Effects of Music Based Intervention (MBI) on Pain Response and Neurodevelopment in Preterm Infants
Phase: NA
Conditions: Preterm Birth, Pain
Sponsor: University of Minnesota

FULL PROTOCOL TITLE 
Effects of Music Based Intervention (MBI) on Pain Response and 
Neurodevelopment in Preterm Infants 
Principal Investigator 
Sonya Wang, MD 
Associate Professor of Neurology and Pediatrics 
University of Minnesota, School of Medicine 
Supported by:  
The National Center for Complementary and Integrative Health  
1 R61 AT010712-01  
 
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Tool Revision History 
Version Number: 2.0b 
Version Date: November 4, 2019 
Summary of Revisions Made:  
Page 11 (Bayley’s III assessment) - Apologies. The statement about Bayley’s III developmental 
assessment was not supposed to be included in R61 study protocol. It is part of the R33 protocol. I am 
deleting it from this section. 
Page 14 (how will they maintain distance of headphones relative to baby’s ears) - In this age group, these 
preterms have very little motor capability to move much – even while awake. In addition, during each 
session of intervention, study staff will be monitoring for infant discomfort. Study staff will also monitor 
to ensure the 1cm distance from the preterms’ ears. 
Page 15 (hearing loss) - Hearing testing is not typically assessed in preterm babies, but rather at term age. 
However, hearing loss in preterm babies typically occurs in those who are medically unstable 
(hyperbilirubinemia, sepsis, or meningitis – approximately 2-4% of the population). That population of 
preterm infants do not meet eligibility criteria for this trial. Hearing will also be confirmed during ERP 
testing. 
Page 22 (blinded statistician) - Yes. This statistician is only aware of the blinded treatment and is thus 
blinded also for the study analysis.  
Page 27 & 28 (why is PIPP considered the primary outcome but not associated with primary study 
objective 1) - This R61 is exploratory with little prior research in all aspects, and thus we had challenges 
regarding sample size/ power considerations. Our main goal is aimed towards meeting Go/NoGo Criteria 
– each with equal weight in mind. Certainly, the order in which all the Go/NoGo criteria is presented can 
be switched around with primary outcome PIPP being Primary objective. With minimal prior research, we 
decided on PIPP to be the primary outcome for statistical calculations. In this exploratory work there is 
not adequate power to assess hypothesis 1 and 2.  
I have put together a version 2.0b study protocol response to these edits with the objectives moved 
around. Version 2b shows PIPP primary outcome to be associated with primary objective. 
Page 28 (eligibility) - Yes. The eligible subjects at the University of Minnesota Neonatal Intensive Care 
Unit meeting inclusion and exclusion criteria. 
Page 29 (missing data) – please see added section 9.6 Missing Data. 
Page 29-30 (clarification about sleep duration measure) - Milestone threshold is reached if any 1 or more 
of the serial EEGs is reached. This threshold was purposely chosen as brain maturation occurs at different 
stages. This exploratory investigation is important to understand whether there might be critical time 
periods during prematurity in which the music intervention may have more impact. If the other time 
points show opposite directions, these may be time periods in which music may be less effective. Because 
no literature is available to describe this, it is important for us to gather this information. 
Page 30-31 (clarification about PIPP/central EEG amplitudes) – similar answers to the above sleep 
measure regarding importance of reaching thresholds at different time points of prematurity. 
 
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Version Number: 2.0c 
Version Date: February 7, 2020 
Updated inclusion criteria to 30 weeks +/- 2 weeks 
Version Number: 3.0 
Version Date: June 18, 2020 
Page 13: PIPP performed during heel lance procedure expanded to include both Minnesota Newborn 
Screen or required heel lance blood tests 
The PIPP is performed when the preterm infant undergoes the heel lance procedure for either the 
Minnesota Newborn Screen or other required blood tests performed during the same approximate time 
period within the scheduled protocol timeline. The PIPP should not pose any additional risk to the subject 
as the heel lance for newborn screening or required blood tests are both standard of care. 
The heel lance procedure for the Minnesota Newborn Screen is the same as the heel lance procedure 
required for other blood tests. Due to new COVID restrictions and limited access to the NICU for 
research purposes, the opportunity to perform the PIPP has been expanded to include other required blood 
tests that utilize the same heel lance procedure performed at approximately the same time period within 
the scheduled protocol timeline. These heel lances are all part of required standard of care and does not 
significantly change the study protocol. 
Page 15 Diagram: 
Changed label to Standard Heel Lance Procedure 
Instead of Newborn Screen Heel Lance 
Page 19: Visit 5/Completion Visit: 
1)
Added Medical History Review to gather information if there are any ongoing medical issues that 
might affect ERP results 
2)
Added music exposure form to gather information on how much music is played at home after 
NICU discharge 
Version Number 4.0 
Version Date: August 31, 2020 
Page 5, 15, 17, 18, 25: COVID efforts: 
Given the need to decrease face to face interactions for COVID precautions, we are slightly decreasing 
the number of music intervention to an average of 4-5 (instead of 5-6) sessions per week. This is still 
 
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Conﬁrm eligibility, obtain consent, enroll 
Standard Heel Lance Procedure
Preterms born at 30 weeks (+/- 2 weeks)

frequent enough music intervention to show changes in pain responses and neurodevelopmental findings 
since the intervention lasts a target of 6 weeks. 
Page 5: …average 4-5 intervention or control sessions per week… 
Page 15: MBI subjects will receive …intervention averaged at 4-5 sessions per week. 
Page 17: …music intervention 4-5 sessions per week... 
Page 18: Adherence …as 80% compliance …average of 4-5 intervention or control sessions per week 
Page 25: Targeted averaged 4-5 sessions of MBI or Control will be played per week. 
Page 16 and 18: Change 6 to 10 playlists: 
To randomize songs properly, our biostatistician created 10 playlists, instead of the 6 originally planned. 
Page 16: stimulation, habituation, and neurological development, we will use ten playlists. 
Page 18: As it is recommended to change song order to increase stimulation, habituation, and neurological 
development, we will use 10 playlists. 
Page 16: “decibel” to “volume”: 
The mp3 player/headphone equipment decibel levels will be checked prior to use. Because the mp3 
players only display volume levels, this word was changed from “decibel” to “volume”. 
Page 16: Daily electronic volume level checks will avoid the need for listening checks of the headphones. 
Version Number 5.0 
Version Date: March 16, 2021 
Pg 14: Clarification that for reliability in scoring, the PIPP will be 
 done by 2 study team members. The 2nd PIPP can be done in-person or virtually. 
Pg 21 and 23 (sec 6.1 and 6.2.2 clarification that HADS is optional for the parent to complete) 
Pg 24 (sec 6.2.4): clarification that EEG will have a video component  
Pg 25: Hearing status of the child will be confirmed through chart review of the child’s clinical BAER 
hearing test. This is done clinically prior to discharge (and/or at 1 month clinical care follow-up). 
Pg 36 (sec 11.3) clarification the confidentiality of the video EEG 
Version Number 6.0 
Version Date: August 11, 2021 
Updated protocol to reflect that ERPs will be conducted between 43-48 weeks corrected gestational age. 
See pgs 10,11,15,16, 26, 30. No impact on analysis. This allows for better compliance with allowing for 
scheduling families with a new born child. 
 
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Version Number 7.0 
Version Date: February 1, 2022 
Pg 20: Updated Section 6.1 Schedule of Evaluations – Screening and Study Visits  to remove the 48 hour 
time period for baseline/visit 1 activities to occur. This change is administrative only as it has no impact 
on study aims, analysis or safety.   
Version Number 8.0 
Version Date: March 8, 2022 
Pg 20-21: Updated Section 6.1 Schedule of Evaluations & Pg 25: Section 6.2.4 Follow up Visits: Visits 
2-5 to move Family Feedback form completion at Visit 4, noting though that this may be completed at 
any time prior to discharge.  
 
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TABLE OF CONTENTS 
Page 
FULL PROTOCOL TITLE ............................................................................................
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1 R61 AT010712-01 ......................................................................................................
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Tool Revision History ....................................................................................................
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TABLE OF CONTENTS ................................................................................................
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STUDY TEAM ROSTER .............................................................................................
 
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PARTICIPATING STUDY SITES .................................................................................
 
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PRÉCIS ..........................................................................................................................
 
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1. STUDY OBJECTIVES ...............................................................................................
 
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1.1 Primary Objective (1) ..............................................................................................
 
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1.2 Primary Objective (2) ..............................................................................................
 
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2. BACKGROUND AND RATIONALE .......................................................................
 
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2.1 Background – Primary Objective (1) .......................................................................
 
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2.1a Study Rationale – Primary Objective (1) ...............................................................
 
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2.2 Background – Primary Objective (2) .......................................................................
 
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2.2a Study Rationale – Primary Objective (2) ...............................................................
 
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3. STUDY DESIGN .........................................................................................................
 
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4. SELECTION AND ENROLLMENT OF PARTICIPANTS ....................................
 
