure 2.26: Case 1 Visit 1 Right Ear  Figure 2.27: Case 1 Visit 1 Left Ear  Y-Tympanogram Recording 1000 Hz  Y-Tympanogram Recording 1000 Hz  Figure 2.28: Case 1 Visit 1 Right Ear B/G  Figure 2.29: Case 1 Visit 1 Left Ear B/G  Tympanogram Recording 1000 Hz  Tympanogram Recording 1000 Hz  100  Figure 2.30: Case 1 Visit 1 Right Ear  Figure 2.31: Case 1 Visit 1 Left Ear  Y- Tympanogram Recording 226 Hz  Y-Tympanogram Recording 226 Hz  Figure 2.32: Case 1 Visit 1 Right Ear B/G  Figure 2.33: Case 1 Visit 1 Left Ear B/G  Tympanogram Recording 226 Hz  Tympanogram Recording 226 Hz 101  2.2.8.2 Case 2: (conductive hearing loss-transient) Case 2, a male was initially tested at 10 days old using otoacoustic emission screen and tympanometry. He did not pass otoacoustic emission screen in either ear. Bilateral 1000 Hz Ya tympanogram (figures 2.34-right ear and 2.35-left ear) and B/G tympanogram (figures 2.36- right ear and 2.37-left ear) recordings were flat with no discernable peak observed. In contrast, 226 Hz Ya tympanograms for both ears showed double-peaked recordings with positive Ytm values of 0.45 mmho and 0.35 mmho for right (figure 2.38) and left ears (figure 2.39), respectively. 226 Hz B/G tympanogram recordings showed a 3B3G in the right ear (figure 2.40) and 5B3G pattern in the left ear (figure 2.41). Ipsilateral middle ear muscle reflex at 1000 Hz and broadband noise signal at 90 dB HL were absent in both ears. He was referred for a diagnostic ABR and was tested at 24 days gestational age.  1000 Hz AC and BC tone-evoked ABR results  indicated mild conductive hearing loss bilaterally. Had 226 Hz tympanometry be used as a screening probe tone, results would have indicated a false negative, suggesting that the infant had normal middle ear function when middle ear abnormalities clearly existed. However, the 1000 Hz probe tone tympanometry correctly identified abnormal middle ear status. In a subsequent follow-up, we have tested this baby and OAEs were present and tympanograms were normal at 1000 Hz probe tone.  102  Figure 2.34: Case 2 Visit 1 Right Ear  Figure 2.35: Case 2 Visit 1 Left Ear  Y-Tympanogram Recording 1000 Hz  Y-Tympanogram Recording 1000 Hz  Figure 2.36: Case 2 Visit 1 Right Ear B/G  Figure 2.37: Case 2 Visit 1 Left Ear B/G  Tympanogram Recording 1000 Hz  Tympanogram Recording 1000 Hz 103  Figure 2.38: Case 2 Visit 1 Right Ear B/G  Figure 2.39: Case 2 Visit 1 Left Ear B/G  Tympanogram Recording 226 H  Tympanogram Recording 226 Hz  Figure 2.40: Case 2 Visit 1 Right Ear  Figure 2.41: Case 2 Visit 1 Left Ear  Y- Tympanogram Recording 226 Hz  104  Y-Tympanogram Recording 226 Hz  2.2.8.3 Case 3: (conductive hearing loss-transient) Case 3, male, was tested using otoacoustic emission screen and tympanometry at 5 separate occasions (Visits 1-5). Results obtained from visits 1-3 (Age 3 weeks, 9 weeks, and 14 weeks) were within normal limits as observed by present TEOAEs and normal tympanometric recordings. At Visit 4 (Age 17 weeks), case 3 did not pass TOAE screen in the right ear, and although within the passing criterion for the left ear, demonstrate low emission amplitudes. Bilateral 1000 Hz tympanograms were flat for both Ya tympanograms (figure 2.42-right ear; figure 2.43-left ear) and B/G tympanograms (figure 2.44-right ear; figure 2.45-left ear) with no discernable peaks. Despite these findings for 1000 Hz probe tone, 226 Hz Ya tympanograms were singlepeaked and normal for both ears (figure 2.46-right ear; figure.2.47-left ear) and B/G tympanograms were 1B1G bilaterally (figure 2.48-right ear; figure 2.49-left ear). Ipsilateral middle ear muscle reflex was absent bilaterally at an intensity of 95 dB HL at 1000 Hz probe tone. For 226 Hz probe tone , middle ear muscle reflex was present at 70 dB HL for the left ear only. Test results were strongly suggestive of conductive hearing loss in at least the right ear. Allowing for any transient middle ear problems to clear, a follow up visit at 21 weeks of age was conducted. At that time, case 3 passed otoacoustic screening for both ears.  The conductive component had resolved revealing normal  tympanometric recordings. 1000 Hz Ya tympanograms were single-peaked with normal admittance at 2.15 mmho and 2.42 mmho for right (figure 2.50) and left (figure 2.51) ears, respectively. 1000 Hz B/G tympanograms were 3B1G configuration bilaterally (figure 2.52-right ear; figure 2.53-left ear). 226 Hz Ya tympanograms were also single  105  peaked (figure 2.54- right ear, figure 2.55-left ear) with B/G tympanograms showing 1B1G patterns for both ears (figure 2.56-right ear; figure 2.57-left ear).  Figure 2.42: Case 3 Visit 4 Right Ear  Figure 2.43: Case 3 Visit 4 Left Ear  Y-Tympanogram Recording 1000 Hz  Y-Tympanogram Recording 1000 Hz  Figure 2.44: Case 3 Visit 4 Right Ear B/G  Figure 2.45: Case 3 Visit 4 Left Ear B/G  Tympanogram Recording 1000 Hz  Tympanogram Recording 100 106  Figure 2.46: Case 3 Visit 4 Right Ear  Figure 2.47: Case 3 Visit 4 Left Ear  Y- Tympanogram Recording 226 Hz  Figure 2.48: Case 3 Visit 4 Right Ear B/G  Y-Tympanogram Recording 226 Hz  Figure 2.49: Case 3 Visit 4 Left Ear B/G  Tympanogram Recording 226 Hz  Tympanogram Recording 226 Hz  107  Figure 2.50: Case 3 follow up Right Ear  Figure 2.51: Case 3 follow up Left Ear  Y-Tympanogram Recording 1000 Hz  Y-Tympanogram Recording 1000 Hz  Figure 2.52: Case 3 follow up Right Ear  Figure 2.53: Case 3 follow up Left Ear  B/G Tympanogram Recording 1000 Hz  B/G Tympanogram Recording 1000 Hz 108  Figure 2.54: Case 3 follow up Right Ear  Figure 2.55: Case 3 follow up Left Ear  Y- Tympanogram Recording 226 Hz  Figure 2.56: Case 3 follow up Right Ear  Y-Tympanogram Recording 226 Hz  Figure 2.57: Case 3 follow up Left Ear  B/G Tympanogram Recording 226 Hz  B/G Tympanogram Recording 226 Hz 109  2.3  Discussion Thirty-one full-term healthy newborns were recruited and were tested  longitudinally in 1-month intervals up to 6 months of age for a total of 6 visits per each infant. The primary goal of this study was to compare multi-frequency, multi-component immittance measures obtained from infants as a function of age from birth to 6 months of age. A secondary goal of our study was to provide age related normative data to use as a guideline within clinic practice. Tympanometric results indicate that the infant middle ear is rapidly changing during this period and is not yet functionally mature by 6 months of age.  These maturational patterns are demonstrated by changes observed through  measures of tympanometric shape, admittance, susceptance, and conductance at multiple probe tone frequencies. In this section, first, a comparison of the current findings to previous published data is discussed.  Second, potential source of differences are  examined. Clinical implications and suggested cut-off values are also discussed. Finally, limitations of the current study and direction for future research are discussed.  110  2.3.1 Comparison of Results Table 2.13 shows a summary comparison of data of admittance at positive tail (+200 daPa) and positive peak compensated admittance between various published works. Table 2.13: Summary Comparison of Data to Previous Published Works Study  Current Study  Mean/ Median  Median  No. Of Ears  57 54 44 47 36 41  Kei et al (2003) Kei et al (2007) Margolis et al (2003) Mazlan et al (2007)  Alaerts et al (2007)  Calandruccio et al (2006)  Mean Mean Mean Mean  Median  Median  Mean Age of Subjects  4 weeks 13 weeks 21 weeks 28 weeks 36 weeks 44 weeks  Admittance (Ya) at +200 daPa in mmho  Positive Peak Compensated (+200 daPa) Admittance (Ytm) in mmho  226 Hz  1000 Hz  226 Hz  1000 Hz  0.50  1.67  0.70  0.85  0.60  1.87  0.80  0.87  0.70  2.25  0.60  0.82  0.60  2.47  0.50  1.06  0.60  2.59  0.50  1.04  0.70  2.73  0.40  1.19  170  3 days  36  2.5 days  30  3 weeks  1.40  1.30  42  2.5days  1.07  0.82  6 weeks  1.33  1.02  1.11  0.95  110  39 39 15 50  < 12 weeks 12- 36 weeks 4-10 weeks 11-19 weeks 20-26 weeks 24-48 weeks  3.10  1.10 0.65  0.56  1.97  0.32  1.88  0.36  1.26  0.40  1.07  0.36  1.43  0.25  1.45  0.29  1.53  0.28  1.41  0.40  1.77  0.31  1.93  111  2.3.1.1  Tympanometric Shapes Our findings indicate that Ya tympanograms obtained from a 226 Hz probe tone in  the current study were predominately multi-peaked at birth and became progressively less complex, with fewer peaks, with age. These observations are in agreement with those reported by Alaerts, Lutz and Woulters (2007) in their <3 month old and 3-9 month old groups and also consistent with findings of Shahnaz et al. (2008) in their NICU and 3 week old infants. In contrast, Ya tympanograms using a 1000 Hz probe tone were predominately single peaked across all 6 visits (95-100% between V1-V5 and 88% at V6). These results were in good agreement with those obtained by Alaerts et al. (2007) of their 0-3 month old infants (91%), Kei et al. (2003) of their 1-6 day old infants (92%), and Shahnaz et al. (2008) of their NICU infants with an age range of 32-51 weeks (95%). Others have also found that the occurrence of multi-peaked tympanograms and the complexity of shapes decreased as the frequency of the probe tone increased (Holte et al., 1991; Kei et al. 2003). Since 226 Hz tympanograms are more likely to show notching or complex patterns in infants, it is easier and more straightforward to quantify tympanometric recordings using 1000 Hz probe tone due to the high prevalence of singlepeak recordings The distribution of Vanhuyse patterns at 226 Hz were exclusively 3B1G (44%) and 3B3G (56%) during the first month with no occurrences of 1B1G. However, by the sixth month, multi-peaked patterns diminished and almost all (98%) tympanograms showed a 1B1G pattern. At birth and during the first few months of life, the healthy infant middle ear is either mass-dominated or at resonance at the standard 226 Hz probe tone. This is reflected by the high prevalence of 3B3G tympanograms; if the susceptance 112  (Ba) notch lies below the tail value, conductance (Ga) also begins to notch, the admittance vector will be below 0º and the middle ear is mass dominated. Furthermore, the shape of the 3B1G tympanograms recorded were of a deep notching pattern where the Ba notch was equal to the tail value suggesting that the middle ear is in resonance and the admittance vector is at 0º.  