Randomized Trial of Maternal Progesterone Therapy to Improve Neurodevelopmental Outcomes in Infants With Congenital Heart Disease
Phase: PHASE2
Conditions: Congenital Heart Disease, Periventricular Leucomalacia, Brain Development, Cardiac Surgery, Neurodevelopmental Disability, Fetal Neuroprotection
Sponsor: Children's Hospital of Philadelphia

 
Template version: July 26, 2012 
  
Title: 
Randomized Trial of Maternal Progesterone Therapy to 
Improve Neurodevelopmental Outcomes in Infants with 
Congenital Heart Disease 
Short Title 
Maternal Progesterone Study 
Drug or Device 
Name(s): 
Vaginal natural progesterone 
FDA IND 
(if applicable)XXX-XX 
Regulatory Sponsor: 
 
eIRB Number 
 
Protocol Date: 4/3/14 
 
Amendment 1 Date:  3/19/15 
Amendment 3 Date: 3/29/2018 
Amendment 2 Date: 12/13/2016 
Amendment 4 Date: 10/26/2020 
Amendment 5 Date: 2/9/2021 
Amendment 6 Date: 5/6/2021 
 
Study Principal Investigator  
J. William Gaynor 
Office Address 
Philadelphia, PA 19104 
Phone 215-590-2708 
email: Gaynor@email.chop.edu 
Site Principal Investigator 
The Children’s Hospital of Philadelphia 
J. William Gaynor 
Office Address 
Division of Cardiac Surgery, Suite 12NW10 
34th St. and Civic Center Blvd. 
Philadelphia, PA 19104 
Phone 215-590-2708 
email: Gaynor@email.chop.edu 
 

  
 
 
 
 
 
 
 
ii
TABLE OF CONTENTS 
Table of Contents .............................................................................................................. ii 
Abbreviations and Definitions of Terms .......................................................................... v 
Abstract ............................................................................................................................vii 
Protocol Synopsis .............................................................................................................ix 
Table 1: Schedule of Study Procedures ........................................................................ xiii 
1.1 
INTRODUCTION ....................................................................................................... 1 
1.2 
NAME AND DESCRIPTION OF INVESTIGATIONAL PRODUCT OR INTERVENTION .............. 1 
1.3 
FINDINGS FROM NON-CLINICAL AND CLINICAL STUDIES ............................................. 2 
1.4 
SELECTION OF DRUGS AND DOSAGES ...................................................................... 6 
1.5 
RELEVANT LITERATURE AND DATA ........................................................................... 7 
1.6 COMPLIANCE STATEMENT ......................................................................................14 
2 
STUDY OBJECTIVES ................................................................................................14 
2.1 
PRIMARY OBJECTIVE (OR AIM) ................................................................................14 
2.2 
SECONDARY OBJECTIVES (OR AIMS) .......................................................................14 
3 
INVESTIGATIONAL PLAN .........................................................................................15 
3.1 
GENERAL SCHEMA OF STUDY DESIGN ....................................................................15 
3.1.1 
Screening Phase...........................................................................................15 
3.1.2 
In Utero Treatment Phase .............................................................................15 
3.1.3 
Post-Natal Phase ..........................................................................................15 
3.1.4 
Neurodevelopmental Evaluation ...................................................................15 
3.2 
ALLOCATION TO TREATMENT GROUPS AND BLINDING...............................................16 
3.2.1 
Randomization ..............................................................................................16 
3.2.2 
Blinding .........................................................................................................16 
3.2.3 
Unblinding .....................................................................................................16 
3.3 
STUDY DURATION, ENROLLMENT AND NUMBER OF SITES .........................................16 
3.3.1 
Duration of Study Participation ......................................................................16 
3.3.2 
Total Number of Study Sites/Total Number of Subjects Projected .................16 
3.4 
STUDY POPULATION ..............................................................................................17 
3.4.1 
Inclusion Criteria ...........................................................................................17 
3.4.2 
Exclusion Criteria ..........................................................................................17 
4 
STUDY PROCEDURES ..............................................................................................17 
4.1 
SCREENING VISIT ..................................................................................................17 
4.2 
IN UTERO TREATMENT PHASE ................................................................................17 
4.2.1 
Prenatal Visit 1 ..............................................................................................18 
4.2.2 
Subsequent Prenatal Visits (every 2 weeks until 36 weeks GA, then weekly)
 
18 
4.2.3 
Follow-up Fetal Brain MRI at 34-36 Weeks GA .............................................18 
4.2.4 
Delivery .........................................................................................................18 
4.2.5 
Post-Natal Phase ..........................................................................................18 
4.2.6 
Post-Natal Hospitalization .............................................................................18 
4.2.7 
Subsequent Hospitalization or Outpatient visit ..……………………………… 18 
4.2.8 
Neurodevelopmental Evaluation ...................................................................18 
4.2.9 
Neurodevelopmental Evaluation and End-of-Study .......................................19 
4.2.10     Subject's Home……………………………...………………………………………..19 
4.3 
CONCOMITANT MEDICATION ...................................................................................19 

 
 
 
 
 
 
 
 
iii
4.4 
RESCUE MEDICATION ADMINISTRATION ...................................................................20 
4.5 
SUBJECT COMPLETION/WITHDRAWAL .....................................................................20 
4.5.1 
Early Termination Study Visit ........................................................................20 
5 
STUDY EVALUATIONS AND MEASUREMENTS ......................................................20 
5.1 
SCREENING AND MONITORING EVALUATIONS AND MEASUREMENTS ..........................20 
5.1.1 
Pre-Natal Medical Record Review ................................................................20 
5.1.2 
Post-Natal Medical Record Review ...............................................................20 
5.1.3 
Physical Examination ....................................................................................20 
5.1.4 
Vital Signs .....................................................................................................20 
5.1.5 
Laboratory Evaluations .................................................................................21 
5.1.6 
Other Evaluations, Measures ........................................................................21 
5.2 
EFFICACY EVALUATIONS ........................................................................................23 
5.2.1 
Obstetric Ultrasound .....................................................................................23 
5.2.2 
Fetal Echocardiography ................................................................................25 
5.2.3 
Fetal Cerebral Blood Flow .............................................................................26 
5.2.4 
Fetal Brain MRI .............................................................................................27 
5.2.5 
Post-Natal Brain MRI ....................................................................................27 
5.2.6 
Neurodevelopmental Evaluation ...................................................................28 
5.3 
PHARMACOKINETIC EVALUATION ............................................................................28 
5.4 
SAFETY EVALUATION .............................................................................................28 
6.1   PRIMARY ENDPOINT......................................................................................................29 
6.2   SECONDARY ENDPOINTS ..............................................................................................29 
6.3   STATISTICAL METHODS .................................................................................................29 
6.3.1   Baseline Data ...................................................................................................29 
6.3.2   Efficacy Analysis ..............................................................................................29 
6.3.3  Safety Analysis ..................................................................................................30 
6.4   SAMPLE SIZE AND POWER ............................................................................................30 
6.5       INTERIM ANALYSIS ....................................................................................................32 
7 
STUDY MEDICATION (STUDY DEVICE OR OTHER STUDY INTERVENTION) .......32 
7.1 
DESCRIPTION ........................................................................................................32 
 
Packaging .....................................................................................................32 
7.1.2 
Labeling ........................................................................................................32 
7.1.3 
Dosing ..........................................................................................................33 
7.1.4 
Treatment Compliance and Adherence .........................................................33 
7.1.5 
Drug Accountability .......................................................................................33 
8 
SAFETY MANAGEMENT ...........................................................................................33 
8.1 
CLINICAL ADVERSE EVENTS ...................................................................................33 
8.2 
ADVERSE EVENT REPORTING .................................................................................33 
8.3 
DEFINITION OF AN ADVERSE EVENT ........................................................................33 
8.4 
DEFINITION OF A SERIOUS ADVERSE EVENT (SAE) ..................................................34 
8.4.1 
Relationship of SAE to Study Drug ...............................................................35 
8.5 
IRB NOTIFICATION OF SAES AND OTHER UNANTICIPATED PROBLEMS ......................35 
8.5.1 
Follow-up report ............................................................................................36 
8.6 
INVESTIGATOR REPORTING OF A SERIOUS ADVERSE EVENT TO SPONSOR ................36 
8.7 
MEDICAL EMERGENCIES ........................................................................................36 
9 
STUDY ADMINISTRATION ........................................................................................36 
9.1 
TREATMENT ASSIGNMENT METHODS ......................................................................36 

  
 
 
 
 
 
 
 
iv
9.1.1 
Randomization ..............................................................................................36 
9.1.3 
Unblinding .....................................................................................................37 
9.2 
DATA COLLECTION AND MANAGEMENT ....................................................................37 
9.3 
CONFIDENTIALITY ..................................................................................................37 
9.4 
REGULATORY AND ETHICAL CONSIDERATIONS.........................................................38 
9.4.1 
Data and Safety Monitoring Plan ...................................................................38 
9.4.2 
Risk Assessment ..........................................................................................38 
9.4.3 
Potential Benefits of Trial Participation ..........................................................42 
9.4.4 
Risk-Benefit Assessment ..............................................................................43 
9.5 
RECRUITMENT STRATEGY (OR CASE ASCERTAINMENT) ............................................43 
9.6 
INFORMED CONSENT/ASSENT AND HIPAA AUTHORIZATION .....................................43 
9.7 
PAYMENT TO SUBJECTS/FAMILIES ..........................................................................44 
9.7.1 
Reimbursement for travel, parking and meals ...............................................44 
9.7.2 
Payments to parent for time and inconvenience (i.e. compensation) .............44 
9.7.3 
Gifts ..............................................................................................................44 
10 PUBLICATION ...........................................................................................................44 
11 REFERENCES ...........................................................................................................45 
Appendix ..........................................................................................................................52 
 

  
 
 
 
 
 
 
 
v
ABBREVIATIONS AND DEFINITIONS OF TERMS 
AC 
AE 
APOE 
ART 
ASO 
Bayley-III 
BCAS 
BDNF 
BMI 
BPD 
CAG 
CPB 
CHD 
CHOP 
CICU 
CNS 
CNV 
CRH 
CTCAE 
CVO 
DMS 
DNA 
DSMB 
DV 
EFW 
FDA 
FL 
FSIQ 
fTMS 
GA 
HC 
HL 
HLHS 
IRB 
ITSEA 
ITT 
IVC 
IVH 
LVO 
MCA 
M-CHAT 
MDI 
 
Abdominal circumference 
Adverse event 
Apo-lipoprotein E 
Assisted reproductive technology 
Arterial switch operation 
Bayley Scales of Infant and Toddler Development-III 
Boston Circulatory Arrest Study 
Brain derived neurotrophic factor 
Body-mass index 
Biparietal diameter 
Center for Applied Genomics 
Cardiopulmonary bypass 
Congenital heart disease 
Children’s Hospital of Philadelphia 
Cardiac Intensive Care Unit 
Central nervous system 
Copy Number Variation 
Corticotrophin releasing hormone 
Common Terminology Criteria for Adverse Events 
Combined ventricular output 
Data management system 
Deoxyribonucleic acid 
Data Safety Monitoring Board 
Ductus Venosus 
Estimated fetal weight 
Food and Drug Administration 
Femoral length 
Full-Scale Intelligence Quotient  
Fetal Total Maturation Score 
Gestational age 
Head circumference 
Humeral length 
Hypoplastic left heart syndrome 
Institutional review Board 
Infant-Toddler Social and Emotional Assessment 
Intention-to-treat 
Inferior vena cava 
Intra-ventricular hemorrhage 
Left ventricular output 
Middle cerebral artery 
Modified Checklist for Autism in Toddlers  
Mental Developmental Index 

  
 
 
 
 
 
 
 
vi
MPA 
MRI 
NEC 
NICU 
PCR 
PDD 
PDI 
PI 
PIQ 
PVL 
RCT 
RDS 
RI 
ROI 
RR 
RVO 
SAE 
SAR 
SD 
SES 
SNP 
SVC 
TBI 
TGA 
TMS 
UA 
UtA 
VMWM 
17-OHP-C 
Medroxyprogesterone acetate 
Magnetic resonance imaging 
Necrotizing enterocolitis 
Neonatal Intensive Care Unit 
Polymerase chain reaction 
Pervasive Developmental Disorder 
Psychomotor Development Index 
Pulsatility Index 
Performance Intelligence Quotient 
Periventricular leucomalacia 
Randomized clinical trial 
Respiratory distress syndrome 
Resistive index 
Region of interest 
Relative risk 
Right ventricular output 
Serious adverse event 
Specific absorption ratio 
Systolic to diastolic ratio 
Socioeconomic status 
Single-nucleotide polymorphism 
Superior vena cava 
Traumatic brain injury 
Transposition of the great arteries 
Total maturation score  
Umbilical artery 
Maternal uterine arteries 
Volume of myelinated white matter  
17 α-hydroxyprogesterone caproate 

 
 
 
 
 
 
 
 