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4.1 Inclusion Criteria .....................................................................................................
 
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Preterm infant born at 30 weeks (+/- 2 weeks) ..............................................................
 
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Medically stable .............................................................................................................
 
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4.2 Exclusion Criteria ....................................................................................................
 
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Treatment for major organ system disease ....................................................................
 
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Significant neurological disorder including, but not limited to, abnormal 
neurological examination, neonatal abstinence syndrome, intraventricular 
hemorrhage, seizures, meningitis, or congenital brain malformations ..........................
 
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Scalp lesions affecting EEG placement .........................................................................
 
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4.3 Study Enrollment Procedures ..................................................................................
 
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5. STUDY INTERVENTIONS .......................................................................................
 
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5.1 Interventions, Administration, and Duration ...........................................................
 
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5.2 Handling of Study Interventions ..............................................................................
 
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5.3 Concomitant Interventions ......................................................................................
 
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5.4 Adherence Assessment  Adherence is defined as 80% compliance in this study 
– average of 4-5 intervention or control sessions per week. If adherence falls below 
80%, the Internal QA Reviewer will discuss with the PI methods for improving 
adherence. ......................................................................................................................
 
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6. STUDY PROCEDURES .............................................................................................
 
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6.1 Schedule of Evaluations – Screening and Study Visits ...........................................
 
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6.2 Description of Evaluations ......................................................................................
 
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6.2.1 Screening Evaluation ........................................................................................
 
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6.2.2 Enrollment, Baseline, and/or Randomization ...................................................
 
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6.2.3 Blinding ............................................................................................................
 
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6.2.4 Followup Visits: Visits 2-5 (See above table for timing of visits) ....................
 
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7. SAFETY ASSESSMENTS ..........................................................................................
 
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7.1 Specification of Safety Parameters ..........................................................................
 
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7.2 Methods and Timing for Assessing, Recording, and Analyzing Safety 
Parameters .....................................................................................................................
 
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7.3 Adverse Events and Serious Adverse Events ..........................................................
 
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7.4 Reporting Procedures ..............................................................................................
 
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7.5 Follow-up for Adverse Events .................................................................................
 
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7.6 Safety Monitoring ....................................................................................................
 
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8. INTERVENTION DISCONTINUATION .................................................................
 
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9. STATISTICAL CONSIDERATIONS ........................................................................
 
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9.1 General Design Issues .............................................................................................
 
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9.2 Sample Size and Randomization .............................................................................
 
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Treatment Assignment Procedures ............................................................................
 
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9.3  Definition of Populations ........................................................................................
 
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9.4 Interim Analyses and Stopping Rules ......................................................................
 
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9.5 Outcomes: Milestone Measures and Data Analyses ................................................
 
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10. DATA COLLECTION AND QUALITY ASSURANCE ........................................
 
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10.1 Data Collection Forms ...........................................................................................
 
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10.2 Data Management ..................................................................................................
 
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10.3 Quality Assurance ..................................................................................................
 
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10.3.1 Training ...........................................................................................................
 
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10.3.2 Quality Control Committee ............................................................................
 
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10.3.3 Metrics ............................................................................................................
 
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10.3.4 Protocol Deviations ........................................................................................
 
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10.3.5 Monitoring ......................................................................................................
 
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11. PARTICIPANT RIGHTS AND CONFIDENTIALITY .........................................
 
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11.1 Institutional Review Board (IRB) Review ............................................................
 
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11.2 Informed Consent Forms .......................................................................................
 
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11.3 Participant Confidentiality .....................................................................................
 
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11.4 Study Discontinuation ...........................................................................................
 
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12. COMMITTEES .........................................................................................................
 
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I. Procedures Schedule 
II. Informed Consent Form Template 
III. Other (add as many appendices as necessary) 
13. PUBLICATION OF RESEARCH FINDINGS .......................................................
 
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14. REFERENCES ..........................................................................................................
 
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15. SUPPLEMENTS/APPENDICES .............................................................................
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STUDY TEAM ROSTER 
1)
Pediatric Neurology (Sonya Wang) – Sonya Wang is the principal investigator 
and will oversee the entire project.  
420 Delaware Street S.E.   
Department of Neurology; MMC 295 
University of Minnesota 
Minneapolis, MN 55455 
Office: 612-301-1454 
Fax: 612-301-1455 
email: sgwang@umn.edu  
2)
Music Therapy (Michael Silverman) - Michael Silverman will be responsible for 
the creation of the recorded lullaby variations and will ensure safety of 
headphones, MP3 players with routine quality control checks on music and 
decibel sound level.  
School of Music 
Room 100 FergH 
7811A (Campus Delivery Code) 
2106 4th St S 
Minneapolis, MN 55455 
Email: silvermj@umn.edu 
3)
Neonatology (Raghavendra Rao) - Raghavendra Rao is a practicing 
neonatologist, and core faculty of the Center for Neurobehavioral Development. 
He will assist in recruitment, data collection, consent, and music based 
intervention administration.  
Pediatric Neonatology 
East Building, MB630 
2450 Riverside Ave 
Minneapolis, MN 55455 
Office: 612-626-0644 
Fax: 612-624-8176 
Email: raghurao@umn.edu 
4)
Biomedical engineering (Theoden Netoff) - Theoden Netoff will supervise EEG 
data analysis and oversee a biomedical engineering graduate student who will 
utilize machine learning tools to identify EEG sleep biomarkers for future 
automatization of EEG interpretation and identification of EEG-sleep wake cycle 
features.  
BiomedicalEng
ineering 
Room 7-105 
NHH 
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Email: tnetoff@umn.edu 
5)
Statistics (Lynn Eberly, Qi Wang) - Lynn Eberly will provide oversight for 
biostatistical support. Qi Wang will provide biostatistical analysis support for this 
project.  
Biostatistics 
A465 Mayo 
420 Delaware St SE 
Minneapolis, MN 55455 
Email: leberly@umn.edu 
6)
Study Coordinators (TBD) – Study coordinators will oversee practical details of 
the project, including coordinating recruitment, implementation of intervention, 
organizing details of data collection, data movement, data storage, data security, 
and data access; data preparation for publications and project reporting.  
Contact info TBD 
PARTICIPATING STUDY SITES 
Sonya Wang, MD 
University of Minnesota (Department of Neurology) 
420 Delaware Street S.E.   
Department of Neurology; MMC 295 
University of Minnesota 
Minneapolis, MN 55455 
Office: 612-301-1454 
Fax: 612-301-1455 
email: sgwang@umn.edu  
PRÉCIS  
Study Title  
Effects of Music Based Intervention (MBI) on Pain Response and 
Neurodevelopment in Preterm Infants  
Objectives  
Primary Objective (1): 
Characterize differences in preterm pain responses between MBI and controls  
Primary Objective (2):  
Office Phone:
612-625-3618
Office 
Phone
+1 612-624-1436 
+1 612-626-0295
 
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Identify differences between MBI and controls in preterm brain maturation and 
early neurodevelopment 
Design and Outcomes   
Study design:  Pilot prospective randomized, double blinded, controlled study 
to test effect of music based intervention (MBI) on pain response and 
neurodevelopment in preterm infants. 
Aim 1 (R61): Characterize differences in preterm pain responses between 
MBI and controls. 
The objective of this aim is to understand the behavioral processes of MBI on 
pain in preterm infants by comparing the PIPP and EEG pain responses in 
the MBI and control cohorts. 
Aim 2 (R61): Identify differences between MBI and controls in preterm brain 
maturation and early neurodevelopment. 
The objective of this aim is to explore biological mechanisms of MBI on 
preterm brain maturation and neurodevelopment using 
electroencephalography (EEG) and event related potentials (ERPs). 
Interventions and Duration  
Specific recorded lullabies will be played for 6 weeks (+/- 1 week). Additional 
follow-up will occur at 43-48 weeks corrected age (~ one month old after the 
original due date). 
Sample Size and Population  
Recruit 60 medically stable preterm infants born at approximately 30 weeks to 
participate in this study. Randomization will be stratified by sex at birth and 
within each stratum use randomly allocated block sizes of 2 and 4. 
1. STUDY OBJECTIVES 
1.1 
Primary Objective (1) 
Aim 1(R61): Characterize differences in preterm pain responses 
between MBI and controls.  
Premature infant pain profiles (PIPP) include physiologic, behavioral, and 
contextual measures which identifies differences in pain responses between 
MBI and controls while still in the neonatal intensive care unit (NICU).  Central 
EEG amplitude changes have been time-locked with painful procedures in 
term infants. We will explore if PIPP scores and central EEG amplitude 
changes are attenuated with MBI in comparison to controls.   
• Hypothesis 1: MBI will show improved pain responses, with lower PIPP 
 
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scores and attenuated central EEG amplitude changes during painful 
procedures, in comparison to the control cohort.  
1.2 
Primary Objective (2) 
 
 
 
Aim 2(R61): Identify differences between MBI and controls in preterm 
brain maturation and early neurodevelopment. 
EEG is a surrogate marker for real time brain function during sleep-wake 
cycles. Because preterm brain networks develop during sleep, sleep duration 
is a strong indicator of brain maturation. Serial biweekly EEGs of preterm 
infants can quantify sleep duration trends and track MBI’s influence on sleep. 
To enhance objectivity, innovative EEG machine-learning tools will be applied 
to the analyses.  
• Hypothesis 2: MBI will enhance preterm EEG brain maturation in 
comparison to controls.  
Due to the natural limitations of evaluating immature neonatal nervous 
systems, ERPs have been utilized to study early neurodevelopment.  ERPs 
quantify electrical brain potentials changes time-locked with a stimulus. 
Auditory ERPs performed at 1 month (43-48 weeks) corrected age evaluates 
attention and discrimination between familiar and novel stimuli - early 
neurodevelopmental signs of recognition memory function and perceptual 
learning.  
• Hypothesis 3: ERPs at 1 month (43-48 weeks) corrected age will show that 
MBI has a greater impact on early neurodevelopment when compared to 
controls. 
   