In a study of NICU and 3 week old infants, Shahnaz et al.  (2008) reported a high proportion of 5B3G (55%), 3B3G (24%), and 3B1G (12%) patterns with a very small proportion of 1B1G (3%) in their NICU infants. The authors reported that the distribution of shapes for the NICU infants were very different compared to the 3 week old infant group; no occurrences of 5B3G were recorded and 1B1G recordings were much higher (13%). These differences between the two age groups indicate some maturational effects. In a longitudinal study of infants 0-2 years of age, Calandruccio et al. (2006) found a high proportion of 1B1G patterns among all their infant age groups with a small proportion of 3B1G tympanograms in their 4-10 week old and 11-19 week old age groups (23.1% and 6.8%, respectively) at 226 Hz probe tone. The differences in distribution of shape between the Calandruccio et al. (2006) study and the current study could be attributed to differences in age ranges. The two youngest age groups included in the Calandruccio et al. (2006) study had greater age ranges (up to 8 weeks) compared to the current study which included 4 groups (V1-V4) with age ranges less than 5 weeks each. patterns.  Differences in age ranges will yield different distribution  Investigating narrow age ranges is important in maturational research as  detailed changes in distribution can be missed if broad age ranges are used. However, despite these differences, the Calandruccio et al. (2006) study also found that, as infants become older, the proportion of 1B1G tympanograms increases at the 226 Hz probe tone. 113  The changes observed between age groups in the current study and the Calandruccio et al. (2006) and Shahnaz et al. (2008) studies indicate maturational effects. At 678 Hz probe tone, infants in the current study yielded a greater variety of tympanometric patterns.  Recorded tympanometric shapes at 678 Hz were equally  scattered across patterns with the majority not conforming to the patterns observed under the Vanhuyse model. Using a probe tone of 630 Hz, Calandruccio et al. (2006) found a higher proportion of 1B1G in their two youngest age groups (between approximately 55% and 80%) compared to the infants at V1 to V4 in the present study (between 16%25%).  These differences could be a result of variance in subject age ranges and  difference in probe tone frequency. The Calandruccio et al. (2006) study used a lower probe tone of 630 Hz compared to a probe tone of 678 Hz used in the current study. However as with the current study, they also noted that occurrence of complex, multipeaked patterns decreased with age at this probe tone frequency. Using a 1000 Hz probe tone, approximately equal proportions of 1B1G (49%) and 3B1G (47%) were recorded during V1 in the current study. These proportions remained relatively constant across the first 5 visits with the proportion of 1B1G dropping slightly by the sixth month (38%). This suggests that at 1000 Hz, the infant middle ear is stiffness dominated at birth and during the first few months of life. It is important to note that the shape of the 3B1G tympanograms recorded at 1000 Hz probe tone was of a different configuration than those recorded using a 226 Hz probe tone. Specifically, the shape of the 3B1G tympanograms recorded at 1000 Hz showed shallow notching of Ba where the notch portion always lies above tail values. This indicates that Ba remains positive between 0º and 45º, and the middle ear is stiffness dominated. Our results were 114  in good agreement with those reported by Alaerts et al. (2007). They reported that in their <3 month old group, occurrences of 1B1G and 3B1G combined was the most dominant accounting for 91% of the observed patterns. The results of their 3-9 month old group were comparable to those collected from our study at V6, with the prevalence of 3B1G at approximately 55% followed by 1B1G at approximately 30%. Similar results were reported by Calandruccio et al. (2006) and Shahnaz et al. (2008). Due to the greater contribution of mass elements in the newborn middle ear system, as opposed to stiffness elements in the adult middle ear, tympanometric patterns observed in newborn infants do not conform to the classic patterns found in older infants, children, and adults. Consistent with findings of Shahnaz et al. (2008), current results support the notion that in the infant ear, admittance tympanograms become simpler in shape as probe frequency increases, the reverse of what is found in adult ears, where admittance tympanograms become more complex as probe frequency increases. Very few studies have evaluated the shape of susceptance and conductance tympanograms in young infants longitudinally.  Consequently, insufficient data is  available to reliably quantify the distribution of shapes that exist within the infant population. However there is a significant value in investigating tympanometric shapes in maturational research of the middle ear. By examining the configuration of the susceptance and conductance portions of tympanograms across age, information on relative contribution of mass and stiffness can be obtained. The changes of shapes across age can demonstrate the frequency specific mechanical changes that occur in the infant ear over time which in turn allows us to understand the efficiency of sound conduction through the middle ear system. 115  2.3.1.2  Equivalent Ear canal volume (Vea) Measurements of admittance and susceptance at both positive and negative tails  increased with age at 226 Hz and 1000 Hz probe tone frequencies in the current study. The increases in magnitude are most likely due to increase in ear canal volume. Keefe, Bulen, Arehart and Burns (2003) found that ear canal diameter increased from 4.4mm for 1 month olds to 6.3 mm for 6 month olds. A larger ear canal volume corresponds to a larger susceptance, and therefore admittance. As a result, the increase in ear canal volume in the growing infant is most likely the cause of increased admittance magnitude over time. The positive admittance tail values of the present study were compared to results obtained by Alaerts et al. (2007) and Calandruccio et al. (2006). Median positive admittance tail value at 226 Hz from infants at V5 (0.60 mmho) in the present study was comparable to results obtained by Alaerts et al. (2007) of 30 ears of 3-9 month old infants (0.56 mmho). However, the median positive admittance tail values at 226 Hz obtained of our infants across all 6 visits were approximately 35% higher compared to infant results from Calandruccio et al. (2006).  Measurements of both the current study and the  Calandruccio et al. (2006) study were taken at +200 daPa. The variation in results could be due to probe tip location in the ear canal. If the probe tip insertion depth in the Calandruccio et al. (2006) study was deeper compared to the current study, admittance tail values would have been lower.  