 
 
vii
ABSTRACT 
Context:   
Neurodevelopmental disability is now recognized as the most common long-term 
complication after cardiac surgery in neonates. White matter injury is the most common 
finding on neuroimaging and pathological studies in this population. Delayed brain maturation 
during fetal development, especially involving white matter, has been identified as an 
important mechanism underlying the risk of neurodevelopmental disability. Progesterone has 
been shown to be neuroprotective by accelerating white matter development and improving 
myelination. 
Objectives:  
Develop preliminary evidence to support a multi-institutional study to determine whether, in 
women carrying fetuses (maternal-fetal dyad) with congenital heart defects (CHD), 
prophylactic vaginal natural progesterone therapy is neuroprotective, and compared to 
placebo: 
1. improves neurodevelopmental outcomes at 18 months, 
2. improves fetal brain growth and maturation, 
3. increases myelination during fetal brain development, 
4. reduces pre-operative brain white matter injury, and 
5. reduces post-operative white matter injury. 
Study Design:  
Phase II single-center placebo-controlled randomized therapeutic exploratory trial. 
Setting/Participants: 
Setting: 
The study will be performed in the Center for Fetal Diagnosis and Treatment/Fetal Heart 
Program, as well as inpatient and outpatient units at CHOP.  
Inclusion Criteria: 
Mother carrying a fetus with CHD (maternal-fetal dyad) requiring surgery with 
cardiopulmonary bypass (CPB) prior to 44 weeks corrected gestational age (GA) identified 
prior to 28 weeks GA. 
Exclusion Criteria: 
1) Major genetic or extra-cardiac anomaly other than 22q11 deletion 
2) Language other than English spoken in the home 
3) Known sensitivity or listed contraindication to progesterone (known allergy or 
hypersensitivity to progesterone, severe hepatic dysfunction, undiagnosed 
vaginal bleeding, mammary or genital tract carcinoma, thrombophlebitis, 
thromboembolic disorders, cerebral hemorrhage, porphyria)  
4) Prescription or ingestion of medications known to interact with progesterone (e.g. 
Bromocriptine, Rifamycin, Ketoconazole or Cyclosporin)  
5) Maternal use of progesterone within 30 days of enrollment 
6) History of preterm birth or short cervix (defined as cervical length ≤ 25 mm at 18-
24 weeks GA  necessitating progesterone therapy 
7) Multiple gestation 
8) Maternal contraindication for magnetic resonance imaging (MRI) 

 
 
 
 
 
 
 
 
 
 
viii
9) Subjects with a known history of non-compliance with medical therapy.. 
Study Interventions and Measures:  
Study drug: 
Vaginal natural progesterone, 90 mg BID as a vaginal gel from 24–39 weeks gestation, 
compared to placebo. 
Main study outcome measures: 
1. Neurodevelopmental evaluation at 18 months of age: 
Bayley Scales of Infant and Toddler Development-III  
Modified Checklist for Autism in Toddlers (M-CHAT)  
Infant-Toddler Social and Emotional Assessment (ITSEA). 
Neurological examination 
2. Fetal brain MRI at 24-28 weeks GA prior to randomization. 
3. Fetal brain MRI at 34-36 weeks GA.  
4. Routine prenatal visits (every 2 weeks until 36 weeks, then weekly). 
5. Obstetric ultrasound (every 4 weeks). 
6. Routine fetal echocardiography (every 4 weeks). 
7. Maternal blood tests: progesterone, genetic testing. 
8. Paternal blood tests: genetic testing. 
9. Cord blood tests: progesterone. 
10. Infant blood testing: genetic testing. 
11. Early post-natal (preoperative) brain MRI (day of surgery). 
12. Post-operative brain MRI (within 10 days of surgery). 
 
 
 

  
 
 
 
 
 
 
 
ix
PROTOCOL SYNOPSIS 
 
Study Title 
Randomized Trial of Maternal Progesterone Therapy to 
Improve Neurodevelopmental Outcomes in Infants with 
Congenital Heart Disease 
Funder 
Hospital and Departmental funds 
Clinical Phase 
Phase II 
Study Rationale 
In the United States, approximately 1 in every 100 newborns is 
diagnosed with congenital heart disease (CHD). Many of these 
newborns (25%-35%) will require either corrective or palliative open 
heart surgery. As recently as the 1960’s, only 20% of newborns with 
critical CHD survived to adulthood. Today, thanks to better 
diagnostic 
technologies 
and 
methods 
(including 
prenatal 
diagnosis), advances in surgery, and improved postoperative care, 
early survival is over 90%. However, with improved early outcomes 
has come the sobering recognition that there is an ongoing risk of 
late mortality, as well as significant morbidity for these children. In 
particular, neurodevelopmental disability is now recognized as the 
most common complication of critical CHD (i.e. those patients 
requiring cardiac surgery in infancy) and has the most negative 
impact on quality of life, academic performance and opportunity for 
independence as an adult.  
The altered fetal hemodynamics secondary to CHD lead to 
decreased blood flow and/or oxygen delivery to the fetal brain. In 
turn, this impairment in blood flow and oxygenation results in 
impaired brain growth and altered structural and cellular maturation, 
particularly of the white matter. Fetal MRI studies have shown that 
during the third trimester, normally a time of rapid brain growth and 
development, brains of infants with CHD fail to grow at the same 
rate as the brains of fetuses without CHD. This growth delay results 
in microcephaly, immature cellular elements of the white matter and 
decreased cortical folding at birth. It has been demonstrated that 
brain immaturity at birth is a primary major risk factor underlying the 
hypoxic-ischemic white matter brain injury and subsequent 
neurodevelopmental disability seen in over 50% of infants following 
surgery for CHD. In addition, there is increasing evidence in the 
CHD population that even late pre-term birth (prior to 39 weeks GA) 
is associated with increased mortality, increased peri-operative 
morbidity, and worse neurodevelopmental outcomes. 
Progesterone is an essential hormone in the occurrence and 
maintenance of pregnancy. Progesterone administration has also 
been shown to be neuroprotective in a variety of clinical situations, 
including traumatic brain injury (TBI).   Sex steroid hormones, 
including progesterone, are critically involved in axonal myelination, 
forming the basis of white matter connectivity in the central nervous 
system (CNS). Progesterone and its metabolites not only promote 
the viability and regeneration of neurons, but also act on myelinating 

  
 
 
 
 
 
 
 
x
glial cell oligodendrocytes in the CNS and play an important role in 
the formation of myelin sheaths. Progesterone has also been shown 
to increase myelination and enhance maturation of immature 
oligodendrocytes progenitor cells to mature oligodendrocytes, 
which are more resistant to hypoxic/ischemic injury. Therapeutic 
administration of progesterone has also been demonstrated to 
prevent preterm birth. Thus, there are two potential mechanisms by 
which 
pre-natal 
progesterone 
therapy 
may 
improve 
neurodevelopmental outcomes in neonates with CHD: 1) a direct 
neuroprotective effect, and 2) decreasing the occurrence of pre-
term birth. 
Study Objective(s) 
Primary  
Develop preliminary evidence to support a multi-institutional study 
to determine whether, in women carrying fetuses (maternal-fetal 
dyad) with CHD, prophylactic vaginal natural progesterone therapy 
is 
neuroprotective, 
and 
compared 
to 
placebo 
improves 
neurodevelopmental outcomes at 18 months of age. 
Secondary 
Develop preliminary evidence to support a multi-institutional study 
to determine whether, in women carrying fetuses (maternal-fetal 
dyad) with CHD, prophylactic vaginal natural progesterone therapy 
is neuroprotective, and compared to placebo: 
1. improves fetal brain growth and maturation, 
2. increases myelination during fetal brain development, 
3. reduces pre-operative brain white matter injury, and 
4. reduces post-operative white matter injury. 
Test Article(s) 
(If Applicable) 
Prophylactic vaginal natural progesterone (90 mg as an 8% 
vaginal gel BID) from 24–39 weeks gestation, compared to 
placebo. 
Study Design 
 
Phase II single-center placebo-controlled randomized therapeutic 
exploratory trial. 
Subject Population 
key criteria for Inclusion 
and Exclusion: 
Inclusion Criteria 
Mother carrying a fetus with CHD (maternal-fetal dyad) requiring 
surgery with cardiopulmonary bypass (CPB) prior to 44 weeks 
corrected gestational age (GA) identified prior to 28 weeks GA. 
Exclusion Criteria 
1. Major genetic or extra-cardiac anomaly other than 22q11 
deletion 
2. Language other than English spoken in the home 
3. Known sensitivity or listed contraindication to progesterone 
(known allergy or hypersensitivity to progesterone, severe 
hepatic 
dysfunction, 
undiagnosed 
vaginal 
bleeding, 
mammary or genital tract carcinoma, thrombophlebitis, 
thromboembolic disorders, cerebral hemorrhage, porphyria) 

  
 
 
 
 
 
 
 
xi
4. Prescription or ingestion of medications known to interact 
with 
progesterone 
(e.g. 
Bromocriptine, 
Rifamycin, 
Ketoconazole or Cyclosporin)  
5. Maternal use of progesterone within 30 days of enrollment 
6. History of preterm birth or short cervix (defined as cervical 
length ≤ 25 mm at 18-24 weeks GA  necessitating 
progesterone therapy 
7. Multiple gestation 
8. Maternal contraindication for MRI 
9. Subjects with a known history of non-compliance with 
medical therapy. 
Number Of Subjects  
 
We plan to enroll sufficient patients to have 80 evaluable 
patients 
(40 
per 
treatment 
group) 
at 
the 
18 
month 
neurodevelopmental testing. Based on known clinical outcomes for 
this patient population and experience with other studies with similar 
follow-up plans, we estimate a 5% early mortality and a further 20% 
attrition from all causes between hospital discharge and the 18 
month evaluation. There may also be dropout prior to birth for a 
variety of reasons: fetal death, intolerance of therapy, withdrawal 
from study. We do not have accurate data to estimate this loss. We 
anticipate needing to enroll 110-130 patients to have a sufficient 
cohort at 18 months (50 per treatment group), with 60 and 52-56 
patients, respectively, per treatment group available for the post-
natal and post-operative brain MRIs.  
Study Duration 
The study begins during the pregnancy at 24 weeks GA and 
continues until the baby is 18 months of age. 
Study Phases 
Screening 
Study Treatment 
Follow-Up   
1. Screening 
2. Enrollment, baseline  evaluation, randomization 
3. Treatment phase (24 to 39 weeks gestational age) 
4. Evaluation Phase (birth to 18 months of age) 
Efficacy Evaluations 
1. Motor Scale of the Bayley-III at 18 months of age. 
2. Cognitive and Language Scales of the Bayley-III at 18 
months of age. 
3. M-CHAT and ITSEA at 18 months of age. 
4. Neurological examination. 
5. Total maturation score (TMS) 
6. Global and regional brain volumes 
7. Volume of myelinated white matter (VMWM) 
8. Presence of periventricular leucomalacia (PVL) on early 
post-natal brain MRI (categorical y/n) 
9. Presence of PVL on early post-operative brain MRI 
(categorical y/n) 
10. PVL Volume on early post-natal brain MRI 
11. PVL Volume on early post-operative brain MRI
Pharmacokinetic 
Evaluations 
(include only if applicable) 

 
 
 
 
 
 
 
 
 
 
xii
Safety Evaluations 
The mother and fetus will be seen every 1-2 weeks after 
enrollment and undergo physical examination, obstetric 
ultrasound. Fetal echocardiography will be performed every 4 
weeks and more frequently if clinically indicated. After birth the 
baby will undergo physical examination, echocardiography, and 
brain MRI.  
Statistical And Analytic 
Plan 
The primary analysis will be intent-to-treat (ITT) and will 
estimate differences between group means and their associated 
confidence intervals.  In parallel we will test the null hypothesis that 
treatment has no effect on each of three outcomes. Because of its 
clinical relevance, the study is primarily designed to provide an 
estimate of possible effect sizes for the Bayley-III Motor Scale at 18 
months. Important information will also be provided on effect sizes 
for the Bayley-III Cognitive and Language Scales at 18 months, M-
CHAT, and ITSEA, as well as total maturation score (TMS) and 
volume of myelinated white matter (VMWM).The analysis will use a 
two-sided two-sample T-interval (estimation) or T-test (hypothesis 
testing). The confidence level will be set at 90% and the Type I error 
at 10%. Normality will be assessed and a transformation, or 
alternatively a non-parametric test, will be used if needed.  
DATA AND SAFETY 
MONITORING PLAN 
Data quality management and ongoing assessment of safety will 
be the responsibility of the PI and multi-disciplinary Steering 
Committee. An independent Data Safety Monitoring Board 
(DSMB) will be established. 

 
xiii  
 
 
 
 
 
 
 
TABLE 1: SCHEDULE OF STUDY PROCEDURES  
Study Phase 
Maternal Interventions and Assessments 
 
Child Assessments 
 
Fetal 
Echocardiograph
y 
Obstetric 
Ultrasound 
Fetal 
Brain 
MRI 
Maternal 
Blood or 
Saliva 
Tests 
Paternal 
Blood or 
Saliva 
Tests 
Institution 
of Study 
Drug 
Prenatal Visit 
(q 2 week 
until 36 
weeks, then 
weekly) 
Termination 
of Study 
Drug 
 
Cord 
Blood 
Tests 
Infant 
Blood 
(from 
existing 
blood 
drawing 
line/routine 
clinical or 
separate 
research 
study 
labwork) or 
Saliva 
Sample 
Tests 
Brain 
MRI 
Neurodevelopme
ntal Evaluation 
Screening and 
Enrollment 
(24 to 28 weeks 
GA) 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Prior to 
Randomization 
 
 
X 
X 
X 
 
X 
 
 
 
 
 
 
Randomization 
 
 
 
 
 
X 
 
 
 
 
 
 
 
After 
Randomization 
q 4 weeks 
q 4 weeks 
 
 
 
 
X 
 
 
 
 
 
 
35 Weeks GA 
 
 
X 
X 
 
 
 
 
 
 
 
 
 
39 weeks GA 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
xiv  
 
 
 
 
 
 
 
Birth 
 
 
 
X 
 
 
 