2. BACKGROUND AND RATIONALE 
2.1 
Background – Primary Objective (1) 
Preterm infants experience up to 14 painful procedures per day during their 
first 2 weeks of life26 and neonatal pain experiences prime adult pain 
responses.27 In rats, painful events in early life can increase the number or 
glucocorticoid receptors in the hippocampus which may affect future stress 
responses.28 Incision of the rat paw in early life has been associated with a 
greater magnitude and duration of hyperalgesia in adulthood.29 Neonatal 
injury has also been associated with increased intensity, spatial distribution 
and duration of ionized calcium-binding adaptor molecule-1-positive microglial 
reactivity in the dorsal horn of the spine (a sensory region of the spine).29 
Anatomical differences in neuronal organization of the sensory active 
subplate zone have been found in preterms, indicating underlying 
developmental changes due to painful procedures.30 Furthermore, greater 
 
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neonatal procedural pain has been associated with reduced white matter and 
subcortical gray matter volumes by term gestation31 and thinner cortices in the 
frontal and parietal lobes by age seven.32  
To control for pain, opioids (including morphine and fentanyl), sedatives 
(benzodiazepines), and sucrose have been widely used in the NICU.33,34 
However, higher cumulative fentanyl doses in preterm infants correlate with a 
higher incidence of cerebellar injury and lower cerebellar diameter at term 
age.35 Midazolam, a benzodiazepine sedative, exposure in preterm infants 
correlates with abnormal hippocampal growth and altered 
neurodevelopmental outcomes in cognitive, language, and motor abilities.36 
Repeated exposure to sucrose for procedural pain in mouse pups reveal 
smaller white matter volumes in the corpus callosum, stria terminalis, and 
fimbria, as well as smaller cortical and subcortical grey matter volumes in 
hippocampus and cerebellum.37  
Music has been presumed to be effective in treating pain by provoking 
feelings of familiarity and security.19 Soothing music redirects attention from 
pain inducers, allows for release of brain endorphins, and reduces stress 
hormones.38 Music has also been shown to relax muscle tone and release 
body tension.39 Prior music intervention studies on pain have shown overall 
positive immediate effects on heart rate, facial expressions, latency to cry, 
and lower pain scores.40,41 However, similar to the MBI studies described 
previously, weaknesses in prior music research on pain in preterm infants 
consist of inconsistent study approaches with heterogenous study groups, 
small sample sizes, and varied interventions with mixed outcomes.20,40,42–48  
2.1a Study Rationale – Primary Objective (1) 
Thus, we propose a novel approach to study immediate and lasting effects of 
MBI on pain responses in the preterm infant utilizing the standardized 
Premature Infant Pain Profile (PIPP) and EEG (Aim 1). With frequent music 
intervention throughout the NICU hospitalization (6 weeks +/- 1 week), pain 
responses are likely to adapt and change over time. PIPP and EEG are 
objective, quantifiable outcomes measures that when performed serially will 
provide pain response trends revealing the cumulative effects of MBI on pain 
responses throughout the NICU stay. Evaluation of MBI in preterm infants will 
provide a deeper understanding of music’s influence on behavioral processes 
of the pain response and offer a low risk treatment alternative to the current 
pain treatment regimen of opioids, benzodiazepines, and sucrose that are 
detrimental to neurodevelopment. 
2.2 
Background – Primary Objective (2) 
In 2018, the World Health Organization reported 15 million (>1 in 10) preterm 
births with rising annual rates. While over 90% of those born at ∼30 weeks 
gestation survive, approximately 50% of these infants suffer from 
neurodevelopmental impairments that span motor, behavior and academic 
 
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performance.1 By 18 months, preterm infants have lower language skills (15 
points) on IQ tests compared to term infants.2 The most active period of 
neurodevelopment occurs from conception to 2 years (“first 1000 days”) 
where the effects of environmental exposures may permanently affect 
functioning throughout life.3,4 Healthy neurodevelopment progresses by a 
complex scaffolding process whereby intricate neural circuits rely on 
successful completion of previous stages of development.4 In particular, the 
3rd trimester of gestation is a critical period of rapid brain growth.5–9 Although 
neurodevelopment continues throughout life, by age 2, the brain has 
undertaken significant restructuring and many developmental changes cannot 
occur beyond this sensitive growth period.4  
Nevertheless, music has the remarkable ability to enhance child 
neurodevelopment. Auditory listening has been found in 27 week fetuses.10 
Fetal auditory learning discriminates low-pitch sound features such as rhythm 
of music and prosodic features of speech11 and fetuses perceive high-pitched 
sounds in a similar manner that adults recognize speech sounds.12 Infants 
exposed to music prenatally have been shown to recognize familiar melodies 
at 4 months old suggesting that early music exposure can form lasting neural 
representations in the brain.13 Preterm music listening has been found to 
increase immediate sleep duration, enhance physiologic functioning (heart 
rate and oxygen saturation levels), improve behavioural states and weight 
gain while decreasing hospital stays and initial weight loss.14,15 Early music 
training is associated with enhanced neural speech processing such as pitch 
changes in speech sounds16 syllable duration17 and improved verbal memory 
skills.18 Music-based interventions (MBIs) for infants are low-risk19, minimally-
invasive, and low-cost,20 and thus, an ideal intervention to trial in the neonatal 
intensive care unit (NICU). The NICU is a necessary but artificial environment 
created for the survival of preterm infants. It is imperative to carefully select 
their exposures in this unique environment which overlaps with the timing of 
the third trimester of pregnancy, a sensitive growth period in which we can 
have a great impact on immediate and future neurodevelopment.  
2.2a Study Rationale – Primary Objective (2) 
Thus, we propose a novel approach to study the effects of a longer duration 
of repeated MBI (6 weeks) by utilizing objective and reproducible measures of 
electroencephalography (EEG) and event related potentials (ERPs). EEG 
captures electrical potential oscillations of the brain which yields valuable 
information about brain function.
25 EEG trends over time reveal brain 
maturation in preterm infants. Utilizing EEG is a novel approach to studying 
the biological mechanisms of MBI effects on preterm infants as EEG is 
objective, captures real-time brain activity, and tracks overall changes in 
neurodevelopment (Aim 2). Furthermore, ERPs test recognition memory 
function and cognitive processing of mother vs stranger’s voice and offers 
 
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another objective measure to further study the early effects of the MBI’s 
biological mechanisms on neurodevelopment of these infants (Aim 2).  
Thus, we propose a novel approach to study immediate and lasting effects of 
MBI on pain responses in the preterm infant utilizing EEG and the 
standardized Premature Infant Pain Profile (PIPP) (Aim 2). With frequent 
music intervention throughout the NICU hospitalization (6 weeks), pain 
responses are likely to adapt and change over time. EEG and PIPP are 
objective, quantifiable outcomes measures that when performed serially will 
provide pain response trends revealing the cumulative effects of MBI on pain 
responses throughout the NICU stay (second clinical primary outcome 
measure). Evaluation of MBI in preterm infants will provide a deeper 
understanding of music’s influence on behavioral processes of the pain 
response and offer a low risk treatment alternative to the current pain 
treatment regimen of opioids, benzodiazepines, and sucrose that are 
detrimental to neurodevelopment. 
Successful completion of studying MBI effects on preterm neurodevelopment 
using rigorous objective measures of EEG and ERPs provides a deeper 
understanding of the biological mechanisms underlying music intervention in 
neurodevelopment and could result in a newly established standard of 
medical care in the NICU to utilize music intervention in the optimization of 
preterm neurodevelopment. Evaluation of MBI in preterm pain provides a 
deeper understanding of music’s influence on behavioural processes of pain 
responses which potentially supports music as a low risk, minimally invasive 
treatment alternative to the current regimen of opioids, benzodiazepines, and 
sucrose that have detrimental side effects. Outcomes of this R61 would 
provide foundational work on music and preterm health which can inform 
future MBI research in optimizing and tailoring different music based 
interventions to specific patient populations. 
Potential Risks  
The planned research procedures include: 1) EEG/ERP, 2) PIPP, and 3) 
Music based intervention or control. These procedures place participating 
infants at no more than minimal risk.   
EEG/ERP  
Risks associated with the EEG and ERP are minimal. The EEG electrode 
placement via EEG caps could cause minor skin irritation or discomfort. 
Routine skin care, involving gentle cleaning, will be maintained to prevent 
these side effects. The ERP data will be gathered using Geodesic Sensor net, 
which may cause similar mild irritation. The EEG/ERPs will utilize EEG caps 
and Sensor nets rather than traditional electrodes which further minimizes the 
risk.  
 