Furthermore, in contrast to the present study,  Calandruccio et al. (2006) did not find an increase in positive tail admittance at 226 Hz for infants between birth and 26 weeks of age. These differences in results could again be due to the larger age ranges included in the Calandruccio et al. (2006) study relative to  116  the present study and the limitations involved with using broad age ranges in maturational studies. Median admittance tail value at 1000 Hz in the present study also showed a steady increase over the 6 visits. This trend was also observed by Mazlan et al. (2007) between their newborn group and 6 week old group. Our median positive tail values at 1000 Hz recorded during V1 (1.67 mmho) were higher than those obtained by Margolis et al. (2003) of their 2-4 week old infants (1.0 mmho) and Mazlan et al. (2007) of their newborn group (1.07 mmho). Mazlan et al. (2007) retested their infants again when they were approximately 6 weeks old and found that median values increased to approximately (1.33 mmho) which were lower than results obtained in the present study of infants at V2 (1.86 mmho). Pressure ranges were the same between the two studies. Differences in results between studies could have been due to depth of probe-tip insertion. There were also differences observed in the rate of change of tail values between 226 Hz and 1000 Hz probe tone frequencies. Admittance at 226 Hz for both positive and negative tails showed an increase from V1 to V3 but remained unchanged thereafter. However, admittance tail values at 1000 Hz showed consistent increase from V1-V6 for both positive and negative tails. This may be due to a reduction of mass in the middle ear system from newborns to young toddlers. The decrease of mass has more pronounced effects at high frequencies than at low frequencies, which causes the susceptance due to mass and therefore admittance, to increase more at 1000 Hz than at 226 Hz. Furthermore, the results of the current study showed that conductance at 1000 Hz is much greater than at 226 Hz for both tails, which agrees with the report from (Qi, Funnel, & 117  Daniel, 2008). They analyzed the tympanogram tails obtained in sixteen, full-term, healthy 3 week old infants and found that the conductance increased from 226 Hz to 1000 Hz by a factor of about 6 for both tails. These large conductance values at high frequencies are presumably associated with vibration of the newborn canal wall and middle ear (Qi et al., 2008). Admittance and susceptance magnitudes at positive tails were greater than at negative tails across all visits for both 226 Hz and 1000 Hz probe tones. Holte et al. (1991) found that the mean diameter changes in the newborns‟ ear canal were much bigger for negative pressures than for positive pressures. These variations between positive and negative compensation are likely due to collapse of the ear canal at high negative pressures. Consequently, the peak-to positive-tail admittance differences were smaller than the peak-to negative-tail differences. Results at V1 in the current study indicate that at 1000 Hz, the mean peak-to negative-tail admittance (1.75 mmho) is greater than the peak-to positive-tail admittance (0.85 mmho) by approximately 0.9 mmho due to the mean positive tail magnitude being approximately 0.98 mmho larger than the negative tail magnitude. Margolis et al. (2003) also reported greater median peak-to negative-tail admittance values (1.7 mmho) than peak-to positive-tail admittance values (1.0 mmho) with a difference of 0.7 mmho for their full-term 3 week old infants which are comparable to the results of the current study. Shahnaz et al. (2008) reported mean peak-to negative-tail and peak-to positive-tail admittance of 0.95 and 0.6 mmho respectively for 3 week old infants with a mean difference of 0.35 mmho. The lower values obtained in their study compared to the present study could be a result of different static pressures used in the measurements. The study of Shahnaz et al. (2008) used static 118  pressures ranging from +250 to -300 daPa compared to the present study which used +200 to -400 daPa. The larger pressure gradient used in the present study, utilizing more extreme negative pressure, may have caused larger displacements of the newborn‟s ear canal wall and tympanic membrane resulting in a greater difference between tail measurements (Holte et al., 2003; Qi et al., 2008). Ideal pressure ranges have not yet been established for infants within this age range and need to be taken into consideration when establishing normative data.  2.3.1.3  Peak Compensated Static Admittance In the present study, median Ytm was calculated using baseline compensation  method from admittance tympanograms and rectangular components compensation method at both tails and at both probe tones. Results indicate that for 226 Hz, the error in estimation of admittance by using the baseline compensation value compared to deriving from rectangular components compensation was large, with a maximum median discrepancy of 0.51 mmho at V1 which was decreased to 0.07 mmho by V6 (positive compensation). For the higher probe tone of 1000 Hz at V1, a median error estimation of 0.31 mmho was recorded which was increased to 0.71 mmho by V6 (positive compensation). In a study of 36 infants (mean age of 62 hours), Kei et al. (2007) also compared static admittance values obtained from the baseline method and rectangular components compensation method. Their results were comparable to ours indicating a mean static admittance estimation error of 0.45 mmho (positive compensation) at 1000 Hz probe tone. This marked error occurs because there is a large discrepancy between phase angle of the admittance magnitude at the tail and the peak/trough of the 119  tympanogram (Kei et al., 2007; Margolis & Shanks, 1991). Subtracting admittance vector data at standard low probe tone of 226 Hz in adults and school-aged children results in negligible error since the phase difference between the Ytm vector and total susceptance is small. As probe tone increases, in adults and children, the admittance vector becomes closer to conductance and phase variation between the Btm and Ytm vector becomes greater. However, the opposite is true for infants. The results of the current study suggest that estimated admittance vector error is large at 226 Hz during the first few months of life and becomes progressively smaller by the time the infant is 6 months of age. However, at 1000 Hz, estimated admittance error is smaller than 226 Hz during the first few months of life and becomes greater with age. Data from the present study demonstrates that, in order to obtain accurate estimates of admittance magnitude, it is necessary to compensate for the effect of ear canal from admittance rectangular components (susceptance and conductance), and then convert the data back to admittance vector for both 226 Hz and 1000 Hz up to at least 6 months of age. However, although it is mathematically more accurate, it is unknown whether component compensation method is diagnostically superior to the baseline compensation method. To date, no sensitivity and specificity data exists to substantiate such a claim. If further research indicates that the mathematical error using the baseline method is diagnostically negligible, then it would be the preferred compensation method due it its simplicity. An overall decrease in Ytm magnitude at 226 Hz probe tone over the period of 6 months was observed from both tails. This trend was observed by Calandruccio et al. (2006) who also investigated positive Ytm derived from rectangular components at 226 Hz. The authors also noted a decrease in admittance between their two youngest age 120  groups (4-10 weeks and 11-19 weeks). No explanation was provided by the authors. Although there is currently no clear explanation of this trend, one possible contribution to decreasing admittance magnitude is increase in resistive elements. In order to further analyze this observation, the following equations were used to calculate resistance (Ra) and reactance (Ba) from susceptance (Ba) and conductance (Ga)  Further analysis of the contribution of susceptance and conductance data suggests that resistance increases with age at 226 Hz from 1.00 mohm to 1.40 mohm. This is opposite to what is found for 1000 Hz probe tone where resistance decreases from 0.70 mohm at V1 to 0.50 mohm at V6. A decrease in resistive components could be attributed to tightening of the ossicular joints and stapes footplate attachment to the oval window during the first few months of life (Saunders, Doant & Cohen, 1983).  Although  resistance overall should be independent of frequency, it seems that the maturational impact on resistance varies differently at 226 Hz and 1000 Hz probe tones. Specifically, resistance at 1000 Hz is affected more than at 226 Hz probe tone. Results of the current study indicate that Ytm magnitude at 1000 Hz probe tone increased over the period of 6 months, with rapid increase between months 3 to 6. Median positive Ytm value at V1 (0.85 mmho) was in good agreement with results obtained by Margolis et al. (2003) of their 3 week old infants (0.80 mmho). The median 121  positive Ytm value derived from rectangular components of infants at V1 to V3 in the present study (1.21 - 1.18 mmho) were greater than those reported by Alaerts et al. (2007) (0.95 mmho) for their <3 month old group. These differences could be due to the larger subject age range used in the Alaerts et al. (2007) study (0-3 months) as well as a smaller sample size (15 ears total). Calandruccio et al. (2006) reported data on positive Ytm derived from rectangular components at 1000 Hz and found no significant difference between their three youngest age groups (4-10, 11-19, and 20-26 weeks) which may be due to the large subject age ranges in their study. Over the 6 month period, the median positive Ytm value obtained from participants of the present study increased by 0.71 mmho, to 1.92 mmho. Our results at V6 are comparable to the mean value reported by Alaerts et al. (2007) of their 3-9 month old group (1.88 mmho) and median value reported by Calandruccio et al. (2006) of their 6-12 month old group (1.93 mmho) despite differences in age range and sample size.  