 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Prior to 
Surgery  
(day of 
surgery) 
 
 
 
 
 
 
 
 
 
 
 
X 
 
After Surgery  
(within 7 days) 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Subsequent 
Hospitalization 
or Outpatient 
Visit 
 
 
 
X 
X 
 
 
 
 
 
X 
 
 
18 months of 
Age 
 
 
 
X 
X 
 
 
 
 
 
X 
 
X 

 
  
 
 
 
1
1    BACKGROUND INFORMATION AND RATIONALE  
1.1 
Introduction 
In the United States, approximately 1 in every 100 newborns is diagnosed with congenital 
heart disease (CHD). Many of these newborns (25%-35%) will require either corrective or 
palliative open heart surgery. As recently as the 1960’s, only 20% of newborns with critical 
CHD survived to adulthood. Today, thanks to better diagnostic technologies and methods 
(including prenatal diagnosis), advances in surgery, and improved postoperative care, early 
survival is over 90%. However, with improved early outcomes has come the sobering 
recognition that there is an ongoing risk of late mortality as well as significant morbidity for 
these children. In particular, neurodevelopmental disability is now recognized as the most 
common complication of critical CHD (i.e. those patients requiring cardiac surgery in infancy) 
and has the most negative impact on quality of life, academic performance and opportunity 
for independence as an adult.  
Altered fetal hemodynamics secondary to presence of CHD may cause decreased blood 
flow and/or oxygen delivery to the fetal brain. In turn, this impairment in blood flow and 
oxygenation results in impaired brain growth and altered structural and cellular maturation, 
particularly of the white matter. Studies using fetal brain MRI have shown that during the third 
trimester, normally a time of rapid brain growth and development, the brains of infants with 
CHD fail to grow at the same rate as the brains of fetuses without CHD; resulting in 
microcephaly, immature cellular elements of the white matter and decreased cortical folding 
at birth. It has been demonstrated that brain immaturity at birth is a primary major risk factor 
underlying 
the 
hypoxic-ischemic 
white 
matter 
brain 
injury 
and 
subsequent 
neurodevelopmental disability seen in over 50% of infants following for CHD. In addition, 
there is increasing evidence in the CHD population that even late pre-term birth (prior to 39 
weeks GA) is associated with increased mortality, increased peri-operative morbidity, and 
worse neurodevelopmental outcomes. 
Progesterone is an essential hormone in the occurrence and maintenance of pregnancy. 
Progesterone administration has also been shown to be neuroprotective in a variety of clinical 
situations, including traumatic brain injury (TBI).   Sex steroid hormones, including 
progesterone, are critically involved in axonal myelination, forming the basis of white matter 
connectivity in the central nervous system (CNS). Progesterone and its metabolites not only 
promote the viability and regeneration of neurons, but also act on myelinating glial cell 
oligodendrocytes in the CNS and play an important role in the formation of myelin sheaths. 
Progesterone has also been shown to increase myelination and enhance maturation of 
immature oligodendrocytes progenitor cells to mature oligodendrocytes, which are more 
resistant to hypoxic/ischemic injury. Therapeutic administration of progesterone has also 
been demonstrated to prevent preterm birth.  
We propose a Phase II single-center placebo-controlled randomized therapeutic 
exploratory trial to determine whether there is evidence that progesterone, administered pre-
natally, improves neurodevelopmental outcomes, improves brain development, and reduces 
perioperative white matter injury in neonates with CHD undergoing cardiac surgery. The data 
provided by this therapeutic exploratory study will aid in selection of appropriate outcomes 
and inform sample size calculations for a future multi-institutional study. 
1.2 
Name and Description of Investigational Product or Intervention 
Natural progesterone (Crinone) Intravaginal gel: 90 mg (8% gel) twice daily 

 
  
 
 
 
2
1.3 
Findings from Non-Clinical and Clinical Studies  
1.3.1 Progesterone Pharmacokinetics 
Progesterone is produced in significant amounts by the luteal phase ovary and the 
fetoplacental unit in the reproductive-age woman.1 It is produced by the adrenal gland in small 
amounts; but in large amounts in the postovulatory ovary. Progesterone is metabolized 
principally in the liver by the 5a-reductase pathway resulting in pregnanediol and 
pregnanetriol; and by conjugation with glucuronide.1 Progesterone’s precursors and 
metabolites do not have significant progestational activity. Progesterone circulates in the 
serum bound to cortisol-binding globulin, bound to albumin, or as free hormone.1 
Oral progesterone produces poorly sustained plasma progesterone concentrations. 
There is considerable first-pass prehepatic and hepatic metabolism.2, 3 The vaginal route for 
systemic drug administration is advantageous for administration of drugs such as 
progesterone, which exert their activity on the uterus and there is evidence of direct vaginal 
to uterus transport of progesterone. 2, 4 Uterine tissue concentrations of progesterone are 
significantly higher after vaginal administration than after systemic administration, despite 
higher blood levels with systemic administration.3, 4 In a randomized trial, women undergoing 
hysterectomy received either vaginal or IM progesterone. Ratios of endometrial to serum 
progesterone concentrations were higher after vaginal administration of progesterone than 
after IM injections.3 The lower blood levels after vaginal administration have been associated 
with a lower prevalence of systemic side effects.3, 5 
Although vaginal progesterone is commonly used in conjunction with assisted 
reproductive technologies (ART) and to prevent preterm birth, there is very little data 
concerning placental levels, transplacental transfer or fetal blood levels. A study using ex vivo 
perfusion of placentas from term pregnancies showed that 17-α-Hydroxyprogesterone 
caproate is metabolized by placental lobules. Both the parent compound and the metabolites 
were transferred to the fetal circuit.6 
1.3.2 Neuroprotective Effects of Progesterone 
Progesterone is classified as a neurosteroid. The enzymes necessary for its synthesis 
are present in the brain and the progesterone receptor is widely distributed in the forebrain, 
limbic system and hypothalamus early in development.7  The potential neuroprotective effects 
of progesterone were discovered because of sex differences in recovery from experimental 
brain injury. Progesterone is neuroprotective in animal models of stroke. Administration of 
progesterone prior to middle cerebral artery occlusion decreases infarct size.8 Post-ischemic 
administration has also been shown to reduce injury. Progesterone reduces cerebral edema 
after experimental TBI.9 Progesterone also has a positive effect after spinal cord contusion.10 
Following peripheral nerve injury, progesterone administration has been shown to induce re-
myelination.11 There is growing evidence, including recently published clinical trials, that 
progesterone and its metabolite allopregnanolone exert neuroprotective effects on the injured 
CNS in humans.12 These positive effects include not only decreased mortality, but also 
reduced morbidity and improved functional outcomes.13-15 
A recent Cochrane review evaluated all randomized controlled trials looking at the effects 
of progesterone in people with TBI.16 The review focused on randomized controlled trials in 
which, in addition to standard treatment, the experimental group received progesterone and 
the control group received non-active treatment (placebo) or no additional treatment. The 
authors identified three studies with a total of 315 subjects. There was evidence of a 
beneficial effect of progesterone on mortality and disability after TBI. Subjects in the 
progesterone group had a risk of death around 40% lower than those in the control group. 

 
  
 
 
 
3
The results of this review indicate that progesterone may reduce mortality and disability 
compared to the control group with no additional adverse events. 
The effectiveness of progesterone as a neuroprotective agent is probably related to its 
role in brain development for both males and females. Fetuses of both sexes are exposed to 
high levels of maternal progesterone during almost the entire period of gestation.14 Many of 
the processes involved in CNS repair after brain injury are thought to recapitulate normal 
brain development.17 Progesterone is synthesized by oligodendrocytes and in excitatory 
neurons in the brains of both males and females in roughly equal amounts.18 Progesterone 
and allopregnanolone reduce excessive excitotoxicity and inflammation while maintaining 
normal levels of cell proliferation and apoptosis in the developing brain.19 Numerous potential 
mechanisms 
likely 
underlie 
the 
neuroprotective 
effects 
of 
progesterone 
and 
allopregnanolone.9 These include regulation of neurotropin production; increased expression 
of brain derived neurotrophic factor (BDNF); activation of specific signaling pathways 
including the cAMP/PKA, MAPK (ERK1/2), and the PI-3K/Akt pathways, previously 
implicated in mediating neuroprotective effects; and regulation of the GABAA receptor.20-24 
The prenatal period is a critical time for brain development and early organization of 
neural circuits.25, 26 White matter development and myelination is essential for the conduction 
of brain signals. Communication between brain regions is established through white matter 
tracts consisting of myelinated axons.26 Disruption of brain myelination processes during 
development can lead to lasting neurological, cognitive and motor effects. In the newborn 
with CHD, white matter injury is the most common lesion identified by neuroimaging studies, 
and is present in up to 50% of patients early after cardiac surgery.27, 28 The primary 
mechanism of white matter injury is thought to be hypoxic/ischemic injury to vulnerable 
oligodendrocyte precursors. Thus the protective effect of progesterone on oligodendrocytes, 
their vulnerable progenitors and myelination is of special interest.  
Progesterone is involved in the regulation of oligodendrocyte precursor proliferation and 
maturation.29 There is evidence that progesterone promotes myelination in the CNS, by 
stimulating the proliferation and maturation of oligodendrocyte precursors into myelinating 
oligodendrocytes.30 Progesterone has also been shown to have positive effects on 
myelination in the developing CNS, likely by enhancement of the maturation of 
oligodendrocytes precursors into myelinating oligodendrocytes. Progesterone has pro-
myelinating effects in cultures of glial cells and brain slice cultures.30 Overall, progesterone 
increases the number of oligodendrocytes, the formation of myelin sheaths, and the synthesis 
of myelin proteins.26, 31 Progesterone has also been shown to increase myelin basic protein 
in organotypic slice cultures of rat cerebellum.32 Further evidence of the critical role of 
progesterone in fetal white matter development is provided by a recent study of chronic 
inhibition of allopregnanolone synthesis in the fetal brain.33 Chronic administration of 
finasteride during late gestation was used to reduce allopregnanolone concentrations in the 
fetal guinea pig brain. Finasteride treatment was associated with significantly reduced 
myelination in the subcortical white matter.33 In addition, neuroactive steroids are important 
in protecting the fetal brain against hypoxia.19 Progesterone and its metabolites reduce 
excitotoxic cell death and may stimulate repair processes.34 The fetal brain responds to acute 
hypoxia by increasing allopregnanolone synthesis, which may ameliorate white matter injury. 
The role of exogenous progesterone administration in protecting against hypoxia induced 
white matter injury is not known. 
The neuroprotective effects are highly dependent on the type of progesterone 
administered. There are important differences in the neurobiology of progesterone and the 
synthetic medroxyprogesterone acetate (MPA), which is the most commonly used progestin 
in hormone therapy regimens.9 MPA is a synthetic progestin derived from 17α-

 
  
 
 
 
4
hydroxyprogesterone. Both natural progesterone and MPA are used to prevent preterm birth. 
Progesterone and MPA are equally effective at reducing the uterotrophic effects of 
unopposed estrogen treatment, but their effects on the brain are not identical.  It has become 
clear that while progesterone is neuroprotective, MPA is not.9 The difference in 
neuroprotective potential may be explained by differential effects on BDNF expression; 
progesterone increases BNDF levels, while MPA has no effect.35 
The post-natal effects of progesterone therapy during fetal life were evaluated in a 
multicenter randomized controlled trial of 17 alpha-hydroxyprogesterone caproate (17-OHP-
C) vs. placebo to prevent preterm birth. Surviving children underwent physical examination 
and developmental screening at 48 months of age. Progesterone therapy was not associated 
with any adverse physical or neurodevelopmental sequelae.36 The lack of improvement in 
neurodevelopmental outcomes is not surprising given the lack of neuroprotective effect of 17-
OHP-C. But no adverse effects of therapy were identified. 
The effects of post-natal progesterone and estradiol supplementation were evaluated in 
a randomized clinical trial of extremely low birth weight infants (n=30).37 Therapy was initiated 
at a median gestational age of 26.6 weeks (corresponding to the third trimester of fetal 
development) and continued for 6 weeks. There were no early adverse effects of the therapy. 
The treatment group showed a trend towards less lung disease and better bone 
mineralization. Neurodevelopmental follow-up was performed at 15 months of age using the 
Bayley Scales of Infant Development-II.38 Median Psychomotor Developmental Index (PDI) 
scores were 101 for the treatment group (n=11) and 71 for the control group (n=10). Median 
Mental Developmental Index scores were 89 and 93 respectively. At a 5 year evaluation, 
there was a trend towards larger head circumference in the treatment group.39 Every 
additional day of therapy was associated with a reduced incidence of cerebral palsy and 
spasticity. The number of patients is small, but the results suggest a potential benefit of 
progesterone therapy.  
1.3.3 Progesterone and Preterm Birth 
In humans, progesterone is necessary for fertilization and maintenance of pregnancy (by 
maintaining myometrial relaxation).40, 41 Progesterone is initially produced by the corpus 
luteum and from 7–8 weeks of gestation increasingly by the placenta. At term, in a singleton 
pregnancy, the placenta produces approximately 250–300 mg progesterone per day.40 
Inhibition of the production/effect of progesterone will induce parturition.40 Progesterone 
seems to have three different potential modes of action which maintain pregnancy: 1) 
functional progesterone withdrawal has been suggested to be essential in humans, i.e. 
changes in receptor levels or metabolism of progesterone40; 2) maternal levels of placental 
corticotrophin releasing hormone (CRH) increase with gestational age and peak at delivery. 
The rise in placental CRH levels is more rapid in women who deliver preterm compared to 
women who deliver at term. Progesterone has an inhibitory effect on placental CRH 
production which may prevent preterm birth; 3) labor is preceded by maturation of the cervix 
through up-regulation of interleukins and increased prostaglandin synthesis. Progesterone 
attenuates these effects, possibly by an anti-inflammatory effect.40 
A characteristic of human pregnancy is that circulating progesterone levels do not fall with 
the onset of labor. The mechanisms leading to functional withdrawal of progesterone effect 
likely involves the progesterone receptor.41 There are several variants arising from 
transcription of the single progesterone receptor gene at alternative start sites.  Progesterone 
receptor B, the most common transcript, mediates many of the actions of progesterone. 
However, there are shorter transcripts, including progesterone receptors A and C. The 
shorter  variant receptors (A and C) lack an N-terminal–activating domain, and can function 