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The PIPP is an observational tool (pain profile) that doesn’t require interaction 
with the subject. The PIPP is performed when the preterm infant undergoes 
the heel lance procedure for either the Minnesota Newborn Screen or other 
required blood tests performed during the same approximate time period 
within the scheduled protocol timeline. The PIPP should not pose any 
additional risk to the subject as the heel lance for newborn screening or 
required blood tests are both standard of care. The PIPP will be performed by 
two study team members concurrently, with the 2nd person either in-person or 
virtually through a HIPAA compliant method. This is in order to ensure 
reliability of data collection. The investigator(s) and study statistician will 
review any discrepancies for statistical analysis.  
Music Based Intervention (MBI)  
MBI has been shown to be minimal risk. The MBI in this trial (lullabies) will be 
played for the infants using headphones placed 1 cm away from the ears to 
avoid discomfort. During music vs control intervention, a study staff personnel 
will be actively monitoring the procedure to ensure that cords from the 
headphones do not interfere with standard of care. The MP3 player has 
bluetooth capability and should not interfere with routine care. Noise levels 
are calibrated before every session. Music amplitude will be at no more than 
70 dB, which has been shown to be a safe noise level. NICU and study staff 
will observe for signs of distress during the music therapy, such as crying or 
finger splay. If these signs continue for more than five minutes, the music 
therapy will be stopped for that day’s session.   
Potential risks regarding confidentiality.   
There is a potential risk for breach of confidentiality, which will be minimized 
by the procedures described in the following section.   
3. STUDY DESIGN 
Pilot randomized, controlled, double blinded music-based intervention (MBI) 
trial of preterm newborns born at 30 weeks (+/- 2 week) in Neonatal Intensive 
Care Unit (NICU) 
We propose a R61 project to explore the behavioral processes underlying 
effects of MBI on pain using the premature infant pain profile (PIPP) and 
EEG. In preterm infants, PIPP scores scale pain responses with painful 
procedures and central EEG amplitudes change when time-locked to a 
painful stimulus. The R61 will also explore biological mechanisms of music 
based intervention (MBI) on preterm brain maturation and neurodevelopment 
using electroencephalography (EEG) and event related potentials (ERPs). 
EEG captures electrical potential oscillations of the brain which yields 
valuable information about brain function. Serial EEGs can track brain 
maturation in preterm infants.  ERPs quantify electrical brain potentials 
changes time-locked with a stimulus. ERPs at 43-48 corrected gestational 
 
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age test recognition memory function and cognitive processing and offers 
another objective measure to study the early effects of the MBI’s on 
neurodevelopment. Specific recorded lullabies with simple arpeggiated 
accompaniment will be played for 6 weeks (+/- 1 week) in a small 
randomized, blinded, controlled study of 60 recruited medically stable 30 
week preterms.  
Primary Objective - Aim 1: Characterize differences in preterm pain 
responses between MBI and controls. 
Hypothesis/Outcomes:  
• 
Hypothesis 1: MBI will show improved pain responses, with lower PIPP 
scores and attenuated central EEG amplitude changes during painful 
procedures, in comparison to the control cohort. 
 Primary Objective – Aim 2: Identify differences between MBI and controls in 
preterm brain maturation and early neurodevelopment.  
Hypothesis/Outcomes:  
• 
Hypothesis 2: MBI cohort will show enhanced preterm EEG maturation 
when compared to controls. 
 
Hypothesis 3: ERPs at 1 month (43-48 weeks) corrected age will show 
that MBI has a greater impact on early neurodevelopment when compared to 
controls.   
 
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1cm
MP3
 
MBI subjects will receive a total of 1.5 hours of music 
intervention averaged at 4-5 sessions per week (Figure 2). 
Music will be alternating: 30 minutes on and 30 minutes off 
and will be played when the subject is awake to cue 
pacification and initiate the sleep process. Voice 
unaccompanied or with single accompanying softly 
arpeggiated instrument66 is best for infants. Lullabies meet 
these criteria and promote language development due to an 
emphasis on vowels, rising and falling melodic phrases, and 
the recognition of soothing sounds. Lullabies will all have 
consistent tempos, with the melody in higher range sung by 
female or children as infants hear these pitches best.66 As it 
is recommended to change song order to increase 
stimulation, habituation, and neurological development, we 
will use ten playlists. MP3 players will be loaded in advance 
with the entire 6 weeks 
of music. Within each week, order of the playlists will be randomly assigned and 
prepared in advance by Dr. Eberly. MBI will have minimal interference with routine 
medical care. Study coordinators will monitor for any distress from music: finger splay, 
grimace, vital sign disturbance, or persistent crying.67 If crying lasts longer than 5 
minutes, the intervention will be stopped for that day’s session. Blinding will be 
maintained by using identical headphones and MP3 players for all subjects. 
Headphones will be placed on each side of infant’s head such that sound stimuli are 
received binaurally.66 Music amplitude will be no more than 70 dB on Scale C.15,66,68 
Screen Daily NICU 
admissions
Screen Maternal records  
Neonatal records
32 weeks (+/1 week) 
Standard Heel Lance   Procedure
32 weeks (+/1 week) 
Standard Heel Lance Procedure 
43-48 weeks corrected gestational age 
Event Related Potentials
EEG, PIPP
Conﬁrm eligibility, obtain consent, enroll 
Standard Heel Lance Procedure
Preterms born at 30 weeks (+/- 2 weeks)
Music Therapy 
(30)
Control 
(30)
34 weeks (+/1 week) 
Standard Heel Lance Procedure
34 weeks (+/1 week) 
Standard Heel Lance Procedure
EEG, PIPP
EEG, PIPP
36 weeks (+/1 week)
36 weeks (+/1 week) 
EEG
Figure 1: 
Study Flow Chart
 
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EEG
Figure 2: Music Intervention

Daily electronic volume level checks will avoid the need for listening checks of the 
headphones. Headphones will be placed 1 cm away from the ears to avoid discomfort 
from headphone compression. The headphones will either play music or silence for the 
control group. 
 
Figure 4: Every two weeks, 30 hour EEG* recordings will capture two sessions of background 
frequencies and changes that occur with Session 1) MBI or Control intervention or Session 2) [MBI + 
pain] or [Control + pain]  
 
4. SELECTION AND ENROLLMENT OF PARTICIPANTS  
4.1 
Inclusion Criteria 
Participants must meet all of the inclusion criteria to participate in this study:   
Preterm infant born at 30 weeks (+/- 2 weeks)  
Medically stable  
4.2 
Exclusion Criteria 
All candidates meeting any of the exclusion criteria at baseline will be 
excluded from study participation: 
 hours EEG* recording
30
 
Session 1 (1.5 hrs)
 
Session 2 (1.5 hrs)
 
MBI or Control
 
MBI or Control
 
+ Pain 
)
(Heel Lance 
Figure 3: 
 hour EEG* records two 
30
intervention sessions 
 
1)
MBI or Control 
 
2)
MBI or Control 
Pain
+
 (Heel Lance) 
  
 
 
 
 
EEG 
 Hrs*
30
 
30 weeks 
/
+ -2weeks 
32 weeks 
+/-1week 
34 weeks 
/
+ -1week 
36 weeks 
/
+ - week
1
 
 
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Treatment for major organ system disease  
Significant neurological disorder including, but not limited to, abnormal 
neurological examination, neonatal abstinence syndrome, intraventricular 
hemorrhage, seizures, meningitis, or congenital brain malformations  
Scalp lesions affecting EEG placement  
4.3 
Study Enrollment Procedures  
Newborns will be recruited from the University of Minnesota Masonic 
Children’s Hospital neonatal intensive care unit (NICU). We will ensure staff in 
the NICU are aware of the study and post flyers in the NICU to alert 
neonatology staff to the study’s purpose, inclusion criteria, and the pager 
number needed to contact the study staff regarding potential subjects. The 
neonatologist will first mention the study to the parents, then ask whether the 
parents would be willing to further discuss the study with the study 
investigators. Only parents who express interest in learning about the 
research study will be approached regarding enrollment of their infant.   
The PI and other selected study investigators listed on the IRB will obtain 
consent. All study investigators who will be obtaining consent will receive 
training on how to determine eligibility of subjects and how to discuss the 
study with parents. The study investigator who obtains consent will ensure the 
parents understand the informed consent process and the requirements of 
the study. Parents will be informed that they can ask questions about the 
study at any time and contact information for the study staff will be provided 
on the consent form. 
Once eligibility has been confirmed and consent obtained, subjects will be 
randomly assigned to the MBI or control treatment cohorts in a 50:50 
allocation ratio using randomization tables, stratified by sex and within each 
stratum use randomly allocated block sizes of 2 and 4, prepared by Dr. Eberly.  
5. STUDY INTERVENTIONS  
5.1 
Interventions, Administration, and Duration  
Specific recorded lullabies with simple arpeggiated accompaniment will be 
played for 6 weeks (+/- 1 week) in a small randomized, blinded, controlled 
study of 60 recruited medically stable 30 week preterms. 
MBI subjects will receive a total of 1.5 hours of music intervention 4-5 
sessions per week. Music will be alternating: 30 minutes on and 30 minutes 
off and will be played when the subject is awake to cue pacification and 
initiate the sleep process. Voice unaccompanied or with single accompanying 
 