The significant increase in Ytm may be  indicative of rapid growth of the middle ear of young infants during this period. In particular, during the first 6 months after birth, length of the cavity from the tympanic membrane to the stapes footplate increases (Eby & Nadol, 1986) and pneumatization of mastoid air cells occurs (Holte et al., 1991). As admittance increases with the volume of air, the increase in space within the middle ear cavity with age is a likely explanation for the admittance increase.  122  2.3.1.4  Peak Compensated Static Susceptance (Btm) and Conductance (Gtm) In the current study, changes in Btm and Gtm were also analyzed. At V1 and V2,  the infant middle ear at 226 Hz is mass dominated as admittance phase angle is below 0 degrees. At V3, Btm becomes positive indicating that admittance phase angle has shifted above 0 degrees and the middle ear is now stiffness dominated. As the infant continues to grow, Btm and Gtm values become closer together until they are approximately equal at V6, suggesting that phase angle is at 45 degrees. The results demonstrate that between the first and sixth month of life, admittance phase angle increases, implying that the middle ear of the infant is progressing towards a more adult-like stiffness dominated system at 226 Hz. At 1000 Hz, Btm remains constant while Gtm increases with age. Specifically, Gtm remains greater than Btm while Btm hovers around 0 mmho from V1 to V6 suggesting that the infant middle ear, between the ages of 1-6 months, is at or near resonance. Furthermore, the increase in Gtm is indicative that resistive elements are decreasing with age resulting in overall increase in admittance magnitude which is also evident in admittance data. As the infant develops, the middle ear becomes more efficient in sound conduction at 1000 Hz. These observations are also verified by plotting Btm, and Gtm as a function of probe tone across the 6 visits (figure 2.19 and figure 2.20 in results section, respectively). For positive compensation, during the first 2 months, Btm lies below 0 mmho across all frequencies indicating a mass dominated system. As age increases, Btm rises above 0 mmho in the low frequencies and becomes stiffness dominated up to 600 Hz by the 6 months of age. However, compared to adult data, the infant middle ear is not 123  yet adult-like at 6 months of age. The overall increase in admittance at 1000 Hz may be due to a reduction of mass elements in the middle ear system as the infant matures. Several possible anatomical factors may be responsible for such changes in mass. Ruah, Schachern, Zelterma, Paparella, and Yoon (1991) investigated the thickness distribution of the human tympanic membrane at different ages using histological images with the help of both light and electron microscopy. Paparella et al. (1980) also reported that residual amounts of amniotic fluid and mesenchyme can remain attached to the tympanic membrane and ossicular chain for several months after birth which may add mass to the middle ear. However, a study on newborn chinchillas suggests that the middle ear cavity is free of debris and seem mature in structure (Hsu, Margolis and Schacher, 2000). If residual amounts of debris within the middle ear exist in human infants, then with time, these materials are gradually absorbed by the middle ear mucosa.  Additionally, a  decreases in the density of stapes due to internal bone erosion occurs (Anson & Donaldson, 1981).  Consequently, the mass component of the middle ear may  significantly decrease during infancy resulting in an increase in overall admittance for higher probe tone frequencies.  2.3.1.5  Individual Longitudinal Data from 5 Infants The longitudinal data from 5 individual infants (Infants A, B, C, D, and E) are  shown in appendix IV. Most group changes that were observed were also consistently shown in longitudinal changes observed within individual subjects. Similar to group data, individual changes demonstrate that the infant ear canal is increasing in volume with age as shown by increased Ya and Ba values at tympanogram tails. Also, individual 124  data shows a progression from a mass towards a stiffness dominated system at 226 Hz and 1000 Hz as Btm values increase. This is also demonstrated through ΔB data. For ΔG, an increase is observed between 1000- 1500 Hz across age. Similar to group data, Ytm at 226 Hz also decreases with age in the individual data while it increases at 1000 Hz. The pattern of Ya and Ba at 226 Hz varies differently across individuals.  2.3.1.6  Tympanometric Differences Observed Between Infants at Visit 6 to School-aged Children and Adults Significant differences in tympanometric findings were observed between infants  at V6 compared to school-aged children and adults. In particular, the results indicate that the equivalent volumes obtained in adults were significantly greater than those obtained in school-aged children which in turn, were also greater than those obtain in 6 month old infants. This suggests that the ear canal continues to grow even past 6 years of age. The significant increase in admittance at tympanometric tails between infants and adults has also been demonstrated by Shahnaz et al. (2008), Alaerts et al. (2007), and Calandruccio et al. (2006). In order to further investigate the differences between age groups, analysis of Ytm, Btm, and Gtm at 226 Hz and 1000 Hz was conducted. Results indicate that for 226 Hz, both the infant and school-age group had significantly lower Ytm and Btm values compared to the adult group. No differences in Gtm values were observed between age groups. These differences could be attributed to the fact that the mature adult ear has a larger ear canal volume and overall greater middle ear cavity compared to the 2 younger age groups. The increase in Ytm and Btm values demonstrated throughout the 6 infant 125  visits and their continued increase when compared to school-aged children and adults shows that the middle ear is undergoing continuous mechanical and acoustic changes over these time period. In contrast, at 1000 Hz probe tone, both Ytm and Gtm values were significantly higher in school-aged children and adults compared to infants at 6 months of age. However, no differences were observed between school-aged children and adults and no differences in Btm were observed between any age groups. This trend was also observed by Shahnaz et al. (2008) and Calandruccio et al. (2006) between infant and adults age groups. It is hypothesized that the observed increase in Ytm and Gtm could be a result of decrease in resistive elements. Although the infant middle ear seems to be changing towards an adult-like system at both 226 Hz and 1000 Hz it is not yet fully mature at 6 months of age.  2.3.2 Clinical Implications There were several significant tympanometric changes observed in the current study between visits and hence indicative of change beyond normal variability. Consequently, these differences highlight the consideration of narrow age ranges in establishing normative tympanometric data. However, clear age dependant guidelines for infants between birth and 6 months of age are currently not available. One of the goals of the current study was to establish age-related guidelines and normative data to characterize the acoustical properties of the normal ear canal and middle ear in young infants. Although limited in sample size, the following information provides age-related  126  guidelines for clinical practice. Cut-off values represent confidence limits with a 95% confidence interval.  2.3.2.1  Tympanometric Shape Consistent with previous published data, the current study has demonstrated that  tympanometry at 1000 Hz probe tone may be more diagnostically appropriate for clinical use in infants below the age of 6 months as recorded tympanograms were almost exclusively single peaked which makes quantification of tympanogram easier. However, tympanometry at 226 Hz probe tone may be used in conjunction with 1000 Hz probe tone after 3 months of age as occurrences of multi-peaked recordings greatly diminished by this age. In the current study, we also analyzed the shapes of susceptance and conductance tympanograms in infants using the Vanhuyse model. It is clear that tympanometric patterns observed in infants do not conform to classic patterns observed in adults and school-aged children. For the purpose of shape analysis, 678 Hz probe tone is not recommended for infants below 6 months of age as it yielded the greatest variation in tympanometric shapes making subjective analysis difficult. 226 Hz probe tone is also not recommended for the same reason for infants below the age of 3 months. However, between 3 and 6 months of age, normal 226 Hz tympanograms are most likely to be of 1B1G or 3B1G pattern. For 1000 Hz probe tone, most normal tympanograms should also conform to 1B1G or 3B1G pattern for infants below the age of 6 months. Population based studies including sensitivity and specificity data needs to be implemented before  127  the clinical utility of tympanometric shapes as a function of probe tone and age can be substantiated.  