 
  
 
 
 
5
as dominant repressors of the function of the progesterone receptor B.41 The proportions of 
progesterone receptors A, B, and C change with the onset of labor in a way that could 
constitute a mechanism of progesterone withdrawal. 
There is increasing evidence that administration of progesterone to women at increased 
risk of preterm birth (those with a short cervix and possibly those with a history of preterm 
birth) is an effective therapy to delay onset of parturition.42 A Cochrane review of 36 
randomized controlled trials involving 8,523 women found that progesterone therapy had 
benefits which include: prolonging the pregnancy, decreased neonatal mortality, decreased 
need for assisted ventilation and decreased risk of necrotizing enterocolitis.43 A recent 
systematic review concluded that in women with a singleton pregnancy and previous preterm 
delivery, progesterone reduces the rates of preterm delivery before 32 weeks, perinatal 
death, as well as respiratory distress syndrome and necrotizing enterocolitis in the newborn.40 
A systematic review of interventions to prevent small-for-gestational age fetuses and to 
prevent perinatal mortality found that progesterone therapy to prevent preterm birth was one 
of the most effective therapies.44 
The optimal progesterone preparation and dosage has not been fully determined. In a 
recent review, the authors concluded that women with a history of prior spontaneous preterm 
birth should be offered progesterone therapy.45 Progesterone supplementation in women with 
a short cervix could be considered. Based on existing evidence, a dose of 250 mg of 17 α-
hydroxyprogesterone caproate (17-OHP-C) given weekly starting between 16/0 and 20/6 
weeks of gestation and continued until 34 to 36 weeks gestation would be reasonable. The 
use of 100 to 200 mg intravaginal progesterone nightly or 90 mg intravaginal progesterone 
gel nightly could be considered.45  A recent study evaluated treatment with vaginal 
progesterone gel of up to 400 mg per day in twin pregnancies.46 No benefit of 400 mg 
compared to 200 mg was found.  Importantly, no serious systemic adverse effects were 
encountered and local tolerance was good in all but one woman. The rate of gestational 
problems was not higher with the larger dose, except for a small increase in the incidence of 
generalized pruritus and a dose-dependent, non-significant, trend towards a higher incidence 
of intrahepatic cholestasis.  
The effects of progesterone on perinatal outcomes and the neonate are of special 
interest. A recent meta-analysis attempted to quantify the effect of progesterone therapy for 
the prevention of preterm birth on perinatal outcomes.42 Sixteen studies (singletons, n=7; 
twins, n=7; triplets, n=2) were included. Outcomes evaluated were neonatal and perinatal 
death, respiratory distress syndrome (RDS), retinopathy, necrotizing enterocolitis (NEC), 
intraventricular hemorrhage (IVH) Grade 3–4, sepsis, admission to the neonatal intensive 
care unit (NICU) and composite adverse outcome. For singleton pregnancies, progesterone 
reduced the rates of neonatal death (risk ratio (RR) 0.487 (95% CI, 0.290–0.818)), RDS (RR 
0.677 (95% CI, 0.490–0.935)), NICU admission (RR 0.410 (95% CI, 0.204–0.823)) and 
composite adverse outcome (RR 0.576 (95% CI, 0.373–0.891)). No favorable effect was 
observed in twin gestations. Progesterone was associated with increased rates of perinatal 
death (RR 1.551 (95% CI, 1.014–2.372)), RDS (RR 1.218 (95% CI, 1.038–1.428)), and 
composite adverse outcome (RR 1.211 (95% CI, 1.029–1.425)) in twin gestations. No 
significant effect was observed in triplet pregnancies. The authors concluded that 
progesterone administration in singleton pregnancies at risk for preterm birth improves 
perinatal outcomes, but may actually have adverse effects in multiple pregnancies.42 Other 
reviews have also found that there do not appear to be any significant maternal or fetal risks 
associated with progestin therapy.45 Longer term outcomes have also been evaluated.36 
Surviving children from a trial of 17-OHP-C vs. placebo to prevent preterm birth underwent 

 
  
 
 
 
6
physical examination and developmental screening at 48 months of age. Progesterone 
therapy was not associated with any adverse physical or neurodevelopmental sequelae.36 
1.3.4 Ongoing Clinical Trial 
The OPPTIMUM study is a double blind randomized placebo controlled trial to determine 
whether progesterone prophylaxis to prevent preterm birth has long term neonatal or infant 
benefit.47 Specifically it will study whether, in women with singleton pregnancy and at high 
risk of preterm labor, prophylactic vaginal natural progesterone, 200 mg daily from 22–34 
weeks gestation, compared to placebo, improves obstetric outcome by lengthening 
pregnancy thus reducing the incidence of preterm delivery (before 34 weeks), improves 
neonatal outcome by reducing a composite of death and major morbidity, and leads to 
improved childhood cognitive and neurosensory outcomes at two years of age. Recruitment 
began in 2009 and is scheduled to close in spring, 2013. As of May 2012, over 800 women 
had been randomized in 60 sites. Fetuses with significant congenital structural (including 
CHD) or chromosomal fetal anomalies are excluded. The primary endpoints are 1) obstetric: 
delivery <34 completed weeks of gestation (Yes/No); 2) neonatal: a composite of death or 
two markers of neonatal morbidity – bronchopulmonary dysplasia in children born at <32 
weeks of gestation and brain injury on cerebral ultrasound; 3) childhood: The Bayley Scales 
of Infant and Toddler Development-III Cognitive score at two years of chronological age. The 
study protocol does not include fetal or post-natal brain MRI, thus limiting the ability to 
quantify brain maturation or white matter injury. 
1.4 
Selection of Drugs and Dosages 
Progesterone therapy has received approval for prevention of preterm birth. On February 
3, 2011, the FDA approved the use of progesterone supplementation (hydroxyprogesterone 
caproate) during pregnancy to reduce the risk of recurrent preterm birth in women with a 
history of at least one prior spontaneous preterm delivery.48 Recently, the Society for 
Maternal-Fetal Medicine reviewed the existing data and concluded that existing randomized 
studies indicate that in women with singleton gestations, no prior preterm birth, and short 
cervical length, vaginal progesterone, either 90-mg gel or 200-mg. suppository, is associated 
with reduction in preterm birth and perinatal morbidity and mortality, and can be offered in 
these cases.49 This was rated as Level I evidence and a level A recommendation. Similarly, 
the Maternal Fetal Medicine Committee of the Society of Obstetricians and Gynaecologists 
of Canada reviewed the existing data and concluded that on the basis of the data from the 
RCTs and a meta-analysis, it is recommended: 1) that in cases where the clinician and the 
patient have opted for the use of progesterone the following dosages should be used: for 
prevention of preterm birth in women with history of previous preterm birth:17 alpha-
hydroxyprogesterone 250 mg IM weekly (level of evidence I-B) or progesterone 100 mg daily 
vaginally (level of evidence I-A); and 2) for prevention of preterm birth in women with short 
cervix, progesterone 200 mg daily vaginally (level of evidence I-A).50 
The proposed study will utilize a transvaginal Bioadhesive Delivery System consisting of 
progesterone in a polycarbophil-based gel (Crinone 8%; Serono Laboratories, Norwell, MA) 
which was designed to provide prolonged and controlled release of progesterone after 
vaginal application.2 The labeled indications include secondary amenorrhea, ART in patients 
who require progesterone supplementation, and ART in patients with partial or complete 
ovarian failure. An unlabeled use is to reduce the risk of recurrent spontaneous preterm birth 
in appropriately selected women. The usual dosage is 90 mg (8% gel) once or twice daily. 
An ongoing multi-institution study to determine if progesterone prophylaxis during pregnancy 
has long-term neonatal or infant benefit uses a daily natural vaginal suppository (200 mg).47 
Therapy will be initiated in the early third trimester (24-28 weeks GA). This is a period of rapid 

 
  
 
 
 
7
fetal brain growth and the period in which brain development in fetuses with CHD lags behind 
that of fetuses without CHD (see below). Therapy will be continued to 39 weeks GA in order 
to continue the potential neuroprotective effects, and potentially decrease the prevalence of 
birth prior to 39 weeks GS. The fetal brain continues to develop throughout gestation and 
there is evidence (see below) that delivery prior to 39 weeks is associated with increased 
mortality, morbidity and worse neurodevelopmental outcomes.51-53 
1.5 
Relevant Literature and Data 
1.5.1 Congenital Heart Disease and Neurobehavioral Disability 
In the United States, approximately 1 in every 100 newborns is diagnosed with CHD. 
Many of these newborns (25%-35%) will require either corrective or palliative open heart 
surgery. As recently as the 1960’s, only 20% of newborns with critical CHD survived to 
adulthood. Today, thanks to better diagnostic technologies and methods (including prenatal 
diagnosis), advances in surgery, and improved postoperative care, early survival is over 90%. 
However, with improved early outcomes has come the sobering recognition that there is an 
ongoing risk of late mortality as well as significant morbidity for these children. 
Neurodevelopmental disability is now recognized as the most common complication of critical 
CHD (i.e. those patients requiring cardiac surgery in infancy) and has the most negative 
impact on quality of life, academic performance and opportunity for independence as an 
adult. 54-57 
On average, children with CHD, compared to children in the normal population, have 
significantly lower scores on IQ and achievement tests, delays in reaching motor milestones, 
as well as higher frequencies of learning disabilities, use of special services, and speech, 
language, and behavioral abnormalities.54, 56 The factors underlying developmental morbidity 
in children with heart disease are complex and multi-factorial; ranging from genetic conditions 
and altered blood flow to the brain in utero to postnatal medical conditions that accompany 
CHD and its medical and surgical therapies.  Regardless of their origin, neurodevelopmental 
problems adversely affect learning and the attainment of academic, social, and vocational 
skills. Ultimately they undermine mental health and employment opportunities in adulthood. 
There is a wealth of disturbing data documenting the impact of neurodevelopmental deficits 
among children and adults affected by CHD.  57 
The most comprehensive data currently available on the neurodevelopmental sequelae 
of neonatal cardiac surgery are provided by the Boston Circulatory Arrest Study (BCAS) 
which examined the incidence of acute neurological injury, as well as later cognitive and 
behavioral outcomes, following the arterial switch operation (ASO) for transposition of the 
great arteries (TGA).58-63 Developmental outcomes were evaluated at 1, 4, and 8 years of 
age. At the 8-year evaluation, Full-Scale Intelligence Quotient (FSIQ) and Performance IQ 
(PIQ), memory, visual-spatial skills, and academic achievement for the entire BCAS cohort 
were lower than population norms. A substantial number of the patients were experiencing 
academic difficulties and 37% had received special education services. There was a 
distinctive pattern of neurodevelopmental dysfunction characterized by mild cognitive 
impairment, impaired executive function, inattention and impulsive behavior, deficits in core 
communication and pragmatic language skills, as well as impaired social cognition.  
Evaluation of the BCAS cohort at 16 years of age has recently been completed.62, 64 The 
evaluation focused on neuropsychological domains that previous evaluations suggested 
were areas of weakness, including executive functions, memory, attention, and visual-spatial 
skills. Not only were the mean scores in the cohort significantly lower than those expected in 
the general population, but the standard deviations were larger, suggesting marked variability 
among children in their outcomes. The deficits are particularly notable because the 

 
  
 
 
 
8
adolescents were of normal birth weight (>2.5 kg), did not have genetic syndromes or 
extracardiac anomalies, and rarely required reoperation or long-term cardiac medications. 
Evaluation of these adolescents with brain MRI revealed a very high prevalence of white 
matter injury and altered white matter microstructure.64 
Most studies of neuroprotection in the CHD population have focused on the intra-
operative period and management of CPB. Yet there is no evidence that any of these 
strategies have resulted in improvements in unadjusted neurodevelopmental outcomes. A 
recent analysis of 1,770 patients (23 centers/5 countries) born 1996-2009  who underwent 
cardiac surgery with CPB at age ≤ 9 mo. and were tested with the Bayley Scales of Infant 
Development-II revealed evidence of only slight improvement over time in Psychomotor 
Development Index (PDI) and Mental Developmental Index (MDI) scores since 1996, even 
after adjustment for patient factors. Surgery in more recent years was not associated with 
better unadjusted PDI or MDI scores.  
 