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softly arpeggiated instrument is best for infants. Lullabies meet these criteria 
and promote language development due to an emphasis on vowels, rising 
and falling melodic phrases, and the recognition of soothing sounds. Lullabies 
will all have consistent tempos, with the melody in higher range sung by 
female or children as infants hear these pitches best. As it is recommended to 
change song order to increase stimulation, habituation, and neurological 
development, we will use 10 playlists. MP3 players will be loaded in advance 
with the entire 6 weeks of music. Within each week, order of the playlists will 
be randomly assigned and prepared in advance by Dr. Eberly. MBI will have 
minimal interference with routine medical care.  
5.2 
Handling of Study Interventions  
N/A 
5.3 
Concomitant Interventions  
N/A 
5.4 
Adherence Assessment  
Adherence is defined as 80% compliance in this study – average of 4-5 
intervention or control sessions per week. If adherence falls below 80%, the 
Internal QA Reviewer will discuss with the PI methods for improving 
adherence. 
6. STUDY PROCEDURES  
 
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6.1 
Schedule of Evaluations – Screening and Study Visits 
*optional element (parent must provide specific consent to complete this 
assessment) 
** Family Feedback may be completed at any point before discharge from the 
hospital 
6.1a Schedule of Evaluations – Music Based Intervention or Control Sessions 
Assessment
Screen
ing 
Baseline/
Visit 1
2 weeks  
(+/- 3 days)  
Visit 2
4 
weeks 
(+/- 3 
days) 
Visit 3
6 weeks  
(+/- 3 
days) 
Visit 4
43-48 weeks 
corrected 
gestational age 
Visit 5/ 
Completion
Informed Consent 
Form
x
Randomization
x
Demographics
x
Medical History
x
x
Physical Exam
x
Vital Signs
x
x
x
x
Baseline Lab Values
x
Inclusions/Exclusion 
Criteria
x
Concomitant Meds
x
x
x
x
x
HADS*
x
Music Exposure
x
x
Baseline Checklist
x
On Study Visit 
Checklist
x
x
x
x
x
EEG
x
x
x
x
PIPP
x
x
x
ERP
x
Adverse Events
x
x
x
x
x
SAE
x
x
x
x
x
Family Feedback**
x**
Study Completion
x
 
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Assessment
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Vital Signs
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
MBI Info Form
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Assessment
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Vital Signs
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
MBI Info Form
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
 
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6.2 
Description of Evaluations  
6.2.1 Screening Evaluation 
Infants’ medical histories will be screened to determine if infant is medically 
stable (no major organ system illnesses or sepsis) with no abnormal  
neurological exams, neonatal abstinence syndrome, intraventricular 
hemorrhage, seizures, meningitis, congenital  brain malformations, or scalp 
lesions affecting the EEG application.     
Consenting Procedure 
The PI and other selected study investigators listed on the IRB will obtain a 
single informed consent that describes both the screening and study 
procedures. All study investigators who will be obtaining consent will receive 
training on how to determine eligibility of subjects and how to discuss the 
study with parents. The study investigator who obtains consent will ensure the 
parents understand the informed consent process and the requirements of 
the study.  
The patient recruitment site will be the University of Minnesota Masonic 
Children’s Hospital neonatal intensive care unit (NICU). Infants born at 30 
weeks (+/- 2 weeks) who meet both inclusion and exclusion criteria will be 
identified by NICU staff. The neonatologist will first mention the study to the 
parent(s)/guardian(s), then ask whether they would be willing to further 
discuss the study with the study investigators. Study investigators include: 
(PI) Sonya Wang, (Music Therapist) Michael Silverman, Raghavendra Rao, 
and the two study coordinators. The PI, Sonya Wang, will oversee the 
consent process and make the final determination of the eligibility. Only 
parents/guardians who consent to hearing about the research study will be 
approached regarding enrollment of their infant. At the time of recruitment, all 
procedures, risks, benefits, and the option to withdraw from the study at any 
time will be explained to the parent(s)/guardian(s) of each participant and 
written, informed consent will be obtained. The voluntary nature of the study 
will be emphasized. The consent form will provide a description of the overall 
purpose of the research, the specific details of the protocol, risks and 
benefits, costs and payments, confidentiality, contact information for the PI, 
the posting of information on ClinicalTrials.gov, and the ability to withdraw 
from participation. At least one parent/guardian will sign the consent form on 
behalf of their infant. Assent will not be obtained because participants are not 
capable of providing it given their very young age (preterm newborn infants). 
A copy of the signed consent form will be kept in the study research files and 
medical records. The parent(s)/guardian(s) of participants will also be given a 
copy of their signed consent form. Parents will be informed that they can ask 
questions about the study at any time and contact information for the study 
staff will be provided on the consent form.  
  
 
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Process for meeting HHS regulatory requirements for parental permission 
and child assent  
This study meets the requirements for 45 CFR 46.404 – Research not 
involving greater than minimal risk. Therefore, according to 45 CFR 46.408, 
the permission of one parent or guardian is sufficient for research to be 
conducted, pending IRB approval. 
Screening 
Both male and female potential subjects will be recruited from the patient 
population admitted to the University of Minnesota Masonic Children’s 
Hospital NICU.  
•
Gestational age will be determined by mother’s last menstrual period, 
fetal ultrasound measurements, and physical examination 
characteristics with a variability of +/- 14 days of the targeted 30 week 
gestational birth age for all subjects. 
•
Eligible infants must be medically stable (no major organ system 
illnesses or sepsis) with no abnormal neurological exams, neonatal 
abstinence syndrome, intraventricular hemorrhage, seizures, 
meningitis, congenital brain malformations, or scalp lesions affecting 
the EEG application. 
6.2.2 Enrollment, Baseline, and/or Randomization 
Enrollment 
All infants for whom a parent or legal guardian has signed consent will be 
considered to be enrolled in the study. 
Baseline Assessments/ Visit 1 
For participants who have successfully been screened for eligibility and are 
enrolled into the study, baseline assessments are performed. These include 
the following: 
•
Demographics 
•
Medical History 
•
Physical Exam 
•
Vital Signs 
•
Baseline Lab Values 
•
Concomitant Meds 
•
HADS (Hospital Anxiety and Depression Scale) for Moms  
o
*optional element (mom must provide specific consent to 
complete this assessment) 
•
Music Exposure during Pregnancy 
 
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•
EEG 
•
PIPP 
•
Adverse Events 
•
SAE 
Randomization 
Once eligibility has been confirmed and consent obtained, subjects will be 
randomly assigned to the MBI or control treatment cohorts in a 50:50 
allocation ratio using randomization tables, stratified by sex, prepared by Dr. 
Eberly. Randomization will occur immediately after screening. 
6.2.3 Blinding 
Blinding will be maintained by using identical headphones and MP3 players 
for all subjects. The PI, any personnel involved in collecting the clinical 
outcomes (e.g., PIPP) or collecting or quantifying EEG outcomes, and the 
statistician will be blinded. The statistician will be aware of blinded treatment 
group, so that e.g. safety summaries by blinded treatment group can be 
prepared for the Data Safety and Monitoring Board (DSMB) closed sessions if 
it so requests. Should the DSMB request unblinded comparisons, the study 
team will contract with the University of Minnesota CTSI’s Biostatistical 
Design and Analysis Center (BDAC) for an independent statistician to work 
with the DSMB using unblinded data. 
The unblinded staff includes Michael Silverman (music therapist) and Erin 
Osterholm (Director of the NICU). Silverman will prepare the music players. 
Both will be unblinded to handle randomization, delivery of interventions, and 
preparations of unblinded reports to the independent study monitors. They will 
not be involved with data monitoring, analyses, or outcome assessments.  
The study coordinators who are assessing the PIPP will wear earplugs and 
noise cancelling headphones, which will ensure that s/he cannot hear what is 
emanating from the infant’s headphones. In addition, family members and 
NICU staff will be reminded frequently to not disclose what they think they 
hear coming from the infant’s headphones. 
6.2.4 Followup Visits: Visits 2-5 (See above table for timing of visits) 
EEG: provides real time information about cerebral function by measuring 
cerebral electrical activity recorded from electrodes placed on the scalp. 
This EEG includes a video component that will be recording the child during 
the EEG, in order to assess if any movement was occurring during the EEG. 
PIPP: The Premature Infant Pain Profile is a 7-item multidimensional measure 
of pain that includes 3 behavioral (brow bulge, eye squeeze, nasolabial 
furrow), 2 physiological (heart rate, oxygen saturation), and 2 contextual 
 