2.3.2.2  Equivalent Ear Canal Volume (Vea) The clinical utility of Vea low cut-off values is to separate normal ear canals from  those containing impacted cerumen and space occupying debris as a result of disease. Furthermore, an accurate Vea measure is also useful for diagnostic and amplification measurements. The utility of high cut-off values is used for separation of normal ear canals from those with perforated tympanic membranes. Due to the asymmetry of the admittance tympanogram, Vea measured at the negative tail is smaller than that measured at the positive tail. Studies have shown that measurement at the negative tail is typically a more accurate estimate of Vea in adults (Shanks & Lilly,1981) and in infants (Kei et al., 2007). However, despite the accuracy, measurements of Vea are most commonly taken at the positive tail (+200 daPa) of the 226 Hz tympanogram as this value has demonstrated to have better test-retest reliability than the negative tail value (-400 daPa) in adults (Margolis & Goycoolea, 1993) and infants (Kei et al., 2007). The present study did not attempt to justify that one measurement was better than the other as there is insufficient clinical data to substantiate the diagnostic benefits between the 2 measurements. The following (table 2.13) are recommended age related low and high cut-off values for Vea taken at +200 daPa and -400 daPa in infants between 1-6 months of age.  128  Table 2.14: Recommended Clinical Cut-off Values for Equivalent Ear Canal Volume for Infants between Birth and 6 months of age Equivalent Ear Canal Volume at  Equivalent Ear Canal Volume at -400daPa  +200daPa (mmho)  (mmho)  Age  226 Hz  226 Hz  (months)  Low cut-off  High cut-off  Low cut-off  High cut-off  1  0.4  0.7  0.2  0.4  2  0.4  0.9  0.2  0.3  3  0.5  0.9  0.4  0.5  4  0.5  0.9  0.4  0.6  5  0.5  0.9  0.4  0.6  6  0.5  0.9  0.4  0.6  2.3.2.3  Peak Compensated Static Admittance The clinical utility of low cut-off Ytm values for infants is most commonly used to  separate normal ears from those with diseases that result in reduced middle ear admittance, namely, OME. Although uncommon within the infant population, high cutoff values are useful for separating normal ears from those with diseases that result in increased admittance such as ossicular discontinuity. While there is a growing amount of literature demonstrating that conventional 226 Hz tympanometry is not suitable for diagnostic use within the newborn population due to high false positive and high false negative rates (Margolis et al., 2003; Meyer, Jardine & Deverson, 1997; Paradise, Smith & Bluestone, 1976; Petrak, 2002; Purdy & Williams, 2002; Shahnaz et al., 2008), our results suggest that 226 Hz tympanometry may be useful for infants above 3 months of age. Therefore, both 226 Hz and 1000 Hz Ytm recommendations will be provided. The use of either positive or negative compensation when calculating Ytm in the infant population has been debatable. Margolis et al. (2003) recommended the use of negative 129  compensation (-400 daPa) for deriving Ytm values as they have shown that mean values are greater than when using positive compensation (+200 daPa). A larger mean Ytm value may permit a better separation of abnormal tympanograms from the normal ones as very shallow or flat tympanograms may be identified more easily (Margolis et al., 2003). However, in a recent study of methodology comparison by Kei et al. (2007) on 36 neonates, the use of negative compensation resulted in lower test-retest reliability as determined by a low intra-correlation coefficient of 0.58 and a high standard error of measurement of 0.33 mmho when compared to positive compensation. Despite this, since no sensitivity or specificity measures currently exist, the following recommended cut-off values will include Ytm using both compensation methods. It has been suggested that the component compensation method approach in obtaining Ytm values in infants is mathematically more accurate than the baseline compensation method because it takes into account the phase differences between two vector quantities (Margolis & Hunter, 2000; Calandruccio et al., 2006). In the present study, mean Ytm values obtained from the component compensation method were significantly greater than those using the baseline compensation method. Theoretically, the larger mean admittance values may allow a better separation of abnormal tympanograms from normal ones. Yet, to date, there has been no population based study to substantiate that the component compensation method yields greater diagnostic power over the baseline method. The following recommended cut-off values (tables 2.14, 2.15, 2.16, and 2.17) will include Ytm calculated from both methods. Further evidence, based on sensitivity and specificity measures of the two approaches, is required to justify their use in clinics. 130  Table 2.15: Recommended Clinical Cut-off Values for 226 Hz Positive Peak Compensated Static Admittance for Infants between 3-6 months of age Positive Peak Compensated Static  Positive Peak Compensated Static Admittance  Admittance (Ytm) Calculated from  (Ytm) Calculated from Peak minus tail Method  Component Method (mmho)  (mmho)  Age  226 Hz  226 Hz  (months)  Low cut-off  High cut-off  Low cut-off  High cut-off  3  0.4  1.4  0.4  1.1  4  0.3  1.8  0.3  1.4  5  0.3  0.9  0.2  0.7  6  0.3  1.0  0.2  0.7  Table 2.16: Recommended Clinical Cut-off Values for 226 Hz Negative Peak Compensated Static Admittance for Infants between 3-6 months of Age Negative Peak Compensated Static  Negative Peak Compensated Static Admittance  Admittance (Ytm) Calculated from  (Ytm) Calculated from Peak minus tail Method  Component Method (mmho)  (mmho)  Age  226 Hz  226 Hz  (months)  Low cut-off  High cut-off  Low cut-off  High cut-off  3  0.5  1.6  0.5  1.4  4  0.4  1.6  0.3  1.4  5  0.4  1.1  0.3  1.1  6  0.4  1.2  0.3  1.0  131  Table 2.17: Recommended Clinical Cut-off Values for 1000 Hz Positive Peak Compensated Static Admittance for Infants between Birth and 6 months of age Positive Peak Compensated Static  Positive Peak Compensated Static Admittance  Admittance (Ytm) Calculated from  (Ytm) Calculated from Peak minus tail Method  Component Method (mmho)  (mmho)  Age  1000 Hz  1000 Hz  (months)  Low cut-off  High cut-off  Low cut-off  High cut-off  1  0.2  2.4  0.1  2.1  2  0.2  2.6  0.0  2.2  3  0.3  2.9  0.0  2.2  4  0.3  3.4  0.0  2.5  5  0.5  3.4  0.2  2.5  6  0.5  3.4  0.3  2.3  Table 2.18: Recommended Clinical Cut-off Values for 1000 Hz Negative Peak Compensated Static Admittance for Infants between Birth and 6 months of age Negative Peak Compensated Static  Negative Peak Compensated Static Admittance  Admittance (Ytm) Calculated from  (Ytm) Calculated from Peak minus tail Method  Component Method (mmho)  (mmho)  Age  1000 Hz  1000 Hz  (months)  Low cut-off  High cut-off  Low cut-off  High cut-off  1  0.9  3.8  0.8  3.5  2  0.9  3.1  0.8  3.2  3  1.0  3.5  0.8  3.7  4  0.6  4.3  0.8  4.2  5  1.0  4.3  0.9  4.0  6  0.7  4.8  1.1  4.5  132  2.3.2.4  Peak Compensated Susceptance (Btm) and Conductance (Gtm) Although the clinical utility of Btm and Gtm has been demonstrated for adults and  school-aged children in separating normal from diseased populations (Beers, Shahaz, Kozak & Westerberg, 2009; Funasaka & Kumakawa, 1988; Harris, Hutchinson, and Moravec, 2005; Shahnaz & Polka, 1997; Shahnaz & Polka, 2002; Shahnaz et al., 2009; Zhao et al., 2002) very little is known about their value for infants. However, if the benefits of using component compensation methods in calculation Ytm can be demonstrated in future research, then clinical significance of Btm and Gtm may be substantiated. The following recommended cut-off values are provided (tables 2.18, and 2.19).  