A recent systematic review evaluated the strength of evidence for current practices for 
neuromonitoring and neuroprotection during cardiac surgery in infants.65 The authors 
reviewed 162 manuscripts and concluded that the level of evidence is insufficient to support 
the effectiveness of most currently used neuroprotective therapies. These findings suggest 
that a new approach and new targets for neuroprotection are essential to improve outcomes. 
1.5.2 Congenital Heart Disease, Brain Development and White Matter Injury 
The utero-placental-fetal circulation is unique in a number of respects that have distinct 
impacts on fetal cerebral blood flow. In the fetus without CHD, gas exchange occurs in the 
placenta with oxygenated blood returning through the umbilical vein and ductus venosus (DV) 
to the portal vein, inferior vena cava (IVC) and right atrium and deoxygenated blood returning 
to the placenta via the umbilical artery. Prior to birth, blood flow to the lungs is very low due 
to elevated pulmonary vascular resistance and relatively low lung volumes. Two connections 
exist between the systemic and pulmonary circulations: 1) the foramen ovale connecting the 
right and left atria, and 2) the ductus arteriosus between the pulmonary trunk and descending 
aorta. Umbilical venous blood is preferentially directed through the DV into the left lobe of the 
liver. As a consequence the oxygen saturation is higher in the left hepatic veins as they join 
the IVC resulting in streams of blood with different saturations. The higher saturated stream 
containing blood from the DV is preferentially directed across the foramen ovale to the left 
atrium. This blood mixes with the limited amount of pulmonary venous blood returning from 
the lungs, resulting in a saturation in the fetal left ventricle of approximately 65%, which is 
then ejected into the ascending aorta and cerebral vasculature. Blood ejected by the right 
ventricle consists of desaturated venous blood from the superior vena cava (SVC) as well as 
the relatively desaturated streams from the IVC and coronary sinus. The resulting saturation 
in the fetal right ventricle is approximately 55%. Blood from the right ventricle is preferentially 
ejected across the ductus arteriosus returning to the placenta via the umbilical arteries.   
In two common forms of congenital heart disease, TGA and hypoplastic left heart 
syndrome (HLHS), alterations in fetal blood flow may lead to decreased brain oxygen delivery 
during fetal life. In TGA, the aorta arises from the right ventricle and thus receives the 
relatively desaturated blood from the superior vena cava and lower saturation stream of blood 
in the inferior vena cava. The higher saturated stream from the left hepatic veins is directed 
normally across the foramen ovale to the morphologic left ventricle. The left ventricle however 
is connected to the pulmonary trunk and this higher saturated blood is delivered to the lungs 
and lower body. In HLHS, the fetal circulation is characterized by increased left atrial 
pressure, resulting in reversal of flow across the foramen ovale. Left ventricular filling is 

 
  
 
 
 
9
impaired and there is either no ventricular output or stroke volume is diminished with reduced 
or absent flow into the ascending aorta. All venous return mixes in the right ventricle and is 
ejected into the pulmonary trunk and ductus arteriosus. Although the resulting saturation is 
not as low as with TGA, a number of additional factors may restrict flow to the cerebral 
circulation. Most importantly, in patients with aortic valve atresia, flow to the head and neck 
vessels only occurs by retrograde flow from the ductus arteriosus via the transverse aorta.  
Brain growth and maturation are delayed in patients with both TGA and HLHS. Despite the 
differences in fetal blood flow and blood oxygen saturations between fetuses with TGA and 
HLHS, post-natal head circumferences and maturation scores are not statistically different. 
This suggests that oxygen delivery (oxygen content x cerebral blood flow) is the critical value 
that determines brain growth and maturation.  
There is convincing evidence that in utero brain development is abnormal in fetuses with 
CHD leading to delayed maturation, poor growth and white matter injury. Altered fetal 
hemodynamics resulting from the presence of CHD may cause decreased in-utero cerebral 
blood flow and oxygenation of the fetal brain.66, 67 In turn, this impairment in blood flow and 
oxygenation results in impaired brain growth and altered structural and cellular maturation, 
particularly of the white matter.68, 69 Fetal imaging studies show that during the third trimester, 
normally a time of rapid brain growth and development, that the brains of infants with CHD 
fail to grow at the same rate as brains of fetuses without CHD.69 During this period of rapid 
brain growth in the normally developing fetal brain, there is a corresponding increase in the 
complexity of brain structure, characterized by cortical folding which progresses as a result 
of white matter volume expansion. During this phase of white matter growth, populations of 
glial cells called oligodendrocytes (the major constituent of white matter) are undergoing rapid 
differentiation and maturation. This increase in maturation and organizational complexity is 
delayed in fetuses with CHD, and is most delayed in those with the most complex defects 
(e.g. aortic atresia).66, 68, 69 The figure shows the relationship between the Fetal Total 
Maturation 
Score 
(fTMS) derived from 
fetal brain MRI for 
normal fetuses and 
those with CHD. A 
lower fTMS implies a 
less 
mature 
brain. 
There is a delay in 
brain maturation in 
the fetuses with CHD 
in late gestation. Thus 
there is convincing 
evidence for global 
brain 
growth 
impairment 
in 
the 
fetus 
with 
CHD 
resulting 
in 
microcephaly, 
immature 
cellular 
elements of the white 
matter and decreased cortical folding at birth. It has been demonstrated in several 
prospective observational studies that this abnormal/delayed development is a primary risk 
factor underlying the hypoxic-ischemic white matter brain injury seen in over 50% of peri-
surgical infants with CHD.27, 28 Pathological examination of this white matter injury 

 
  
 
 
 
10
demonstrates that this injury (PVL) is identical to that seen in infants born prematurely. Using 
TMS, it has been demonstrated that risk for pre-operative and postoperative injury seen in 
infants with is highly correlated with the maturational state of the brain prior to surgery. 67, 68 
In addition, brain immaturity at the time of cardiac surgery is a strong predictor of functional 
outcome at 2 years. 70, 71 
1.5.3 Assessment of Cerebral Blood Flow in the Fetus 
Fetal brain growth and development depends upon an adequate supply of oxygen and 
other substrates. Autoregulatory mechanisms in the fetus alter cerebrovascular resistance to 
counteract changes in oxygen delivery.72 Animal models and studies in human fetuses have 
shown a decrease in cerebrovascular resistance to compensate for fetal hypoxia. This 
redistribution of fetal circulation has been termed the ‘brain-sparing effect’ and may be an 
adaptive response to intrauterine hypoxia. 
There is increasing evidence that cerebral blood flow (CBF) in the fetus is altered in the 
presence of CHD. Middle cerebral artery (MCA) blood flow velocities (peak systolic velocity, 
peak diastolic velocity, and mean velocity) and vascular resistance can be assessed using 
pulsed-wave Doppler. Pulsatility index (PI) is a measure of vascular resistance in the 
circulatory bed downstream from the point of Doppler sampling. It is calculated according to 
the equation, PI = (peak systolic velocity – end-diastolic velocity)/ time-averaged mean 
velocity.72 The impact of CHD on fetal cerebrovascular flow patterns is dependent on the 
specific type of CHD. Studies at CHOP have shown that cerebrovascular resistance is lower 
than normal in fetuses with HLHS.72 In fetuses with right sided obstructive lesions (tetralogy 
of Fallot), cerebrovascular resistance is significantly higher than in fetuses with HLHS and is 
higher than in normal fetuses, but not to a significant degree. The fetus with HLHS and aortic 
atresia has absent antegrade blood flow in the ascending aorta, with CBF supplied retrograde 
via the ductus arteriosus, likely limiting cerebral perfusion. The decreased cerebral vascular 
resistance seen in the fetus with HLHS may be an attempt to attract a greater amount of CBF 
to compensate for decreased oxygen delivery.  
Fractional moving blood volume (FMBV) is an alternative method to assess the fetal 
cerebral circulation which uses Power Doppler to estimate quantitatively brain tissue 
perfusion.73-76 Using defined regions of interest, FMBV allows assessment of regional 
differences in brain perfusion.76 The method has been validated in animal models and is 
potentially a more sensitive method of brain blood flow redistribution in fetal growth 
restriction.76  FMBV analyzes changes in amplitude (power) on the backscattered Doppler 
ultrasound signals originated from red blood cells.  FMBV estimation aims to compensate for 
the effect that depth, tissue interfaces, and attenuation have on the power Doppler ultrasound 
signals. FMBV is more sensitive than MCA flow velocity or cerebral placental ratio (CPR) to 
identify SGA fetuses at risk of abnormal neonatal neurobehavioral performance.  SGA 
fetuses with increased frontal perfusion showed a 30% risk of abnormal neurobehavior 
expressed in social interactive organization, state organization, and attention capacity.75 
FMBV has not been used to assess the fetal cerebral circulation in fetuses with CHD. 
CBF in infants with CHD can also be assessed using a magnetic resonance imaging 
technique, pulsed arterial spin-label perfusion magnetic resonance imaging. At CHOP, early 
post-natal (pre-operative) CBF was measured under standard ventilation and repeated after 
increased carbon dioxide in 25 terms infants with CHD.67 Preoperative structural brain 
abnormalities were seen in more than 50% of the cohort, with the most common being PVL 
(28%) and microcephaly (24%). Observed CBF values were much lower than what is 
suggested by limited normal data and critically decreased in some patients. The presence of 
PVL was strongly associated with low baseline CBF and poor CO2 reactivity.67 The poor 

 
  
 
 
 
11
vascular responsiveness to inspired CO2 may be due to maximal baseline vasodilation in 
response to low CBF in these patients. Impaired CO2 reactivity has been associated with 
poor neurodevelopmental outcome and a higher risk of death.67  
1.5.4 Serum Progesterone Levels 
In humans, progesterone is necessary for fertilization and maintenance of pregnancy 
(by maintaining myometrial relaxation).40, 41 Progesterone is initially produced by the corpus 
luteum and from 7–8 weeks of gestation increasingly by the placenta. At term, in a singleton 
pregnancy, the placenta produces approximately 250–300 mg progesterone per day.40 Prior 
to ovulation, serum levels are less than 1 ng/ml. Serum rise after ovulation for 6-10 days, 
then fall if fertilization does not occur. If a viable pregnancy occurs, serum levels rise 
throughout gestation. A characteristic of human pregnancy is that circulating progesterone 
levels do not fall with the onset of labor. 
There is no data concerning fetal serum progesterone levels either during a normal 
pregnancy, or in response to exogenous progesterone. Although vaginal progesterone is 
commonly used for in conjunction with assisted reproductive technologies (ART) and to 
prevent preterm birth, there is very little data concerning placental levels, transplacental 
transfer or fetal blood levels. A study using ex vivo perfusion of placentas from term 
pregnancies showed that 17-α-Hydroxyprogesterone caproate is metabolized by placental 
lobules. Both the parent compound and the metabolites are transferred to the fetal circuit.6  
There is no data on serum progesterone levels during pregnancy in mothers carrying a fetus 
with CHD. Uterine tissue concentrations of progesterone are significantly higher after vaginal 
administration than after systemic administration, despite higher blood levels with systemic 
administration.3, 4 In a randomized trial, women undergoing hysterectomy received either 
vaginal or IM progesterone. The ratio of endometrial to serum progesterone concentrations 
was higher after vaginal administration of progesterone than after IM injections.3 The lower 
blood levels after vaginal administration have been associated with a lower prevalence of 
systemic side effects.3, 
5 Thus maternal serum progesterone levels after vaginal 
administration will not reflect endometrial or fetal serum concentrations. 
1.5.5 Placental Function in Normal and CHD Pregnancies 
The placenta is connected to the fetus by the umbilical cord and to the mother via the 
uterine wall. The umbilical cord contains two arteries and one vein. The placenta is primarily 
a vascular organ in which the uterine arteries bring maternal blood in contact with the 
placental villi in the intervillous space, where the trophoblastic epithelium surrounding the villi 
mediates the transfer of oxygen and other maternally-derived substances to the fetus. The 
normal placenta is long and thin, and contains numerous placental villi. The combination of 
a placental villous tree and its decidual artery is called a placentome (also a cotyledon). The 
diffusion barrier for transfer between mother and fetus is normally very small, made up by the 
trophoblast basement membrane and the fetal villous capillary endothelium. Fetal capillaries 
receive fetal deoxygenated blood and fetal waste products from arterioles arising from the 
umbilical arteries. Oxygenated and nutrient-enriched blood is returned to the fetus via a 
network of veins that drain into the umbilical vein. The maternal uterine arteries dilate 
significantly during the course of a normal pregnancy to increase maternal blood flow to the 
placenta and developing fetus; for example, 50 mL/minute of maternal blood enters the non-
gravid uterus, increasing to approximately 700 mL/minute at near-term. This large increase 
is made possible by increased maternal blood volume, increased cardiac output, uterine 
artery dilation, and low uterine and placental vascular resistance. Appropriate placental 
functioning at the anatomic/histologic level of the placentome requires: 1) maternal uterine 
and decidual artery integrity, patency, flow, and dilation during pregnancy; 2) increased 

 
  
 
 