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(gestational age and behavioral state) measurements. The measurements 
are scaled on a 4 point scale (0,1,2,3) with increasing change of each 
variable from baseline. The 7 items are summed for a total pain intensity 
score. 
Visit 2 and 3: 
•
Vital Signs 
•
Concomitant Meds 
•
On Study Visit Checklist 
•
EEG 
•
PIPP 
•
Adverse Events 
•
SAE 
Visit 4: 
•
Vital Signs 
•
Concomitant Meds 
•
On Study Visit Checklist 
•
EEG 
•
Adverse Events 
•
SAE 
•
Family Feedback** may be completed at visit 4 or any time before 
discharge from the hospital 
Prior to discharge the study team will review the child’s medical chart to 
collect the clinical BAER score. The BAER stands for the 
Brainstem auditory evoked response (BAER) test which evaluates a child’s 
hearing and is done as part of clinical care. If the child does not pass while 
hospitalized, the clinical care team may conduct a follow-up BAER test at 
their clinical care follow-up appointment. For a child that has a follow-up 
BAER test, the study team will review the medical chart and collect this 
information to ensure that hearing is confirmed prior to the child’s study ERP, 
as part of Visit 5/Completion Visit. If the child does not pass this 2nd hearing 
test, the PI may withdraw the family from the ERP portion of the study visit.    
Visit 5/Completion Visit: 
 
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•
Concomitant Meds 
•
Medical History 
•
Music Exposure 
•
On Study Visit Checklist 
•
Adverse Events 
•
SAE 
•
Study Completion 
•
ERP:  Event related potentials quantify changes in electrical brain 
potentials timelocked in milliseconds with a stimulus and they are 
widely used to study cognitive abilities such as discrimination, 
attention, and memory. 
Event related potential (ERP) testing will occur on follow-up at 43-48 weeks 
corrected gestational age in all subjects to evaluate early neurodevelopment. 
6.2.5 Music Based Intervention or Control Sessions 
Targeted averaged 4-5 sessions of MBI or Control will be played per week. 
With each session of intervention, evaluations of vital signs and MBI 
information will be obtained. Target 6 weeks of MBI or control (+/- 1 week). 
•
Vital Signs 
•
MBI Information 
7. SAFETY ASSESSMENTS  
7.1 
Specification of Safety Parameters 
Study coordinators will monitor for any distress from music: finger splay, 
grimace, vital sign disturbance, or persistent crying.  If crying lasts longer 
than 5 minutes, the intervention will be stopped for that session. 
7.2 
Methods and Timing for Assessing, Recording, and Analyzing Safety 
Parameters 
 
 
NA 
7.3 
Adverse Events and Serious Adverse Events  
An adverse event (AE) is generally defined as any unfavorable and 
unintended diagnosis, symptom, sign (including an abnormal laboratory 
finding), syndrome or disease which either occurs during the study, having 
been absent at baseline, or if present at baseline, appears to worsen. 
Adverse events are to be recording regardless of their relationship to the 
 
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study intervention.   
A serious adverse event (SAE) is generally defined as any untoward 
medical occurrence that results in death, is life threatening, requires inpatient 
hospitalization or prolongation of existing hospitalization, results in persistent 
or significant disability/incapacity, or is a congenital anomaly. 
The main adverse effects that may occur during the study are: 
Distress resulting from music based intervention and mild skin irritation 
or discomfort from EEG/ERP electrodes. NICU and research staff will be 
instructed to observe for signs of distress lasting more than five minutes 
during music based intervention that do not appear to be associated with 
other co-occurring interventions or conditions. Signs of distress will include 
finger splay, grimace, vital sign disturbance, or persistent crying. If these signs 
of distress last longer than 5 minutes, music based intervention will be 
stopped for that session and this event will be reported to the PI. Routine skin 
monitoring and cares will be performed by NICU staff and research 
coordinators while EEG leads are in place. Care will be taken when placing 
the brain net for EEG or 128-channel Geodesic Sensor net for ERP to avoid 
discomfort.  
7.4 
Reporting Procedures 
A core group from the research team consisting of the PI, co-investigators, 
and statisticians will be responsible for ongoing monitoring of the trial. They 
will also be responsible for reporting any issues regarding the safety of the 
study or threats to data integrity to the IRB.   
All adverse events will be reported to the PI immediately or within 24 hours. 
The PI will report adverse events that meet University of Minnesota IRB 
reporting requirements to the IRB per IRB guidelines.  We do not foresee any 
serious adverse events from participation in this minimal-risk music-based 
intervention study. For each adverse event (serious or non-serious), the 
investigator will provide the onset, duration, intensity, treatment, action taken, 
and outcome. The investigator will determine the relationship of the adverse 
event to the study-related procedures. Study coordinators will enter safety 
monitoring and adverse event data into the electronic database. Eligibility 
verification and data completion will be monitored by the research coordinator 
and PI. 
7.5 
Follow-up for Adverse Events 
The PI will record all reportable events with start dates occurring any time 
after informed consent is obtained until 7 (for non-serious AEs) or 30 days (for 
SAEs) after the last day of study participation.  At each EEG/ERP study visit, 
the investigator will inquire about the occurrence of AE/SAEs since the last 
visit.  Events will be followed for outcome information until resolution or 
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7.6 
Safety Monitoring  
This research study involves no greater than minimal risk to participants. All 
participants will be monitored by the Principal Investigator (PI) and study 
coordinators.  
A Data Safety Monitoring Board (DSMB) has been convened for this study. It 
is an independent group of experts charged with reviewing study data for 
subject safety, study conduct and progress, and providing formal 
recommendations regarding study continuation, modification, and termination.  
The University of Minnesota CTSI Independent Monitor(s) will review study 
materials (documents, records, drug/device accountability, Case Report 
Forms, etc.) to assure that the study is conducted, recorded, and reported in 
compliance with FDA Good Clinical Practice. 
8. INTERVENTION DISCONTINUATION  
Safety findings that would prompt temporary suspension of enrollment and/or 
study interventions until a complete safety review is convened (either routine 
or ad hoc) include the following – lasting longer than 5 minutes: 
1) Finger splay 
2) Grimacing 
3) Vital Sign disturbances associated directly with intervention 
4) Crying 
Should these findings last longer than 5 minutes in duration during 
intervention, the intervention will be stopped for that intervention session. If 
that preterm displays these findings >50% of their sessions within any one 
week (i.e., in 4, 5, or 6 sessions in that week), the intervention will be halted 
for 3 days and then re-initiated. During the 3 days of intervention halting for 
that infant, the NICU staff will be consulted on whether they recommend 
protocol modification for this preterm upon re-initiation. If this happens again 
(i.e., findings at 4, 5, or 6 sessions within one week) for this preterm after re-
initiation, the intervention will be discontinued for that preterm but outcome 
measures will still be collected for that infant’s complete follow-up. 
After the first 3 preterms have been enrolled, if 2 or 3 of the 3 enrolled 
preterms have this temporary halting of the intervention, the DSMB/ CTSI 
Independent Monitor(s) will be consulted regarding whether the protocol 
should be modified. After at least 10 infants have been enrolled, if >40% of 
infants have this temporary halting of the intervention, the DSMB/ CTSI 
Independent Monitor(s) will be consulted regarding whether the protocol 
should continue as is, proceed with enhanced monitoring, be further 
investigated, be discontinued, or be modified and then proceed.  Suspension 
of enrollment (for a particular group, a particular study site or for the entire 
 
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study) is a potential outcome of a DSMB/ CTSI Independent Monitor(s) safety 
review. 
Subsequent review of serious, unexpected, and related AEs by the DSMB/ 
CTSI Independent Monitor(s), IRB, and NCCIH or relevant local regulatory 
authorities may also result in suspension of further study interventions/
administration of study product at a site. NCCIH retain the authority to 
suspend additional enrollment and study interventions/administration of study 
product for the entire study, as applicable. 
9. STATISTICAL CONSIDERATIONS  
9.1 
General Design Issues  
The R61 phase of this clinical trial - Effects of Music Based Intervention (MBI) 
on Pain Response and Neurodevelopment in Preterm Infants – is exploratory 
and designed to inform planning of the R33 phase. 
Primary Objective (1): Characterize differences in preterm pain responses 
between MBI and controls. Premature infant pain profiles (PIPP) include 
physiologic, behavioral, and contextual measures which identifies differences 
in pain responses between MBI and controls while still in the neonatal 
intensive care unit (NICU).  Central EEG amplitude changes have been time-
locked with painful procedures in term infants. We will explore if PIPP scores 
and central EEG amplitudes change with MBI compared to controls.   
This primary objective(1) addresses the first hypothesis and the milestone 
measures (3 and 4) described in Appendix A. 
• 
Hypothesis 1: MBI will show improved pain responses, with lower PIPP 
scores and attenuated central EEG amplitude changes during painful 
procedures, in comparison to the control cohort. 
Measure 3) PIPP scores comparing MBI to controls during a painful 
procedure. 
Measure 4) Central EEG amplitude changes comparing MBI to controls 
during a painful procedure. 
Primary Objective (2): Identify differences between MBI and controls in 
preterm brain maturation and early neurodevelopment.  
EEG is a surrogate marker for real time brain function during sleep-wake 
cycles. Because preterm brain networks develop during sleep, sleep duration 
is a strong indicator of brain maturation. Serial biweekly EEGs of preterm 
infants can quantify sleep duration trends and track MBI’s influence on sleep.  
Due to the natural limitations of evaluating immature neonatal nervous 
systems, ERPs have been utilized to study early neurodevelopment.  ERPs 
quantify electrical brain potentials changes time-locked with a stimulus. 
 