Table 2.19: Recommended Clinical Cut-off Values for Compensated Susceptance for Infants between Birth and 6 months of age Positive Compensated Susceptance  Negative Compensated Susceptance  (Btm) (mmho)  (Btm) (mmho)  Age  1000 Hz  1000 Hz  (months)  Low cut-off  High cut-off  Low cut-off  High cut-off  1  -1.1  0.7  -0.1  2.1  2  -1.3  0.9  -0.4  1.8  3  -0.1  1.0  0.2  2.3  4  -1.3  0.8  -0.1  2.0  5  -1.2  1.1  0.3  2.5  6  -1.6  0.9  -0.2  2.3  133  Table 2.20: Recommended Clinical Cut-off Values for Compensated Conductance for Infants between Birth and 6 months of age Positive Compensated Conductance  Positive Compensated Conductance  (Gtm) (mmho)  (Gtm) (mmho)  Age  1000 Hz  1000 Hz  (months)  Low cut-off  High cut-off  Low cut-off  High cut-off  1  0.2  2.2  0.5  2.6  2  0.1  2.5  0.4  3.0  3  0.3  2.8  0.5  3.1  4  0.2  3.1  0.3  4.1  5  0.6  3.6  0.8  3.6  6  0.4  3.3  0.5  4.0  2.3.3 Directions for Future Research As a first step toward understanding tympanograms of infants, we have longitudinally analyzed tympanometric recordings at 226, 678, and 1000 Hz probe tones from birth to 6 month old infants. Although the current study brought forth both new information and added to an existing pool of data, our results raised several unanswered questions. The results of the current study indicate that, in addition to changes in middle ear characteristics, equivalent ear canal volume increased between birth and 6 months of age. As a result, SPL values at the level of the tympanic membrane may be different at various ages as the infant grows. Since accurate SPL measures affect both diagnostic and amplification measures utilizing inserted transducers, it is important to understand how the changes in the external and middle ear of the infant affect SPL values. Bingham, 134  Jenstad, and Shahnaz (2009) investigated SPL values by taking RECD measurements of 15 infants in the current study at V1 and V2 and found no significant changes between the 2 visits. However, the authors did find that the SPL levels measured in infant ears were more variable and significantly higher than the SPL levels present in average adult ears. Hence, it would be important to further investigate at what age range SPL value differences can be observed in the infant and when they become adult-like. Future research should include longitudinal measures of RECD in conjunction with ear canal and middle ear measures in order to better understand how SPL varies with changes in external and middle ear transmission properties. Since only healthy, normal hearing infants were included in the present study, it is not clear if and how pathology of the external and middle ear affects functional maturation between birth to 6 months of age. Hence an area for further research should involve longitudinal investigation of pathological ears including population studies of sensitivity and specificity. Some researchers have found differences in middle ear transmission properties between certain races (Shahnaz & Davies, 2008) and gender (Swanepoel et al., 2007). Shahnaz and Davies (2006) found significant race differences on 4 tympanometric measures between 40 Chinese and Caucasian young adults. Swanepoel et al. (2007) reported significantly higher static admittance recorded from young infant males compared to females. Although these factors were beyond the scope of the current study and were not controlled for explicitly, other studies highlight the possible need for consideration of race and gender in establishing normative tympanometric data in future research. 135  3  References  Abdala, C., & Keefe, D.H. (2006). Effects of middle ear immaturity on distortion product otoacoustic emission suppression tuning in infant ears. Journal of the Acoustical Society of America, 120, 3832-3832.  Alaerts, J., Lutz, H., & Woulters, J. (2007). 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Ear and Hearing, 23(2), 150-8.  159  Appendices  Appendix I: ANOVA tables of Tympanometric Measures for Infants across Visits  Table A1.1: Summary of ANOVA for Infants Admittance at tails at 226 Hz with two within-subject factors (6 levels: number of visits) and (2 levels: pos versus neg tail) Ya at Positive/Negative Tail at 226 kHz from Ya Intercept  SS  DF  MS  F  P  103.2551  1  103.2551  2435.614  0.000000  Errors  1.2294  29  0.0424  Visits  1.1624  5  0.2325  24.005  0.000000  Errors  1.4043  145  0.0097  Tail Pressure  5.2562  1  5.2562  308.284  0.000000  Errors  0.4945  29  0.0171  Interaction Between Visits & Tail Pressure Errors  0.2029  5  0.0406  10.150  0.000000  0.5797  145  0.0040  Table A1.2: Summary of ANOVA for Infants Admittance at tails at 1000 Hz with two within-subject factors (6 levels: number of visits) and (2 levels: pos versus neg tail) Ya at Positive/Negative  SS  DF  MS  F  P  Intercept  1232.729  1  1232.729  2439.974  0.000000  Errors  14.651  29  0.505  Visits  63.239  5  12.648  77.815  0.000000  Errors  23.568  145  0.163  Tails Pressure  74.138  1  74.138  372.149  0.000000  Errors  5.777  29  0.199  0.140  5  0.028  0.592  0.705885  6.863  145  0.047  Tail at 1000 Hz from Ya  Interaction Between Visits & Tail Pressure Errors  160  Table A1.3: Summary of ANOVA for infants Susceptance and Conductance at Positive Tails at 226 Hz with two within-subject factors (6 levels: number of visits) and (2 levels: Rectangular component) Ba/Ga at Positive Tail at  SS  DF  MS  F  P  Intercept  65.35692  1  65.35692  759.9939  0.000000  Errors  2.49390  29  0.08600  Visits  0.13856  5  0.02771  1.2391  0.293846  Errors  3.24277  145  0.02236  Rectangular Component  12.55520  1  12.55520  782.5821  0.000000  Errors  0.46526  29  0.01604  0.48113  5  0.09623  13.1089  0.000000  1.06437  145  0.00734  226 Hz  Interaction Between Visits & Rectangular Component Errors  Table A1.4: Summary of ANOVA for infants Susceptance and Conductance at Negative Tails at 226 Hz with two within-subject factors (6 levels: number of visits) and (2 levels: Rectangular component) Ba/Ga at Negative Tail at  SS  DF  MS  F  P  Intercept  26.10917  1  26.10917  1114.652  0.000000  Errors  0.67928  29  0.02342  Visits  0.56152  5  0.11230  11.199  0.000000  Errors  1.45407  145  0.01003  Rectangular Component  3.42030  1  3.42030  407.863  0.000000  Errors  0.24319  29  0.00839  1.02913  5  0.20583  41.346  0.000000  0.72183  145  0.00498  226 Hz  Interaction Between Visits & Rectangular Component Errors  161  Table A1.5: Summary of ANOVA for infants Susceptance and Conductance at Positive Tails at 1000 Hz with two within-subject factors (6 levels: number of visits) and (2 levels: Rectangular component) Ba/Ga at Positive Tail at  SS  DF  MS  F  P  Intercept  886.9245  1  886.9245  1716.063  0.000000  Errors  14.9883  29  0.5168  Visits  21.4054  5  4.2811  43.806  0.000000  Errors  14.1707  145  0.0977  Rectangular Component  53.5151  1  53.5151  213.742  0.000000  Errors  7.2608  29  0.2504  17.8372  5  3.5674  30.144  0.000000  17.1600  145  0.1183  1000 Hz  Interaction Between Visits & Rectangular Component Errors  Table A1.6: Summary of ANOVA for infants Susceptance and Conductance at Negative Tails at 1000 Hz with two within-subject factors (6 levels: number of visits) and (2 levels: Rectangular component) Ba/Ga at Negative Tail at  SS  DF  MS  F  P  Intercept  320.8467  1  320.8467  1745.855  0.000000  Errors  5.3295  29  0.1838  Visits  29.0024  5  5.8005  68.724  0.000000  Errors  12.2383  145  0.0844  Rectangular Component  0.0041  1  0.0041  0.016  0.900270  Errors  7.5018  29  0.2587  13.1216  5  2.6243  24.945  0.000000  15.2548  145  0.1052  1000 Hz  Interaction Between Visits & Rectangular Component Errors  162  Table A1.7: Summary of ANOVA for Infants Ytm from Ya Tympanograms at 226 Hz with two within-subject factors (6 levels: number of visits) and (2 levels: positive versus negative tail) Ytm from Ya at 226 Hz  SS  DF  MS  F  P  Intercept  196.2490  1  196.2490  912.1229  0.000000  Errors  6.2395  29  0.2152  Visits  11.0216  5  2.2043  12.3431  0.000000  Errors  25.8952  145  0.1786  Compensation Method  5.2563  1  5.2563  308.2845  0.000000  Errors  0.4945  29  0.0171  0.2029  5  0.0406  10.1500  0.000000  0.5797  145  0.0040  Interaction Between Visits & Compensation Errors  Table A1.8: Summary of ANOVA for Infants Ytm from Ya Tympanograms at 1000 Hz with two within-subject factors (6 levels: number of visits) and (2 levels: positive versus negative tail) Ytm from Ya at 1000 Hz  SS  DF  MS  F  P  Intercept  848.8244  1  848.8244  127.2890  0.000000  Errors  193.3859  29  6.6685  Visits  9.9149  5  1.9830  9.1689  0.000000  Errors  31.3594  145  0.2163  Compensation Method  74.1382  1  74.1382  372.1490  0.000000  Errors  5.7773  29  0.1992  0.1402  5  0.0280  0.5923  0.705885  6.8629  145  0.0473  Interaction Between Visits & Compensation Errors  163  Table A1.9: Summary of ANOVA for Infants Ytm from B/G Tympanograms at 226 Hz with two within-subject factors (6 levels: number of visits) and (2 levels: positive versus negative tail) Ytm from Btm/Gtm at  SS  DF  MS  F  P  Intercept  306.1087  1  306.1087  501.7358  0.000000  Errors  17.6929  29  0.6101  Visits  21.4270  5  4.2854  25.7531  0.000000  Errors  24.1285  145  0.1664  Compensation Method  1.7745  1  1.7745  90.2023  0.000000  Errors  0.5705  29  0.0197  0.3007  5  0.0601  2.9300  0.014993  2.9760  145  0.0205  226 Hz  Interaction Between Visits & Compensation Errors  Table A1.10: Summary of ANOVA for Infants Ytm from B/G Tympanograms at 1000 Hz with two within-subject factors (6 levels: number of visits) and (2 levels: positive versus negative tail) Ytm from Btm/Gtm at  SS  DF  MS  F  P  Intercept  1181.829  1  1181.829  139.4442  0.000000  Errors  245.783  29  8.475  Visits  20.598  5  4.120  12.1662  0.000000  Errors  49.099  145  0.339  Compensation Method  20.803  1  20.803  68.5911  0.000000  Errors  8.795  29  0.303  0.793  5  0.159  2.9158  0.015397  7.888  145  0.054  1000 Hz  Interaction Between Visits & Compensation Errors  164  Table A1.11: Summary of ANOVA for Infants Positive Compensated Susceptance and Conductance at 226 Hz with two within-subject factors (6 levels: number of visits) and (2 levels: Rectangular Component) Btm /Gtm at 226 Hz for  