 
12
maternal blood flow and low resistance through the intervillous space; 3) normal exposure of 
the villous trophoblastic endothelium to maternal blood; 4) low diffusion distance between 
maternal and fetal blood from the intervillous space across the trophoblast and villous 
endothelium; 5) integrity and patency of the villous vessels; 6) unobstructed flow in the 
umbilical cord.  
There is increasing awareness of the importance of normal function and regulation of a 
placental synthetic, metabolic, and inflammatory functions to maintain placental integrity, 
maternal and fetal well-being, and normal fetal development.  A variety of acute, sub-acute, 
and chronic abnormalities of placental anatomy and vascular function have been linked to 
maternal abnormalities such as preeclampsia and to fetal events such as fetal demise, IUGR, 
and perinatal brain injury; the types of injury have included white matter injury, stroke, or 
neonatal encephalopathy.77-80 Overall, associations between the development of early and 
mid-term placental abnormalities and subsequent events such as IUGR, stroke, 
preeclampsia, and abnormal post-natal brain anatomy and neurodevelopmental outcomes 
are increasing and becoming more firmly established. Despite this information, examination 
of the placenta—histopathological or otherwise—is not routine, even in pregnancies 
complicated by IUGR or early evidence of neonatal brain injury.  
There is a significant lack of information about placental anatomy and function in infants 
born with CHD, despite the increased frequencies of IUGR, pre-term birth, relative brain 
immaturity, reduced brain size, and brain imaging abnormalities (prior to any cardiac or other 
interventions), etc. that occur in these infants.  A recent study found significantly reduced 
fetal somatic growth velocity in the later stages of pregnancy as well as reduced birth weight 
in HLHS infants and suggested suboptimal energy balance due to placental vascular 
pathology as a potential cause.51  The increased risk of IUGR in fetuses with CHD was also 
suggested by a recent retrospective cohort study which found an approximately 2-to-3-fold 
increased risk of IUGR accompanied the fetal diagnosis of CHD (without chromosomal or 
extracardiac anomalies.77 Preliminary data collected recently at CHOP suggests that various 
placental abnormalities with the potential to alter placental function and fetal homeostasis 
frequently occur in pregnancies complicated by CHD.  Taken together, these data indicate 
that pregnancies complicated by fetal CHD may be at increased risk for abnormalities of 
placental anatomy and/or function that can compromise fetal growth and development and 
lead to increased risk of perinatal brain injury.  Thus, a comprehensive and multimodal 
analysis of placental anatomy, vascularity, and biochemical function is warranted. 
1.5.6 CHD and Preterm Birth 
There is increasing evidence that even late preterm and early term birth is associated 
with worse perinatal and long-term neurodevelopmental outcomes in both the normal fetus 
and the fetus with CHD.  In the fetus without CHD, there is significant neonatal morbidity 
associated with elective delivery at 37–38 weeks compared to deliveries occurring at 39–40 
weeks.81 Neonatal death, respiratory complications, hypoglycemia, sepsis, seizures, 
necrotizing enterocolitis, hypoxic-ischemic encephalopathy, cardiopulmonary resuscitation or 
ventilator support within 24 hours after birth, umbilical-cord-blood arterial pH below 7.0, a 5-
min Apgar of 3 or less, admission to the neonatal intensive care unit (NICU) and prolonged 
hospitalization of at least 5 days were significantly less likely to occur as gestational age 
increased from 37 (15.3%) to 39 weeks (8%), and plateaued at 40 weeks (7.3%).81 For the 
fetus without CHD, postponing elective delivery to 39 weeks may prevent 48% of cases of 
complications among deliveries at 37 weeks and 27% among 38-week deliveries.  
Preterm birth is common for mothers carrying fetuses with CHD. Among 397 babies with 
CHD delivered in the Special Delivery Unit (SDU) at CHOP in in fiscal years 2011 to 2013, 

 
  
 
 
 
13
53% were delivered at < 39 weeks gestational age and 16% at ≤ 37 weeks. Among 971 
neonates treated at Boston’s Children Hospital from 2002 through 2008, 56% were born at < 
39 weeks gestational age and 19% at ≤ 37 weeks.52 Data from the National Vital Statistics 
Report (http://www.cdc.gov/nchs/data/nvsr/nvsr62/) show that in 2011, among all births in the 
United States, 38% were at < 39 weeks gestational age and 12% were at < 37 weeks. These 
data suggest that the risk of preterm birth is increased for the fetus with CHD. 
Late preterm or early term birth also has serious adverse effects for the fetus with CHD. 
Investigators from Boston Children’s Hospital evaluated 971 consecutive neonates with CHD 
and a known gestational age born from 2002 through 2008.52 Compared with the referent 
group of neonates who were delivered at 39 to 40 completed weeks gestation, neonates born 
at 37 to 38 weeks had increased mortality (6.9% vs. 2.6%; adjusted P=0.049) and morbidity 
(49.7% vs. 39.7%; adjusted P=0.02) rates and tended to require a longer duration of 
mechanical ventilation (adjusted P=0.05). They concluded that for neonates with critical CHD, 
delivery before 39 weeks gestation is associated with greater mortality and morbidity rates 
and more resource use and that the ideal gestational age for delivery of these patients may 
be 39 to 40 completed weeks. Evidence from CHOP suggests that younger gestational age 
is associated with worse neurodevelopmental outcomes in children with CHD.53 
Neurodevelopmental outcomes were evaluated in 351 preschool children with CHD born at 
36 weeks or greater gestational age. Performance for cognition, language, executive 
function, social skills, visual-motor, and fine-motor skills was better for those born at 39 to 40 
weeks of GA or more versus those born at less than 39 weeks (all P<.05). These findings are 
consistent with the hypothesis that delivery before 39 to 40 weeks gestational age is 
associated with worse outcomes in patients with CHD. Early delivery of a child with CHD is 
often indicated because of maternal or fetal health issues. In the absence of these concerns, 
these data suggest that elective (or spontaneous) delivery at 39 to 40 weeks gestational age 
is associated with better neurodevelopmental outcomes. 
The reasons for an increased incidence of preterm birth for fetuses with CHD have not 
been fully delineated. Early delivery is sometimes required for maternal or fetal health issues. 
However, there is evidence that placental structure and function are often abnormal in 
pregnancies where the fetus has CHD, which may contribute to the risk of preterm birth. 
Preliminary data from CHOP shows that there are multiple abnormalities in the placenta of 
the fetus diagnosed with CHD, which may impact growth and development, timing of delivery 
and long-term outcomes such as neurodevelopment and late vascular health. Placentas from 
72 pregnancies were examined. Insertion of the umbilical cord into the placenta was eccentric 
in 61 (84%) and velamentous in 3 (4%). Abnormal maturation of villi was present in 29%; 
chorioangiosus in 14%. An abnormal fetal/placental weight ratio (as compared to published 
norms) was seen in 46%. Evidence for inflammation was present overall in 19% and vascular 
thrombosis in 16%. Infarction was present overall in 14%, but most commonly in HLHS and 
TGA (25%). There is evidence that the fetus with CHD is at increased risk for intra-uterine 
growth retardation (IUGR), which may be due in part to uteroplacental insufficiency.77 In a 
retrospective cohort study of 67,823 patients, there were 193 cases of fetal CHD (0.3%) and 
5,669 cases of IUGR (8.4%). Prenatal diagnosis of CHD was associated with an increased 
risk of IUGR (23.8% vs. 8.5%, adjusted odds ratio [aOR] 3.3, 95% confidence interval [CI] 
2.4–4.6), and the risk was greatest in fetuses with major CHD (16.5% vs. 8.5%, aOR 2.1, 
95% CI 1.3–3.2).77 Isolated CHD was also associated with an increased risk of IUGR (17.8% 
vs. 8.5%, aOR 2.2, 95% CI 1.4–3.7). The increased risk persisted after excluding extracardiac 
and chromosomal anomalies. There was a significantly increased incidence of single 
umbilical artery in fetuses with CHD, consistent with structural placental abnormalities. There 
is no data concerning progesterone levels and metabolism in pregnancies with a fetus with 

 
  
 
 
 
14
CHD. Overall, the existing data suggest that pregnancies with CHD are at increased risk for 
uteroplacental insufficiency and may be considered stressed or compromised pregnancies. 
 
1.6 COMPLIANCE STATEMENT 
This study will be conducted in full accordance all applicable Children’s Hospital of 
Philadelphia Research Policies and Procedures and all applicable Federal and state laws 
and regulations including 45 CFR 46, 21 CFR Parts 50, 54, 56, 312, 314 and 812 and the 
Good Clinical Practice: Consolidated Guideline approved by the International Conference on 
Harmonization (ICH). All episodes of noncompliance will be documented. 
The investigators will perform the study in accordance with this protocol, will obtain 
consent and assent, and will report unanticipated problems involving risks to subjects or 
others in accordance with The Children’s Hospital of Philadelphia IRB Policies and 
Procedures and all federal requirements. Collection, recording, and reporting of data will be 
accurate and will ensure the privacy, health, and welfare of research subjects during and 
after the study.  
2 
STUDY OBJECTIVES 
We propose a Phase II single-center placebo-controlled randomized therapeutic 
exploratory trial to determine whether there is evidence that progesterone, administered pre-
natally, improves neurodevelopmental outcomes, improves brain development, and reduces 
perioperative white matter injury in neonates with CHD undergoing cardiac surgery. The data 
provided by this therapeutic exploratory study will aid in selection of appropriate outcomes 
and inform sample size calculations for a future multi-institutional study.  
2.1 
Primary Objective (or Aim) 
Primary Aim: Develop preliminary evidence to support a multi-institutional study to 
determine whether, in women carrying fetuses with CHD (maternal-fetal dyad), prophylactic 
vaginal natural progesterone therapy is neuroprotective, and compared to placebo improves 
neurodevelopmental outcomes at 18 months of age. 
Hypothesis: 
Compared to placebo, progesterone administration results in a higher score on the Motor 
Scale of the Bayley Scales of Infant and Toddler Development-III (Bayley-III) at 18 months of 
age. 
2.2 
Secondary Objectives (or Aims) 
Specific Aims: Develop preliminary evidence to support a multi-institutional study to 
determine whether, in women carrying fetuses with CHD (maternal-fetal dyad), prophylactic 
vaginal natural progesterone therapy is neuroprotective, and compared to placebo: 
1. improves fetal brain growth and maturation, 
2. increases myelination during fetal brain development, 
3. reduces pre-operative brain white matter injury, and 
4. reduces post-operative white matter injury. 
Hypotheses: 
Compared to placebo, progesterone administration results in 
1. higher scores on the Cognitive and Language Scales of the Bayley Scales of Infant    
and Toddler Development-III (Bayley-III) at 18 months of age, 

 
  
 
 
 
15
2. a higher total maturation score (TMS) on early post-natal MRI, 
3. Increased global and regional brain volumes 
4. a greater volume of myelinated white matter (VMWM) in the posterior limb of the 
internal capsule on early post-natal, pre-operative MRI, 
5. lower periventricular leucomalacia (PVL) volume on early post-natal, pre-operative 
MRI, 
6. lower PVL volume on post-operative MRI. 
3 
INVESTIGATIONAL PLAN 
3.1 
General Schema of Study Design 
Phase II single-center placebo-controlled randomized therapeutic exploratory trial 
3.1.1 Screening Phase 
Potential subjects will be identified by clinically indicated fetal echocardiography in the Fetal 
Heart Program Clinic by one of the investigators (J.R. or A.S.). Potential subjects will be 
screened for eligibility at 24-28 weeks GA by a study nurse using the protocol inclusion and 
exclusion criteria.  Subject permission (informed consent) will be obtained prior to any study 
related procedures being performed. 
3.1.2 In Utero Treatment Phase  
After informed consent is obtained, the subject will undergo baseline evaluation including 
medical history review, physical examination, obstetric ultrasound, fetal echocardiography, 
and fetal brain MRI. Maternal and paternal blood samples will be obtained. Progesterone 
levels will be assessed in the mother. Serum samples will be banked for later assessment of 
progesterone metabolites and biomarkers. The maternal and paternal samples will be used 
for genetic testing.  Subjects will be randomized to study drug or placebo. For scheduling 
reasons, off site SD initiation may occur. After randomization, the subjects will return for 
routine prenatal visits every 2 weeks until 36 weeks GA and weekly thereafter. Obstetric 
ultrasound and fetal echocardiography will be performed every 4 weeks and as clinically 
indicated. Maternal blood tests will be repeated every 4 weeks.  Fetal brain MRI will be 
obtained at 34-36 weeks GA. The study drug will be continued until 39 weeks GA, the onset 
of spontaneous labor if prior to 39 weeks GA, or the time of induction of labor or cesarean 
section for maternal or fetal health concerns. At the time of delivery, maternal and cord blood 
samples will be obtained. A portion of the placenta will be obtained. 
3.1.3 Post-Natal Phase 
After birth, the child will undergo clinically indicated evaluation and treatment. Data will 
be collected from the medical record concerning diagnosis, associated anomalies, medical 
and surgical management and the hospital course. A blood (either from the cord or from an 
existing blood drawing line during a hospitalization, routine clinical labwork or at the time of 
a research draw for a separate study) or saliva sample will be obtained for genetic testing. 
An early post-natal brain MRI will be obtained on the day of cardiac surgery, during the same 
anesthetic but prior to surgery.  Post-operative brain MRI will be obtained within 10 days of 
surgery. 
3.1.4 Neurodevelopmental Evaluation 
All surviving infants will undergo neurodevelopmental evaluation at 18 months of age 
(corrected for prematurity).  An interim medical history will be obtained, including details of 
any hospitalizations and surgical procedures. A neurological examination will be performed. 
Growth parameters will be assessed. The neurodevelopmental evaluation will include the 

 
  
 
 