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Auditory ERPs performed at 43-48 weeks corrected age evaluates attention 
and discrimination between familiar and novel stimuli - early 
neurodevelopmental signs of recognition memory function and perceptual 
learning.  
This primary objective(2) addresses the below two hypotheses and the 
milestone measures (1 and 2) described in Appendix A. 
• 
Hypothesis 2: MBI will enhance preterm EEG brain maturation in 
comparison to controls.  
Measure 1) Average sleep (REM + non-REM) duration difference between 
MBI and controls using EEG. 
• 
Hypothesis 3: ERPs at 1 month (43-48 weeks) corrected age will show 
that MBI has a greater impact on early neurodevelopment when compared to 
controls.   
Measure 2) Late Slow Wave amplitude difference in auditory ERPs 
comparing MBI to controls. 
9.2 
Sample Size and Randomization 
The R61 phase is exploratory. Based on the number of eligible infants and 
assuming 60% of eligible subjects enrollment, we can feasibly enroll 60 
infants in 1.5 years.. This sample size is similar to or larger than other MBI 
studies in infants.21,23,68,107  
The clinical measure Premature Infant Pain Profile (PIPP) at 4 weeks is the 
primary outcome for the R61-phase pilot clinical trial. With expected 
recruitment of at least 60 preterms, expected 50 completed PIPPs at 4 weeks 
represents a loss to follow-up of (60-50)/60 = ~17%. Such a loss rate is 
slightly more conservative than previous studies completed in the University 
of Minnesota NICU (which has had ~15% loss rates). This sample size is 
similar to or larger than other previous MBI studies in infants. 
With 50 completed PIPPs at 4 weeks, for comparing MBI to control (25 per 
group), there is 80% power to detect a group difference in PIPP of 2.9 and 
85% power to detect a group difference in PIPP of 3.1. These calculations 
assume a between-infant standard deviation of 3.6, estimated from the 
placebo group of a recent study of sucrose as an analgesic for pain from a 
standard-of-care heel lance. These calculations were carried out in SAS with 
the following code: 
proc power; 
twosamplemeans test=diﬀ 
         meandiﬀ = . 
         stddev = 3.6 
         npergroup = 25 
 
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         power = 0.80 0.85 
         alpha = 0.05 
; 
run; 
The R61 exploratory phase data from primary outcome clinical measure PIPP 
at 4 weeks will be utilized to assist in planning of the R33 phase. The 
associations of baseline characteristics (infant, e.g. birthweight, and maternal 
pregnancy history, etc) may inform the design of the R33 phase. For example, 
these secondary analyses might indicate that we should stratify 
randomization in the R33 phase by a dichotomized infant birthweight. These 
analyses, using linear models or non-parametric alternatives if needed (e.g., 
measures are highly skewed), will use both intervention groups and we will 
examine the magnitudes of associations in each group and, if appropriate, in 
the pooled groups. 
Treatment Assignment Procedures 
Once eligibility has been confirmed and consent obtained, Randomization 
will be accomplished through the use of randomization tables, stratified by 
sex and within each stratum use randomly allocated block sizes of 2 and 4. 
These will be prepared by the study statistician (Dr. Eberly). 
Blinding will be maintained by using identical headphones and MP3 players 
for all subjects. The PI, any personnel involved in collecting the clinical 
outcomes (e.g., PIPP) or collecting or quantifying EEG outcomes, and the 
statistician will be blinded. The statistician will be aware of blinded treatment 
group, so that e.g. safety summaries by blinded treatment group can be 
prepared for the Data Safety and Monitoring Board (DSMB) closed sessions if 
it so requests. Should the DSMB request unblinded comparisons, the study 
team will contract with the University of Minnesota CTSI’s Biostatistical 
Design and Analysis Center (BDAC) for an independent statistician to work 
with the DSMB using unblinded data. 
The unblinded staff includes Michael Silverman (music therapist) and Erin 
Osterholm (Director of the NICU). Silverman will prepare the music players. 
Both will be unblinded to handle randomization, delivery of interventions, and 
preparations of unblinded reports to the independent study monitors. They will 
not be involved with data monitoring, analyses, or outcome assessments.  
The study coordinators who are assessing the PIPP will wear earplugs and 
noise cancelling headphones, which will ensure that s/he cannot hear what is 
emanating from the infant’s headphones. In addition, family members and 
NICU staff will be reminded frequently to not disclose what they think they 
hear coming from the infant’s headphones.  
 
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9.3  
Definition of Populations 
N/A 
9.4 
Interim Analyses and Stopping Rules 
N/A 
9.5 
Outcomes: Milestone Measures and Data Analyses 
Measure 1: Average sleep (REM+nonREM) duration difference between MBI 
and controls using EEG. Normal preterm brain development is dependent on 
cycling between wakefulness and sleep with sleep durations gradually 
increasing by about 5% increments every two weeks until term age. EEG will 
be used to measure the average duration of sleep (REM + non-REM) 
between MBI and controls. Average sleep duration measurements will 
determine if MBI promotes/enhances brain maturation.  
Statistical Analysis: Four quality sleep durations from within each EEG 
session will be averaged per-infant per-timepoint (one EEG session for 
each of the 4 age timepoints, 30 weeks, 32 weeks, 34 weeks, 36 
weeks). At each timepoint, average sleep duration will be compared 
between MBI infants and controls. Milestone threshold will be reached 
if >5% longer average durations of sleep are found in the MBI cohort 
compared to controls in any 1 or more of the serial EEGs. 
Measure 2: Late Slow Wave amplitude difference in auditory ERPs 
comparing MBI to controls. Due to immature nervous systems, early infant 
neurodevelopment studies have centered on attentional capture paradigms 
using ERPs. ERPs quantify changes in electrical brain potentials time-locked 
to a stimulus and are widely used to study cognitive abilities such as learning, 
discrimination, attention, and memory. At one month age, the ERP Late Slow 
Wave of a mom/stranger stimulus is considered a reflection of perceptual 
learning and memory. ERP Late Slow amplitude measurements will determine 
if MBI enhances neurodevelopment. 
Statistical Analysis: ERP Late Slow Wave amplitudes from the 100 trials of the 
auditory recognition task will be averaged. Within-infant average Late Slow 
Wave amplitudes across mother’s voice trials minus the same average across 
stranger’s voice trials will be computed and averaged. Mother-vs-stranger 
Late Slow Wave average difference between MBI and controls will be 
compared. Milestone threshold will be reached if ≥1 µV greater amplitude 
difference in ERPs of the MBI cohort compared to controls is found at one 
month adjusted age. 
Measure 3: PIPP scores comparing MBI to controls during a painful 
procedure. The premature infant pain profile (PIPP) is well established and 
has undergone extensive psychometric testing. Music creates a sense of 
 
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familiarity and security in preterm infants and redirects their attention away 
from the painful stimulus; thus, PIPP scores will be used to measure if MBI 
has an effect on pain response in comparison to controls. 
Statistical Analysis: The PIPP will be scored at 3 age timepoints (30 weeks, 
32 weeks, 34 weeks). At each timepoint, average PIPP among MBI infants 
will be compared to average PIPP among control infants. Milestone threshold 
will be reached if MBI cohort PIPP scores are ≥1 point lower than controls at 
any 1 or more of the scoring points. 
Measure 4: Central EEG amplitude changes comparing MBI to controls 
during a painful procedure. The heel lance painful procedure has been shown 
to evoke central EEG activity with significantly higher amplitudes, ≥10 µV in 
infants.  EEG will be used to measure amplitude changes in central EEG 
activity to determine if MBI alters responses to a painful procedure. 
Statistical Analysis: Three quantifiable changes in central EEG amplitudes will 
be collected - one for each age timepoint at which a heel lance is done (at 30, 
32, and 34 weeks). Within-infant central EEG amplitude differences between 
heel sticks and resting states will be computed. Average central EEG 
amplitude change (heel-stick minus resting-state) among MBI infants will be 
compared to controls. Milestone threshold is reached if ≥10 µV attenuation in 
central EEG amplitudes occurs with a painful procedure in the MBI cohort 
compared to controls in any of the serial EEGs.  
9.6  Missing Data 
With regards to missing data, the built-in loss rate of 17% includes loss of 
subjects and also loss due to missing data. Thus, with our recruitment of 60 
subjects, we should still be able to reach our 50 subject target completing 
PIPP data at 4 weeks of MBI. 
This pilot clinical trial in the R61 phase is sufficiently small in sample size that 
imputation methods (e.g., missing week 4 PIPP value or sleep duration) are 
likely to perform poorly, specifically are likely to add substantial noise to the 
quantification of the Go/NoGo outcomes. The Go/NoGo quantifications will be 
computed without imputation and compared to the declared thresholds. 
Detailed infant and maternal characteristics from baseline assessments will 
be able to well describe the characteristics of those infants with missing data 
compared to those with complete data.  
 