SS  DF  MS  F  P  Intercept  58.24178  1  58.24178  505.7695  0.000000  Errors  3.33949  29  0.11515  Visits  3.18381  5  0.63676  7.0783  0.000006  Errors  13.04412  145  0.08996  Rectangular Component  30.53174  1  30.53174  78.3806  0.000000  Errors  11.29643  29  0.38953  30.86319  5  6.17264  67.1964  0.000000  13.31965  145  0.09186  Positive Compensation  Interaction Between Visits & Rectangular Component Errors  Table A1.12: Summary of ANOVA for Infants Negative Compensated Susceptance and Conductance at 226 Hz with two within-subject factors (6 levels: number of visits) and (2 levels: Rectangular Component) Btm /Gtm at 226 Hz for  SS  DF  MS  F  P  Intercept  110.0138  1  110.0138  377.0733  0.000000  Errors  8.4610  29  0.2918  Visits  5.5502  5  1.1100  8.6791  0.000000  Errors  18.5452  145  0.1279  Rectangular Component  15.2317  1  15.2317  38.9743  0.000001  Errors  11.3336  29  0.3908  28.9824  5  5.7965  64.4517  0.000000  13.0406  145  0.0899  Negative Compensation  Interaction Between Visits & Rectangular Component Errors  165  Table A1.13: Summary of ANOVA for Infants Positive Compensated Susceptance and Conductance at 1000 Hz with two within-subject factors (6 levels: number of visits) and (2 levels: Rectangular Component) Btm/Gtm at 1000 Hz for  SS  DF  MS  F  P  Intercept  168.7977  1  168.7977  56.02587  0.000000  Errors  87.3727  29  3.0129  Visits  13.9165  5  2.7833  10.04849  0.000000  Errors  40.1632  145  0.2770  Rectangular Component  190.5759  1  190.5759  86.77245  0.000000  Errors  63.6919  29  2.1963  5.0732  5  1.0146  3.16517  0.009645  46.4818  145  0.3206  Positive Compensation  Interaction Between Visits & Rectangular Component Errors  Table A1.14: Summary of ANOVA for Infants Negative Compensated Susceptance and Conductance at 1000 Hz with two within-subject factors (6 levels: number of visits) and (2 levels: Rectangular Component) Btm/Gtm at 1000 Hz for  SS  DF  MS  F  P  Intercept  618.0842  1  618.0842  163.7685  0.000000  Errors  109.4499  29  3.7741  Visits  7.6414  5  1.5283  5.1696  0.000214  Errors  42.8662  145  0.2956  Rectangular Component  41.2835  1  41.2835  24.1312  0.000032  Errors  49.6129  29  1.7108  4.2229  5  0.8446  2.6418  0.025622  46.3557  145  0.3197  Negative Compensation  Interaction Between Visits & Rectangular Component Errors  166  Table A1.15: Summary of ANOVA for Infants Tympanometric Peak Pressure with two within-subject factors (6 levels: number of visits) and (2 levels: Probe Tone) Tympanometric Peak Pressure (TPP) at 226 Hz  SS  DF  MS  F  P  Intercept  103372.4  1  103372.4  23.62135  0.000041  Errors  122534.4  28  4376.2  Visits  20604.2  5  4120.8  2.14767  0.063295  Errors  268625.0  140  1918.7  Probe Tone  1.4  1  1.4  0.00144  0.969987  Errors  27514.8  28  982.7  5833.5  5  1166.7  1.70274  0.137843  95926.4  140  685.2  & 1 kHz  Interaction Between Visits & Probe Tone Errors  167  Appendix II: Descriptive Statistics and ANOVA Tables for change in Susceptance and Conductance for Infants across Visits  Table A2.1: The 5th, 50th, and 95th percentiles of change in susceptance (ΔB) between 260 Hz -900 Hz across age Change of susceptance (ΔB) in mmho Visit V1 V2 Frequency Median 0.13 0.24 260 th 5 -0.746 -0.6845 th 95 0.415 0.5695 Median -0.27 0.11 300 th 5 -0.939 -0.9785 th 95 0.2985 0.4265 Median -0.37 -0.135 350 th 5 -1.175 -1.3005 th 95 0.081 0.3785 Median -0.535 -0.45 400 th 5 -1.194 -1.353 th 95 -0.252 0.2535 Median -0.73 -0.63 500 th 5 -1.284 -1.292 th 95 -0.343 -0.115 Median -0.64 -0.62 600 th 5 -1.077 -1.245 th 95 -0.306 -0.193 Median -0.54 -0.49 700 th 5 -1.0225 -1.2465 th 95 -0.1815 -0.21 Median -0.39 -0.425 800 th 5 -0.97 -1.186 95th -0.073 0.0065 Median -0.32 -0.35 900 th 5 -1.1435 -1.222 th 95 0.0195 0.2265  V3 0.355 -0.2055 0.5275 0.32 -0.3515 0.5445 0.13 -0.576 0.438 -0.18 -0.823 0.3355 -0.445 -1.003 0.096 -0.55 -1.182 -0.112 -0.775 -1.0935 -0.142 -0.735 -1.1525 0.1455 -0.63 -1.358 0.331  168  V4 0.41 -0.1825 0.5965 0.39 -0.4905 0.5365 0.28 -1.0835 0.543 0.2 -1.231 0.6485 -0.24 -1.242 0.4565 -0.325 -1.1485 0.263 -0.45 -1.0695 0.229 -0.435 -1.0745 0.442 -0.44 -1.333 0.704  V5 0.325 0.0785 0.6325 0.38 -0.002 0.6935 0.35 -0.4875 0.684 0.27 -0.34 0.6815 0.01 -0.7725 0.4235 -0.185 -0.68 0.235 -0.36 -0.6735 0.1815 -0.28 -0.554 0.277 -0.25 -0.6525 0.1715  V6 0.33 0.25 0.6425 0.37 0.14 0.705 0.38 -0.19 0.6675 0.395 -0.6325 0.6925 0.25 -0.7325 0.735 0.045 -0.835 0.565 -0.095 -0.8075 0.4175 -0.055 -0.755 0.5075 -0.385 -1.0775 0.5975  Table A2.2: The 5th, 50th, and 95th percentiles of change in susceptance (ΔB) between 1000 Hz- 2000 Hz across age Change of susceptance (ΔB) in mmho Visit V1 V2 Frequency Median -0.61 -0.39 1000 th 5 -1.391 -1.527 th 95 -0.059 0.362 Median -0.77 -0.71 1100 th 5 -1.647 -1.6715 th 95 -0.095 -0.054 Median -0.98 -0.845 1200 th 5 -2.069 -1.952 th 95 0.043 -0.0385 Median -1.51 -1.215 1300 th 5 -2.2605 -2.7405 th 95 -0.2345 -0.1045 Median -1.83 -1.495 1400 th 5 -2.473 -3.573 th 95 -0.379 -0.007 Median -2.01 -1.76 1500 th 5 -2.8955 -4.3145 95th -0.475 -0.4245 Median -2.09 -2.1 1600 th 5 -3.281 -5.1545 th 95 -0.574 -0.519 Median -2.46 -2.425 1700 th 5 -3.91 -5.346 th 95 -0.616 -0.699 Median -2.86 -2.63 1800 th 5 -4.149 -5.9465 th 95 -1.193 -1.411 Median -2.99 -3.12 1900 th 5 -5.223 -5.7265 th 95 -1.3635 -1.6095 Median -3.35 -3.83 2000 th 5 -5.78 -5.716 th 95 -1.345 -1.4545  V3 -0.67 -1.078 0.25 -0.665 -1.7625 0.1505 -0.73 -1.618 0.304 -0.805 -2.437 0.195 -1.015 -3.175 0.176 -1.375 -4.0175 0.342 -1.705 -4.6365 0.701 -2.06 -5.4645 0.093 -2.3 -5.7835 -0.5775 -2.49 -5.7725 -0.7795 -3.77 -6.8745 -0.746 169  V4 -0.43 -1.604 0.63 -0.59 -1.9155 0.0305 -0.605 -2.124 0.063 -0.875 -2.7375 0.0055 -1.06 -2.926 0.011 -1.555 -3.0735 -0.171 -1.64 -3.615 -0.0015 -2.1 -4.355 -0.546 -2.1 -5.136 0.277 -2.845 -7.3355 -0.4175 -4.335 -8.421 -1.1825  V5 -0.25 -0.8275 0.574 -0.555 -1.814 0.022 -0.525 -1.84 0.2115 -0.795 -3.3135 -0.169 -0.57 -2.6875 -0.1505 -1.47 -4.3405 -0.1795 -1.72 -5.8245 -0.084 -2.29 -6.72 -0.408 -2.485 -7.8245 -0.644 -3.375 -6.817 -0.7255 -3.82 -7.807 -0.478  V6 -0.6 -1.5275 0.5025 -0.91 -2.52 0.62 -0.51 -1.885 0.8 -1.35 -3.13 -0.035 -1.195 -4.0225 -0.24 -1.955 -6.0675 -0.5125 -1.91 -6.885 -0.4225 -2.705 -7.43 -0.8325 -2.53 -8.4625 -0.37575 -3.405 -7.565 -0.8525 -4.575 -8.9075 -0.7725  Table A2.3: The 5th, 50th, and 95th percentiles of change in conductance (ΔG) between 260 Hz- 900 Hz across age Change of Conductance (ΔG) in mmho  Frequency Median 260 5th 95th Median 300 5th 95th Median 350 5th 95th Median 400 5th 95th Median 500 5th 95th Median 600 5th 95th Median 700 5th 95th Median 800 5th 95th Median 900 5th 95th  Visit V1 0.97 0.601 1.327 1.025 0.5625 1.3955 0.845 0.342 1.1185 0.745 0.3445 1.065 0.535 0.153 0.6985 0.25 -0.124 0.534 0.29 -0.068 0.494 0.27 -0.163 0.556 0.32 0 0.852  V2 0.9 0.41 1.7515 1.065 0.544 1.611 1.02 0.68 1.568 0.935 0.5725 1.344 0.7 0.192 1.0895 0.565 -0.0325 0.8995 0.5 -0.1355 0.8385 0.405 -0.253 0.763 0.525 -0.162 1.188  V3 0.595 0.38 1.1705 0.81 0.4385 1.411 0.94 0.568 1.5005 1.005 0.7185 1.3705 0.965 0.396 1.182 0.78 0.14 1.0335 0.54 0.009 1.013 0.425 -0.0945 1.0925 0.41 -0.3565 1.4865  170  V4 0.445 0.1935 1.871 0.57 0.22 1.7195 0.71 0.257 1.442 0.82 0.35 1.452 0.835 0.3375 1.12 0.77 -0.043 1.073 0.74 -0.149 1.26 0.7 -0.094 1.2655 0.67 -0.0095 1.6955  V5 0.41 0.119 1.1825 0.505 0.0725 1.287 0.705 0.1765 1.1815 0.77 0.2175 1.164 0.875 0.21 1.321 0.845 0.055 1.3345 0.9 -0.033 1.411 0.85 -0.011 1.481 0.84 0.0675 2.0415  V6 0.31 0.135 0.945 0.39 0.1825 0.9875 0.51 0.22 1.1525 0.685 0.2725 1.225 0.795 0.28 1.3425 0.78 0.21 1.49 0.97 0.0525 1.5975 0.99 0.14 1.715 1.15 0.205 2.365  Table A2.4: The 5th, 50th, and 95th percentiles of change in conductance (ΔG) between 1000 Hz-2000 Hz across age Change of Conductance (ΔG) in mmho  Frequency Median 1000 5th 95th Median 1100 5th 95th Median 1200 5th 95th Median 1300 5th 95th Median 1400 5th 95th Median 1500 5th 95th Median 1600 5th 95th Median 1700 5th 95th Median 1800 5th 95th Median 1900 5th 95th Median 2000 5th 95th  Visit V1 0.37 0.003 1.447 0.39 -0.061 1.406 0.49 -0.154 1.517 0.83 -0.3715 2.3395 0.88 -0.312 2.2465 1.055 -0.2165 1.745 0.39 -0.3024 2.17355 0.39 -0.3110 2.19635 0.43 -1.396 2.307 0.12 -4.8955 2.262 -0.22 -6.881 2.177  V2 0.75 -0.1965 1.701 0.675 -0.25 2.0695 0.91 -0.266 2.6015 0.605 -0.2265 1.4965 0.44 -0.466 1.276 0.41 -0.467 1.811 0.35 -0.961 1.797 0.455 -1.112 1.687 0.03 -1.713 1.842 -0.04 -1.618 1.462 -0.92 -3.044 2.153  V3 0.48 -0.1725 2.13 0.47 -0.428 2.513 0.7 -0.2915 3.235 0.83 -0.616 2.8825 0.93 -0.613 2.896 0.88 -0.7605 2.4545 1.1 -0.534 2.467 0.965 -0.6485 1.9415 0.88 -0.4795 2.5115 0.49 -1.037 1.9355 0.165 -2.4155 2.414  171  V4 0.855 -0.0365 2.2895 0.85 -0.2195 3.0155 0.85 -0.363 3.432 1.185 -0.377 3.3345 0.985 -0.208 3.099 0.97 -0.389 3.27 1.12 -0.2405 3.4935 0.975 -1.2085 2.791 1.28 -1.7245 3.4305 1.005 -2.8405 2.563 1.07 -5.074 2.918  V5 0.96 0.283 1.9955 1.045 0.2825 3.214 1.205 0.347 3.9695 1.475 0.0645 4.0995 1.21 0.1615 4.5935 1.04 0.1115 3.627 1.64 0.1465 3.95 1.344 -0.1135 3.261 1.84 -2.002 3.579 1.325 -3.119 2.8995 1.855 -5.735 3.4735  V6 1.275 