 
16
Bayley Scales of Infant and Toddler Development-III, the Modified Checklist for Autism in 
Toddlers (M-CHAT), and the Infant-Toddler Social and Emotional Assessment (ITSEA). 
3.2 
Allocation to Treatment Groups and Blinding 
3.2.1 Randomization 
The study biostatistician, Dr. Putt, will generate a block randomization schedule based 
on stratification by fetal CHD diagnosis: TGA, HLHS, and other CHD. The randomization 
schedule will be based on equal allocation of subjects to the progesterone and placebo arms 
within each block. To ensure blinding, the randomization schedule will be generated using 
variable block sizes. Upon confirmation of eligibility in the DMS, the randomization module 
will be programmed to generate a randomization number that corresponds to a unique kit 
number of progesterone or placebo treatment. This kit number will be used to distribute 
treatment. 
3.2.2 Blinding 
All primary study team members will be blinded to treatment arm. The database 
administrator will have access to the information that links the kit number to the treatment 
arm. He/she will provide this information to the Investigational Pharmacy so that kits are 
prepared according to this scheme. Progesterone and placebo treatment kits will appear 
identical. They will be labeled with the following information: 
1. Kit number 
2. Study name and number 
3. Subject ID # [blank line to record this number] 
3.2.3 Unblinding 
Unblinding can occur if there are concerns over maternal or fetal health, or if requested 
by the DSMB. A direct request from the PI to the Investigational Pharmacy will be used to 
initiate unblinding. All requests for unblinding will be documented and reported to the DSMB. 
3.3 
Study Duration, Enrollment and Number of Sites 
3.3.1 Duration of Study Participation 
The study duration for each mother will consist of the in utero treatment phase beginning 
at 24-28 weeks GA and continuing until 30 days after stopping study drug or after delivery. 
The study duration for each fetus/child will include the in utero treatment phase, the post-
natal phase and the neurodevelopmental evaluation. The study will begin at 24-28 weeks GA 
and continue to 18 months of age (corrected for prematurity). 
3.3.2 Total Number of Study Sites/Total Number of Subjects Projected 
The study will be conducted at a single site, the Children’s Hospital of Philadelphia. We 
plan to enroll sufficient patients to have 80 evaluable patients (40 per treatment group) at the 
18 month neurodevelopmental testing. Based on known clinical outcomes for this patient 
population and experience with other studies with similar follow-up plans, we estimate a 5% 
early mortality and a further 20% attrition from all causes between hospital discharge and the 
18 month evaluation. There may also be dropout prior to birth for a variety of reasons: fetal 
death, intolerance of therapy, withdrawal from study. We do not have accurate data to 
estimate this loss. We anticipate needing to enroll 110-130 patients to have a sufficient cohort 
at 18 months (40 per treatment group), with 60 and 52-56 patients, respectively, per treatment 
group available for the post-natal and post-operative brain MRIs. Based on current patient 

 
  
 
 
 
17
volume in the Fetal Heart Program Clinic, we anticipate there will be 70-75 eligible patients 
each year. If the consent rate is 65%, we will be able to complete enrollment within 3 years. 
3.4 
Study Population 
3.4.1 Inclusion Criteria 
1) Mothers carrying a fetus with CHD (maternal-fetal dyad) requiring surgery with CPB 
prior to 44 weeks corrected GA identified prior to 28 weeks GA will be eligible.  
2) Informed consent 
 
3.4.2 Exclusion Criteria 
1) Major genetic or extra-cardiac anomaly other than 22q11 deletion 
2) Language other than English spoken in the home 
3) Known sensitivity or listed contraindication to progesterone (known allergy or 
hypersensitivity to progesterone, severe hepatic dysfunction, undiagnosed vaginal 
bleeding, mammary or genital tract carcinoma, thrombophlebitis, thromboembolic 
disorders, cerebral hemorrhage, porphyria)  
4) Prescription or ingestion of medications known to interact with progesterone (e.g. 
Bromocriptine, Rifamycin, Ketoconazole or Cyclosporin)  
5) Maternal use of progesterone within 30 days of enrollment 
6) History of preterm birth or short cervix (defined as cervical length ≤ 25 mm at 18-24 weeks 
GA  necessitating progesterone therapy 
7) Multiple gestation 
8) Maternal contraindication for MRI 
Subjects with a known history of non-compliance with medical therapy. 
Subjects that do not meet all of the enrollment criteria may not be enrolled. Any violations of 
these criteria must be reported in accordance with IRB Policies and Procedures.  
4 
STUDY PROCEDURES 
4.1.1 
Screening Visit 
 Confirmation of Eligibility 
 Informed Consent 
 Medical Record Review 
 Fetal Echocardiography (including assessment of CBF) 
 
4.1.2 Randomization Visit (for scheduling reasons subjects may be offered off site 
study drug initiation) 
 If not previously obtained at the screening visit, Informed Consent will be obtained 
prior to study procedures being performed 
 Maternal Blood Tests  
 Maternal and Paternal Blood for Genetic Testing 
 Fetal Brain MRI 
 
4.2 
In Utero Treatment Phase 
Subjects will be randomized to study drug or placebo. After randomization, the subjects will 
return for routine prenatal visits every 2 weeks until 36 weeks GA and weekly thereafter. 

 
  
 
 
 
18
Obstetric ultrasound and fetal echocardiography will be performed every 4 weeks and as 
clinically indicated. Maternal blood tests will be repeated every 4 weeks. 
4.2.1 Prenatal Visit 1 
 Medical Record Review 
 Fetal Echocardiography (including assessment of CBF) 
 Dispense Study Drug 
4.2.2 Subsequent Prenatal Visits (every 2 weeks until 36 weeks GA, then weekly) 
 Medical Record Review 
 Fetal Echocardiography (including assessment of CBF) (every 4 weeks or as 
clinically indicated) 
 Maternal Blood Tests (every 4 weeks) 
 Collect unused study drug 
 Dispense study drug 
 Assess possible adverse events  
4.2.3 Follow-up Fetal Brain MRI at 34-36 Weeks GA 
4.2.4 Delivery 
 Medical Record Review 
 Maternal and Cord Blood Tests  
 Collect unused study drug 
 Assess possible adverse events  
 
4.2.5 Post-Natal Phase 
After birth, the child will undergo clinically indicated evaluation and treatment. Data will 
be collected from the medical record concerning diagnosis, associated anomalies, medical 
and surgical management and the hospital course. An early post-natal brain MRI will be 
obtained on the day of cardiac surgery, during the same anesthetic but prior to surgery.  Post-
operative brain MRI will be obtained within 10 days of surgery. 
If blood sample for genetic testing was not previously obtained during the post-natal 
hospitalization as a result of the discontinuation of all nonessential on-campus research due 
to COVID-19 or it was determined there was insufficient DNA for analysis, consent will be 
obtained using the consent addendum prior to obtaining additional samples. 
4.2.6 Post-Natal Hospitalization 
 Evaluation by dysmorphologist 
 Infant Blood for Genetic Testing 
 Medical Record Review 
 Assess possible adverse events  
 Early post-natal brain MRI (day of surgery) 
 Post-operative brain MRI (within 10 days of surgery) 
 
4.2.7 Subsequent Hospitalization/Outpatient visit 
 
Infant Blood for Genetic Testing from an existing blood drawing line or drawn at the 
time of routine clinical labwork (if cord or infant blood not previously obtained during 

 
  
 
 
 
19
the post-natal hospitalization as a result of the discontinuation of all nonessential on-
campus research due to COVID-19 or if it was determined there was insufficient 
DNA for analysis) 
 
Maternal and Paternal Blood or Saliva for Genetic Testing (if blood not previously 
obtained as a result of the discontinuation of all nonessential on-campus research 
due to COVID-19 or if it was determined there was insufficient DNA for analysis) 
4.2.8 Neurodevelopmental Evaluation 
All surviving infants will undergo neurodevelopmental evaluation at 18 months of age 
(corrected for prematurity).  An interim medical history will be obtained, including details of 
any hospitalizations and surgical procedures. Growth parameters will be assessed. A 
neurological examination will be performed. The neurodevelopmental evaluation will include 
the Bayley Scales of Infant and Toddler Development-III, the Modified Checklist for Autism 
in Toddlers (M-CHAT), and the Infant-Toddler Social and Emotional Assessment (ITSEA). 
4.2.9 Neurodevelopmental Evaluation and End-of-Study 
 Physical and neurological examination 
 Evaluation by dysmorphologist (may be performed via telephone or video 
conference due to the potential risks of COVID-19) 
 Vital signs and growth parameters  
 Medical record review 
 Assess possible adverse events  
 Bayley Scales of Infant and Toddler Development-III 
 Modified Checklist for Autism in Toddlers (M-CHAT) 
 Infant-Toddler Social and Emotional Assessment (ITSEA). 
 Child saliva sample for genetic testing (if cord or infant blood not previously 
obtained during the post-natal hospitalization as a result of the discontinuation of all 
nonessential on-campus research due to COVID-19 or if DNA quantity was 
determined to be insufficient) 
 Maternal and paternal blood or saliva for genetic testing (if blood not previously 
obtained as a result of the discontinuation of all nonessential on-campus research due 
to COVID-19 or insufficient DNA for analysis) 
 
4.2.10 Subject’s Home 
 
Maternal, paternal or child saliva or buccal epithelial cell sample for genetic testing 
(if cord or infant blood not previously obtained during the post-natal hospitalization 
as a result of the discontinuation of all nonessential on-campus research due to 
COVID-19 or if DNA quantity was determined to be insufficient) 
 
4.3 
Concomitant Medication 
All prior and concomitant medications used by the mother within 30 days prior to the 
screening visit and through the end of the study will be recorded. The dates of administration, 
dosage, and reason for use will be included.  

 
  
 
 
 
20
4.4 
Rescue Medication Administration 
In previous studies, the vaginal progesterone gel has been well tolerated with few adverse 
effects. The most common effect is vaginal irritation which will be treated with sitz baths; and 
for severe cases a low potency topical steroid cream (hydrocortisone 1 % cream). If a woman 
goes into preterm labor, this will be treated according to standard clinical protocols. 
4.5 
Subject Completion/Withdrawal 
Subjects may withdraw from the study at any time without prejudice to their care.  They 
may also be discontinued from the study at the discretion of the Investigator for lack of 
adherence to study treatment or visit schedules and adverse events (AE).  The Investigator 
may also withdraw subjects who violate the study plan, or to protect the subject for reasons 
of safety or for administrative reasons.  It will be documented whether or not each subject 
completes the clinical study. If the Investigator becomes aware of any serious, related 
adverse events after the subject completes or withdraws from the study, they will be recorded 
in the source documents and on the CRF. For women without a history of preterm birth, there 
are no known risks to progesterone withdrawal. 
4.5.1  Early Termination Study Visit 
No procedures are required to be completed prior to withdrawal 
5 
STUDY EVALUATIONS AND MEASUREMENTS 
5.1 
Screening and Monitoring Evaluations and Measurements 
5.1.1 Pre-Natal Medical Record Review 
Please see Appendix 
5.1.2 Post-Natal Medical Record Review 
Medical Chart Review 
Please see Appendix. 
5.1.3 Physical Examination 
Maternal Examination: 
 
General physical examination (at initial visit). 
 
Blood pressure/ pulse (at each visit). 
 
Height/Weight/BMI /Fundal height (at each visit). 
 
Pelvic examination /Clinical pelvimetry (third trimester). 
Infant Evaluation: 
CICU Admission: Each child will undergo clinically indicated physical examination, 
laboratory evaluation and imaging studies. 
Genetic Screening by a Dysmorphologist: All children entered into the study will be 
evaluated for other syndromic diagnosis by Dr. Elaine Zackai, a dysmorphologist.  In any 
study of the neurologic outcome of patients with CHD, it is important to determine whether 
there is an underlying syndromic diagnosis that in and of itself would impact on outcome. 
5.1.4 Vital Signs 
Vital signs will be monitored and recorded as clinically indicated. 

 
  
 
 
 
21
5.1.5 Laboratory Evaluations 
Maternal Evaluation: As part of routine prenatal care, each mother will undergo clinically 
indicated testing. The laboratory tests listed below should have been obtained by the 
referring physician at the initial prenatal care visit. If not previously collected, clinically 
indicated testing will be completed as standard of care with the timing determined by the 
clinicians caring for the mother. 
Clinical Laboratory Tests: 
 
Hemoglobin/hematocrit /platelet count (to be repeated in the third trimester-36 
weeks). 
 
Blood type and antibody screen (to be repeated at 24-28 weeks). 
 
Rubella titer.  
 
Urine (protein, sugar, blood, leukocyte esterase at each visit). 
 
Diabetes screen (at 24-28 weeks). 
 
Hepatitis B surface antigen. 
 
Hepatitis C antibodies (optional). 
 
HIV (to be repeated in the third trimester-36 weeks). 
 
Syphilis test (to be repeated in the third trimester-36 weeks). 
 
Pap smear. 
 
Urinary drug screen (as clinically indicated). 
 
Urine culture. 
 
Gonorrhea and Chlamydia cultures (to be repeated in the third trimester-36 weeks). 
 
Group B streptococcus culture (to be obtained at 36 weeks). 
Other Evaluation (only if clinically indicated): 
 
Cytomegalovirus, herpes simplex, toxoplasmosis, and varicella maternal titers, 
hemoglobin electrophoresis, Tay-Sacks, fragile X, cystic fibrosis and spinomuscular 
carrier screen, and parental karyotype will be offered to some. 
 
Screening for chromosomal abnormalities: first trimester screen, Quad screen, non-
invasive prenatal testing (NIPT). 
 
Fetal karyotype (by chorionic villous sampling or amniocentesis). 
 