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10. DATA COLLECTION AND QUALITY ASSURANCE 
10.1 Data Collection Forms  
NICU-based study activities. PI, Sonya Wang, with the assistance of Co-
Investigators, Raghavendra Rao and Lynn Eberly, will be responsible for 
overseeing study implementation and all NICU-based data collection. 
Specifically, they will oversee the research coordinators who provide day-to-
day supervision of participant informed consent and enrollment, MBI vs 
control intervention, and outcome assessments including EEGs and PIPPs. 
Neurodevelopmental testing. Tracking of study participants after NICU 
discharge and coordination of follow-up visits will be overseen by PI, Sonya 
Wang with the assistance of study coordinators. ERP testing will be 
conducted by Neely Miller at the Center for Neurobehavioral Development. 
Bayley’s III Neurodevelopmental testing will be conducted by the 
psychometrician at the Center for Neurobehavioral Development. 
Data coordination and management will involve design of data collection 
forms in REDCap for NICU and follow-up data. These will be managed by the 
study coordinators with PI, Sonya Wang, and Co-Investigator, Lynn Eberly, 
oversight. REDCap data analysis will be conducted by PI, Sonya Wang, Co-
Investigators, Lynn Eberly, and Biostatistician, Qi Wang. EEG data will be 
stored in password protected encrypted hard drives by study coordinators. 
EEG data will be managed by study coordinators and PI, Sonya Wang. EEG 
data analysis will be conducted by PI, Sonya Wang, and Co-Investigators, 
Theoden Netoff, Lynn Eberly, and the biomedical engineering graduate 
student. 
Data collected specifically for this study will be for clinical trial research 
purposes only and will include clinical data regarding the subject’s medical 
history and hospital course including clinical characteristics such as age, daily 
weight including birthweight, race and any underlying neonatal, perinatal and 
maternal health conditions. Most clinical data will be obtained from subjects’ 
medical records. In addition, EEG, PIPP, ERP, and neurodevelopmental 
assessment data will be collected. Study staff will have access to individually 
identifiable information about the infants enrolled in the study. Data will be 
collected by the study coordinator and recorded onto case report forms 
(CRFs). Data from the CRFs will subsequently be entered into the research 
database located on the encrypted study laptops. The CRFs and the 
electronic database will only have de- identified information to preserve 
confidentiality of patients’ protected health information. Files that link the 
study identification number for each subject to the subject’s personal 
information (such as name, medical record number, parents’ names) will filed 
separately and only be accessible to the PI and study coordinators.   
10.2 Data Management  
See section 10.1 above.  
 
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10.3 Quality Assurance  
10.3.1 Training 
The PI will ensure all staff are qualified for their study roles and trained on the 
protocol. 
10.3.2 Quality Control Committee  
This study will also be monitored semi- annually via independent monitoring 
services from the Clinical and Translational Science Institute (CTSI) at the 
University of Minnesota.  
CTSI Independent Monitor(s) review study materials (documents, records, 
Case Report Forms, etc) to assure that the study is conducted, recorded, and 
reported in compliance with FDA Good Clinical Practice. 
10.3.3 Metrics 
CTSI Independent Monitor(s) will also ensure that the study is conducted in 
accordance with the protocol and inclusion/exclusion criteria as approved by 
the IRB. The goal is to promote and facilitate compliance with Good Clinical 
Practice through: 
•
Regular monitoring visits 
•
Quality assurance 
•
Data query resolution 
•
Review of study regulatory files 
•
Adverse Event/Serious Adverse Event (AE/SAE) reviews 
•
Compliance consultation services 
•
Typical review of subject specific documents includes but is not limited to: 
o
Signed informed consent/HIPAA documents 
o Case Report Forms (CRFs) 
o
Medical records (for AE/SAE) 
o
Regulatory binders 
o
Communications with FDA/IRB 
10.3.4 Protocol Deviations 
Protocol deviations will be documented in the study file and those that meet 
IRB reporting criteria will be reported accordingly. 
10.3.5 Monitoring 
This research study involves no greater than minimal risk to participants. See 
above 10.3.2 for above details of monitoring. 
 
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11. PARTICIPANT RIGHTS AND CONFIDENTIALITY  
11.1 
Institutional Review Board (IRB) Review  
The IRB at the University of Minnesota is a fully authorized Institutional 
Review Board that provides oversight to research conducted at the university. 
It functions in compliance with the congressional statutes governing 
Assurance of Compliance with Health and Human Services (HHS) 
Regulations for Protection of Human Research Subjects. This board will be 
providing oversight to the current study. 
11.2 
Informed Consent Forms 
The PI and other selected study investigators listed on the IRB will obtain 
consent. All study investigators who will be obtaining consent will receive 
training on how to determine eligibility of subjects and how to discuss the 
study with parents. The study investigator who obtains consent will ensure the 
parents understand the informed consent process and the requirements of 
the study. Parents will be informed that they can ask questions about the 
study at any time and contact information for the study staff will be provided 
on the consent form. 
11.3 
Participant Confidentiality  
Every effort will be made during this study to ensure confidentiality. Only 
authorized study personnel and others authorized for regulatory purposes as 
described in the consent form will have access to this information. Study data 
will be de-identified and entered by the study coordinators into a password 
protected, encrypted electronic database for storage, retrieval, and analysis. 
EEG files will be de-identified and recorded onto a password protected, 
encrypted computer workstation. Each EEG file will be downloaded onto an 
encrypted hard drive with one extra backup copy. The EEG files will include 
video of the child and those who may be caring for the child during the EEG, 
such as a parent or staff member. There is also sound captured during the 
EEG but this is not listened to by the investigator. All confidentiality 
processes, as outlined above, will be followed for all video recordings. In all 
records, presentations, and manuscripts to be made public, the videos taken 
during the EEG will be de-identified so not to show the infant’s face. 
Families will be approached by research staff in private settings e.g. the 
mother’s or infant’s private hospital room. All information and data collected 
for the sole purpose of this study will be kept confidential. Only authorized 
study personnel and others authorized for regulatory purposes as described 
in the consent form will have access to this information. Medical information 
collected during this study will become part of the subject’s hospital record, if 
the information is determined to be pertinent to the subject’s medical care or 
is a usual part of the subject’s care (e.g., results of tests ordered by treating 
clinicians that are also recorded for research purposes). Medical records are 
available to other health care professionals at the hospital and may be 
 
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reviewed by hospital staff in their course of carrying out their responsibilities. 
However, they are required to maintain confidentiality in accordance with 
applicable laws and hospital policies. Study information that is not included in 
the medical record and could be personally identifiable will be under restricted 
access and viewable only by members of the research team (PI and study 
coordinators). These research records will not be made available to anyone 
not on the research team except upon a parent’s request or as required by 
law. Deidentified EEG data will be recorded onto a password protected, 
encrypted computer workstation. Each EEG file will be downloaded onto an 
encrypted hard drive with one extra backup copy. Data from various domains 
will be entered by the study coordinators into an electronic database for 
storage, retrieval, and analysis. Additional measures needed to ensure 
restricted access and confidentiality will be considered and implemented as 
deemed necessary. In all records, presentations, and manuscripts to be made 
public, information or data will not be personally identifiable. In the case that 
data or references to specific study patients are used, the patients will only be 
identified by code in order to maintain confidentiality. 
11.4 
Study Discontinuation  
The study may be discontinued at any time by the IRB, the NCCIH, the 
OHRP, the FDA, or other government agencies as part of their duties to 
ensure that research participants are protected.  
12. COMMITTEES 
N/A. 
13. PUBLICATION OF RESEARCH FINDINGS  
We will ensure that this trial “Effects of Music Based Intervention 
(MBI) on Neurodevelopment and Pain Response in Preterm 
Infants” is appropriately registered and results information is 
submitted to ClinicalTrials.gov, as outlined in the NIH policy and 
according to the specific timelines stated in the policy. Any 
presentation, abstract, or manuscript will be made available for 
review by the sponsor and the NCCIH prior to submission.  
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15. SUPPLEMENTS/APPENDICES
 
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