0.3075 3.1425 1.39 0.375 3.5225 1.32 0.43 4.1925 1.545 0.445 3.7675 1.43 0.43 2.58 1.43 0.085 2.6125 1.66 0.1375 3.0525 1.02 -1.255 2.6775 1.295 -1.62 3.5275 0.76 -3.935 3.1275 1.05 -6.92 4.6  Appendix III: ANOVA tables of Tympanometric Measures for Infants at Visit 6 Compared to School-aged Children and Adults  Table A3.1: Summary of ANOVA for Infants at V6/School-aged Children/Adults Positive Ytm from Ya tympanograms at 226 Hz with two within-subject factors (3 levels: age groups) and (1 level: Ytm) Positive Peak Compensated SS  Static Admittance (Ytm) at  DF  MS  F  P  226 Hz Intercept  54.16853 1  54.16853 949.1957 0.000000  Age  2.12207  1.06103  Errors  17.34862 304  2  18.5925  0.000000  0.05707  Table A3.2: Summary of ANOVA for Infants at V6/School-aged Children/Adults Admittance at positive tail from Ya tympanograms at 226 Hz with two within-subject factors (3 levels: age groups) and (1 level: Ytm) Admittance at +200 (Y+) at 226 Hz Intercept  SS 184.089 4  DF  1  MS  F  184.089  3829.15  4  2 167.031  Age  16.0602  2  8.0301  Errors  14.7593  307  0.0481  172  P  0.00 0.00  Table A3.3: Summary of ANOVA for Infants at V6/School-aged Children/Adults Positive Peak Compensated Static Admittance/Susceptance/Conductance at 226 Hz with two within-subject factors (3 levels: age groups) and (3 levels: Rectangular Components and Ytm) Ytm/ Btm/Gtm at 226 Hz for Positive  SS  DF  MS  F  P  Compensation Intercept  147.4797 1  147.4797  972.4247 0.000000  Age  3.2181  2  1.6090  10.6095  Errors  47.0152  310  0.1517  COMPONENT  7.9953  2  3.9976  397.0953 0.000000  COMPONENT* AGE  0.9384  4  0.2346  23.3023  Errors  6.2417  620  0.0101  0.000035  0.000000  Table A3.4: Summary of ANOVA for Infants at V6/School-aged Children/Adults Positive Peak Compensated Static Admittance/Susceptance/Conductance at 1000 Hz with two within-subject factors (3 levels: age groups) and (3 levels: Rectangular Components and Ytm) Ytm/ Btm/Gtm at 1000 Hz for Positive  SS  DF  MS  F  P  Compensation Intercept  1503.891 1  1503.891  562.4714 0.000000  Age  17.360  2  8.680  3.2463  Errors  828.853  310  2.674  COMPONENT  1155.035 2  577.517  302.7333 0.000000  COMPONENT* AGE  34.303  8.576  4.4954  Errors  1182.760 620  4  173  1.908  0.040243  0.001378  Appendix IV: Individual Data from 5 Subjects  Figure A1.1: Overview of Positive Tail and Positive Peak Compensated Admittance and Susceptance Longitudinal Data for Subject A Admittance (Ya) at Positive Tail 226 Hz  Positive Peak Compensated Static Admittance (Ytm) 226 Hz  1  1.00  0.5 0  0.00 1  2  3  4  5  6  1  2  3  4  5  6  Positive Peak Compensated Static Susceptance (Btm) 226 Hz  Susceptance (Ba) at Positive Tail 226 Hz 1 1 0.5 0  0  Magnitude (mmho)  1  2  3  4  5  6  1  2  3  4  5  6  -1  Admittance (Ya) at Positive Tail 1000 Hz  Positive Peak Compensated Static Admittance (Ytm) 1000 Hz  4  4.00 2  2.00  0  0.00 1  2  3  4  5  6  1  2  3  4  5  6  Positive Peak Compensated Static Susceptance (Btm) 1000 Hz  Susceptance (Ba) at Positive Tail 1000 Hz 4 3 2 1 0  1 0 -1 1  2  3  4  5  6  -2  Visits (1-6) 174  1  2  3  4  5  6  Figure A1.2: Variation of Susceptance (ΔB) and Conductance (ΔG) as a Function of Probe Tone Frequency for Subject A  Variation of Conductance (ΔG) as a Function of Probe Tone Frequency  4 Visit 1  2  Visit 2 Visit 3 Visit 4 Visit 5 Visit 6  -4 -6  2000  1500  1000  -2  500  0 250  Compensated Conductance (mmho)  6  Frequency (Hz)  175  Figure A1.3: Overview of Positive Tail and Positive Peak Compensated Admittance and Susceptance Longitudinal Data for Subject B Admittance (Ya) at Positive Tail 226 Hz  Positive Peak Compensated Static Admittance (Ytm) 226 Hz  1  1.50 1.00  0.5  0.50 0  0.00 1  2  3  4  5  6  1  Susceptance (Ba) at Positive Tail 226 Hz  Magnitude (mmho)  2  3  4  5  3  4  5  6  Positive Peak Compensated Static Susceptance (Btm) 226 Hz  0.8 0.6 0.4 0.2 0 1  2  1 0.5 0 -0.5 -1  6  Admittance (Ya) at Positive Tail 1000 Hz  1  2  3  4  5  6  Positive Peak Compensated Static Admittance (Ytm) 1000 Hz  4 3 2 1 0  2.00 1.00 0.00 1  2  3  4  5  6  1  Susceptance (Ba) at Positive Tail 1000 Hz 0.5 0 -0.5 -1 -1.5 -2  2 1 0 2  3  4  5  3  4  5  6  Positive Peak Compensated Static Susceptance (Btm) 1000 Hz  3  1  2  6  Visits (1-6) 176  1  2  3  4  5  6  Figure A1.4: Variation of Susceptance (ΔB) and Conductance (ΔG) as a Function of Probe Tone Frequency for Subject B Variation of Susceptance (ΔB) as a Function of Probe Tone Frequency 0.5 2000  1500  1000  -0.5  500  0  250  Compensated Susceptance (mmho)  1  Visit 1  -1  Visit 2  -1.5  Visit 3  -2  Visit 4  -2.5  Visit 5  -3  Visit 6  -3.5 -4  Frequency (Hz)  Variation of Conductance (ΔG) as a Function of Probe Tone Frequency  1.5 1  Visit 1 Visit 2  0.5  Visit 3 0 2000  1500  1000  500  -0.5  Visit 4  250  Compensated Conductance (mmho)  2  Visit 5 Visit 6  -1 -1.5  Frequency (Hz)  177  Figure A1.5: Overview of Positive Tail and Positive Peak Compensated Admittance and Susceptance Longitudinal Data for Subject C Admittance (Ya) at Positive Tail 226 Hz  Positive Peak Compensated Static Admittance (Ytm) 226 Hz  1.1  1.50  0.9  1.00  0.7  0.50  0.5  0.00 1  2  3  4  5  6  1  3  4  5  6  Positive Peak Compensated Static Susceptance (Btm) 226 Hz  Susceptance (Ba) at Positive Tail 226 Hz  Magnitude (mmho)  2  0.85 0.8 0.75 0.7 0.65  1 0.5 0 1  -0.5 1  2  3  4  5  6  2  3  4  5  6  -1  Admittance (Ya) at Positive Tail 1000 Hz  Positive Peak Compensated Static Admittance (Ytm) 1000 Hz  4 3 2 1 0  4.00 2.00 0.00 1  2  3  4  5  6  1  Susceptance (Ba) at Positive Tail 1000 Hz  2  3  4  5  6  Positive Peak Compensated Static Susceptance (Btm) 1000 Hz  4 3 2 1 0  2 0 -2 1  2  3  4  5  6  -4  Visits (1-6) 178  1  2  3  4  5  6  Figure A1.6: Variation of Susceptance (ΔB) and Conductance (ΔG) as a Function of Probe Tone Frequency for Subject C Variation of Susceptance (ΔB) as a Function of Probe Tone Frequency  2000  1500  1000  -2  500  0 250  Compensated Susceptance (mmho)  2  Visit 1 Visit 2  -4  Visit 3 -6  Visit 4  -8  Visit 5 Visit 6  -10 -12  Frequency (Hz)  Variation of Conductance (ΔG) as a Function of Probe Tone Frequency 6 4 Visit 1  2  Visit 2  -4  Visit 3 Visit 4 Visit 5  -6  Visit 6  -8 -10  2000  1500  1000  -2  500  0 250  Compensated Conductance (mmho)  8  Frequency (Hz)  179  Figure A1.7: Overview of Positive Tail and Positive Peak Compensated Admittance and Susceptance Longitudinal Data for Subject D Admittance (Ya) at Positive Tail 226 Hz  Positive Peak Compensated Static Admittance (Ytm) 226 Hz  1.5  0.80 0.60 0.40 0.20 0.00  1 0.5 0 1  2  3  4  5  6  1  3  4  5  6  Positive Peak Compensated Static Susceptance (Btm) 226 Hz  Susceptance (Ba) at Positive Tail 226 Hz 0.8 0.6 0.4 0.2 0  Magnitude (mmho)  2  1 0 1  -1 1  2  3  4  5  6  2  3  4  5  6  -2  Admittance (Ya) at Positive Tail 1000 Hz  Positive Peak Compensated Static Admittance (Ytm) 1000 Hz  4 3 2 1 0  1.50 1.00 0.50 0.00 1  2  3  4  5  1  6  2  3  4  5  6  Positive Peak Compensated Static Susceptance (Btm) 1000 Hz  Susceptance (Ba) at Positive Tail 1000 Hz 4 3 2 1 0  1 0 -1 1  2  3  4  5  6  -2  Visits (1-6) 180  1  2  3  4  5  6  Figure A1.8: Variation of Susceptance (ΔB) and Conductance (ΔG) as a Function of Probe Tone Frequency for Subject D Variation of Susceptance (ΔB) as a Function of Probe Tone Frequency  2000  1500  1000  -1  500  0 250  Compensated Susceptance (mmho)  1  Visit 1  -2  Visit 2  -3  Visit 3 Visit 4  -4  Visit 5 -5  Visit 6  -6 -7  Frequency (Hz)  Variation of Conductance (ΔG) as a Function of Probe Tone Frequency  4 3  Visit 1 Visit 2  2  Visit 3 1  Visit 4 Visit 5  -2  Frequency (Hz)  181  2000  1500  1000  -1  500  0 250  Compensated Conductance (mmho)  5  Visit 6  Figure A1.9: Overview of Positive Tail and Positive Peak Compensated Admittance and Susceptance Longitudinal Data for Subject E Admittance (Ya) at Positive Tail 226 Hz  Positive Peak Compensated Static Admittance (Ytm) 226 Hz  0.8 0.6 0.4 1  2  3  4  5  6  0.2  0.80 0.60 0.40 0.20 0.00 1  0  Susceptance (Ba) at Positive Tail 226 Hz 0.6 0.55 0.5  Magnitude (mmho)  0.45 2  3  4  5  3  4  5  6  Positive Peak Compensated Static Susceptance (Btm) 226 Hz  0.65  1  2  6  0.6 0.4 0.2 0 -0.2 -0.4  Admittance (Ya) at Positive Tail 1000 Hz  1  2  3  4  5  6  Positive Peak Compensated Static Admittance (Ytm) 1000 Hz  3  1.00  2 1  0.50  0  0.00 1  2  3  4  5  6  -0.50  Susceptance (Ba) at Positive Tail 1000 Hz  1  2  3  4  5  6  Positive Peak Compensated Static Susceptance (Btm) 1000 Hz  3  1  2 1  0.5  0  0 1  2  3  4  5  6  -0.5  Visits (1-6) 182  1  2  3  4  5  6  Figure A2.0: Variation of Susceptance (ΔB) and Conductance (ΔG) as a Function of Probe Tone Frequency for Subject E  2 1  2000  1500  500  -1  1000  0 250  Compensated Susceptance (mmho)  Variation of Susceptance (ΔB) as a Function of Probe Tone Frequency  -2 -3 -4 -5  Frequency (Hz)  Variation of Conductance (ΔG) as a Function of Probe Tone Frequency  4 3  Visit 1 Visit 2  2  Visit 3  -1 -2  Frequency (Hz)  183  2000  Visit 5 1500  0 1000  Visit 4  500  1  250  Compensated Conductance (mmho)  5  Visit 6  

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