Chromosomal or genetic evaluation through maternal serum assessment for free fetal 
DNA. 
Infant Evaluation: As part of routine care, each infant will undergo clinically indicated testing. 
5.1.5.1 Pregnancy Testing 
Not applicable 
5.1.6 Other Evaluations, Measures 
Neonatal Admission Questionnaires 
Demographic, Ethnicity and Socioeconomic Status (SES) Forms: demographic 
information will be collected in order for the study team to maintain contact for 18-month 
follow-up.  In addition self-report ethnicity, level of education, and type of employment will be 

 
  
 
 
 
22
obtained on both mother and father.  This information will serve as data to calculate the 
Hollingshead score, a measure of SES.  
Maternal and Cord Blood Tests 
Maternal blood will be obtained at the first visit (unless off site study drug initiation has 
occurred), each subsequent prenatal visit and at the time of delivery. Maternal blood for 
genetic testing (if blood not previously obtained as a result of the discontinuation of all 
nonessential on-campus research due to COVID-19 or insufficient DNA for analysis) will be 
obtained during a child’s subsequent hospitalization or outpatient visit to CHOP. Cord blood 
will be obtained at the time of delivery. Infant blood will be obtained during a hospitalization 
through the infant’s existing blood drawing line or during routine clinical bloodwork if cord 
blood was not obtained at the time of delivery or DNA quantity was determined to be 
insufficient for analysis. Maternal blood will be collected into four plasma preparation tubes 
(K-EDTA, 4 mL whole blood each) on ice, centrifuged at 600 X g for 5 min at 4 oC, and the 
plasma supernatant aliquoted into separate 2 mL cryovials (to avoid repeated freeze-thaw 
events) and the red blood cells returned to each of the four preparation tubes (to avoid 
repeated freeze-thaw events).  The cryovials containing separates plasma samples are 
rapidly frozen in liquid nitrogen and then stored at –80 oC (Clinical and Translational Research 
Center and CHOP Clinical Laboratory) for subsequent assay (ELISA or qRT-PCR).  The 
same procedures will be used to collect and store umbilical venous blood at the time of 
delivery.  One aliquot will be sent for assessment of progesterone levels. Additional samples 
will be stored at -80 C for later assessment of progesterone metabolites and biomarkers of 
placental function. The serum progesterone levels will be performed by the CHOP Clinical 
Laboratory. The total blood volumes over the course of the study will be: Maternal: 90 ml; 
Paternal: 5 ml; Cord Blood: 22 ml; Infant 5 ml (only if cord blood not obtained). If DNA quanitity 
was determined to be insufficient an additional 5 mL maternal, paternal and infant sample 
may be obtained. If blood will be obtained at the time of a research draw for a separate study 
the amount drawn will not exceed the lesser of 50 mL or 3 mL/kg, even when a participant is 
providing samples for multiple studies. 
Saliva Samples 
Blood is the preferred specimen for DNA collection. However, at times a blood draw will 
not be possible to obtain. In those instances, we will obtain a saliva sample or a sample from 
a swab of the inner aspect of the cheek. The saliva may be obtained from enrolled subjects 
by the study team either in person or by mail. 
 
Placenta 
Gross Examination: 
The placenta will be weighed and examined visually to determine the location of umbilical 
cord insertion, the types and number of umbilical vessels, and to identify and quantify 
macroscopic areas, size, and location of abnormalities such as calcification, infarction, 
hemorrhage, etc. 
Tissue Histology and Biochemistry: 
The placenta is a heterogeneous organ with regard to normal functions and 
pathophysiologic injury.  Specific tissue sampling strategies to limit bias and maximize 
analytical accuracy and precision have been described.82-84 In brief, it is necessary to analyze 
multiple samples to ensure that the result is an adequate representation of the entire 
organ.   To this end, a systematic uniform random stereologic sampling design will be 

 
  
 
 
 
23
employed to generate full-thickness samples (approximately 8-12 per placenta, depending 
upon the size of the organ the random orientation of the sampling grid).82, 83  Each specimen 
will be divided in equal portions for 1) fixation and histochemical analyses and 2) rapid 
freezing and subsequent molecular and biochemical determinations. 
Genetic Testing 
Sample Procurement and Preparation: Whole blood will be obtained from the biological 
parents and from the infant through an existing blood drawing line during a hospitalization or 
during routine clinical bloodwork. The blood samples will be sent directly to Center for Applied 
Genomics (CAG) for DNA extraction and Cell Line preparation.  The total blood volumes over 
the course of the study will be: Maternal: 86 ml; Paternal: 5 ml; Cord Blood: 22 ml; Infant 5 
ml (only if cord blood not obtained). If DNA quanitity was determined to be insufficient an 
additional 5 mL maternal, paternal and infant sample may be obtained. 
APOE Genotyping: Genomic DNA for analysis will be obtained from each peripheral 
blood sample or saliva collection kit.  Crude leukocyte nuclei are prepared from peripheral 
blood by standard techniques.  High molecular weight DNA will be extracted via the Genepure 
automated nucleic acid extractor (Applied Biosystem Inc.) utilizing the supplied reagents and 
protocol.  A sample of genomic DNA will be used to determine APOE genotypes using a 
polymerase chain reaction (PCR) based restriction enzyme isoform genotyping protocol. The 
remaining DNA will be banked at   20 °C for future analyses.  The APOE genotype will be 
used as a covariate in the analysis of the neurodevelopmental testing results. 
Genome Wide Association Testing: Genome Wide Association Testing will be 
performed by CAG, at The Children’s Hospital of Philadelphia. CAG is experienced in SNP 
genotyping at an industrial scale. The Illumina technology uses single tube whole genome 
amplification followed by primer-extension without PCR or ligation. Infant and biological 
parent blood samples will be processed by CAG.  The GWAS will be used to evaluate the 
parent-child trios for possible copy number variations (CNV). The CNV data will be used as 
a covariate in the analysis of the neurodevelopmental testing results. We will ask the parents 
for permission to bank the DNA for future studies and to share the results with other 
investigators. 
 
5.2 
Efficacy Evaluations 
5.2.1 Obstetric Ultrasound 
The ultrasound examination will be conducted with real-time scanners, using a 
transabdominal or a transvaginal approach or both. All examinations will be performed by 
one of three experienced perinatal sonographers using the same ultrasound equipment, who 
will be blinded to the patient group assignment. The abdominal and transvaginal transducers 
to be used will include frequencies ranging from 3 MHz to 10 MHz or higher to provide 
superior resolution while allowing adequate penetration.  The detailed obstetric ultrasound 
examination to be performed at the initial evaluation will include cardiac activity, fetal 
presentation, amniotic fluid index (AFI), placental position, fetal biometry, an anatomic 
survey.  The maternal cervix (normal, funneled, short or long (in centimeters) and adnexa will 
also be examined (presence, location and size of uterine or adnexal masses). 
Cardiac activity: fetal heart rate and rhythm. 
Fetal presentation: cephalic, breech, transverse or variable. 

 
  
 
 
 
24
Amniotic fluid index: less than 5 cm, oligohydramnios and more than 24 cm, 
polyhydramnios or deepest amniotic fluid vertical pocket (less than 2 cm, oligohydramnios 
and more than 8 cm, polyhydramnios). 
Placental position: anterior, posterior, fundal, lateral, low-lying or previa.  
Placental cord insertion: central, marginal, eccentric, velamentous. 
Fetal Biometry at initial Examination: 
 
Biparietal diameter (BPD) will be obtained at the level of the thalami and cavum septi 
pellucid, and the measurement will be taken from the outer edge of the proximal skull 
to the inner edge of the distal skull. 
 
Head circumference (HC) will be measured at the same level as the BPD, around the 
outer perimeter of the calvaria. 
 
Femoral length (FL) and humeral length (HL) will be measured with the beam of 
insonation perpendicular to the shaft, excluding the distal femoral or humeral 
epiphysis. 
 
Abdominal circumference (AC) will be determined at the skin line on a true transverse 
view at the level of the junction of the umbilical vein, portal sinus, and fetal stomach 
when visible. 
 
Estimated fetal weight (EFW) will be obtained using a combination of measurements 
such as BPD, HC, AC and FL from previously described formulas, expressed in 
grams, and compared with weight percentiles from published nomograms. 
Elements of the Fetal Anatomic Survey: 
 
Head, Face and Neck: Cerebellum, choroid plexus, cisterna magna, lateral ventricles, 
midline falx, cavum septi pellucidi, third ventricle, corpus callosum, posterior fossa, 
cerebellum, transcerebellar diameter (in centimeters), profile, nose, nasal bone, 
orbits,  lips and palate. 
 
Chest-Heart: Four-chamber view, outflow tracts, situs, fetal heart rate and rhythm. 
 
Abdomen: Stomach (presence, size and situs), kidneys, bladder, umbilical cord 
insertion into the fetal abdomen, umbilical cord vessel number. 
 
Spine: Cervical, thoracic, lumbar and sacral spine. 
 
Extremities: Legs and arms (presence or absence). Femoral and humeral length in 
centimeters. Other long bone lengths will be obtained as clinically indicated. 
 
Genitalia (male, female, ambiguous). 
Fetal Biometry at Repeat Examination: (ultrasound for growth to be performed at 4 week 
intervals) 
 
BPD, HC, FL, HL, AC, EFW. 
 
Fetal presentation. 
 
Amniotic fluid index and/or amniotic fluid deepest vertical pocket. 
 
Placental position (placental position at the initial evaluation may not correlate with its 
location in the third trimester). 

 
  
 
 
 
25
 
Maternal cervix (cervical length will be measured transabdominally or transvaginally 
at each sonographic evaluation until 32 weeks gestation) and presence, location and 
size of uterine and adnexal masses.  
Doppler Ultrasonography: 
Doppler color flow mapping with reduced color scale will be used to identify the ductus 
venosus (DV) and the middle cerebral artery (MCA) and average color scale to locate the 
umbilical artery (UA) and maternal uterine arteries (UtA).  Pulsed- wave Doppler will be used 
to determine blood flow velocities of the UA, UtA, DV and MCA.  The peak systolic velocity, 
peak diastolic velocity, mean velocity, systolic to diastolic ratio (SD), pulsatility index (PI) 
resistive index (RI) and UA PI/MCA PI with a mixture from stable signals during fetal apnea 
will be obtained for each of the aforementioned vessels at the initial evaluation at 24-28 
weeks and then every 4 weeks. 
5.2.2 Fetal Echocardiography 
All patients will have standard fetal echocardiographic examinations and Doppler flow 
studies at the time of enrollment and then every 4 weeks (or as clinically indicated) until 37-
39 weeks of gestation. The studies will be performed by two to three dedicated cardiac 
sonographers under direct supervision of a pediatric cardiologist. The examinations will be 
performed using a Siemens ACUSON Sequoia 512 ultrasound system or equivalent coupled 
with either a 6 C2 or an 8V3 MHz transducer (Siemens, Erlangen, Germany). Multiple two-
dimensional views will be obtained to evaluate fetal heart anatomy. Doppler interrogation will 
be performed to evaluate valve competence, stenosis and shunting. M-mode will be used to 
assess cardiac rhythm. Color-flow mapping will be used to verify the anatomy, as well as 
intracardiac flow.  The two-dimensional images will be recorded on standard video clips for 
subsequent analysis.  Doppler samples will be obtained at the following sites: aortic valve, 
pulmonic valve, atrioventricular valve inflows, transverse aortic arch, ductus arteriosus, 
umbilical vein, DV, MCA, UA, and UtA.   
For the aortic outflow, the sample volume will be placed at the level of the aortic valve on 
either a 5 chamber view of the fetal heart or on a long axis view of the fetal heart. For the 
pulmonic outflow, the sample volume will be placed at the level of the pulmonic valve on 
either a right ventricular outflow tract view or on a ductal view. The transverse aortic arch will 
be sampled on the aortic arch view while the ductus arteriosus will be sampled on the ductal 
view. The MCA will be sampled in the mid-portion of the vessel while the umbilical artery will 
be sampled in a free-floating loop. The UtA will be sampled below the crossing point of the 
internal iliac artery. The velocity waveforms will be obtained using an angle of insonation 
lower than 20°.  The length of the sample volume will range between 1 and 4 mm.  All 
recordings will be performed during periods of both fetal rest and apnea.  Aortic valve 
diameters will be assessed from the two-dimensional images of apical five-chamber views or 
long axis views of the fetal heart. Pulmonic valve diameters will be assessed from the two-
dimensional images of the right ventricular outflow tract view or the ductal view of the fetal 
heart. The measurements will be performed from video clips during systole and only the 
maximum diameters will be selected.  Measurements will be repeated on a minimum of 3 
different cardiac cycles for each valve and the values obtained will be averaged. Valvular 
area of the aortic and pulmonic valves will be calculated assuming a circular cross-section, 
according to the formula π X (valve diameter/2)2. The velocity time integral across both the 
aortic and pulmonic valve will be calculated at the area under the curve of the aortic and 
pulmonic outflow Doppler samples, as described above. Absolute right (RVO) and left (LVO) 
ventricular output (mL/min) will be derived by multiplying aortic and pulmonic valvular area, 
fetal heart rate and time velocity integral across the respective semilunar valve.  The 

 
  
 
 
 
26
combined ventricular output (CVO= RVO + LVO) and the ratio between RVO and LVO 
(RVO/LVO) will then be calculated. 
The pulsatility index (PI) for the MCA, UA, UtA, and ductus arteriosus will be calculated 
according to the formula: (peak systolic velocity – end-diastolic velocity)/time-averaged mean 
velocity. The direction of flow in the transverse aortic arch will be recorded as all antegrade, 
all retrograde, or bidirectional. Any ductal constriction will be noted. A qualitative assessment 
of left and right ventricular cardiac function will be made. Finally, the presence or absence of 
hydrops fetalis will be noted.  
5.2.3 Fetal Cerebral Blood Flow 
MCA Flow Velocities and Pulsatility Index 
Doppler color flow mapping with reduced color scale will be used to identify the circle of 
Willis and the MCA.  Pulsed-wave Doppler will be used to determine blood flow velocities in 
the UA, ductus venosus (DV) and MCA.  The peak systolic velocity, peak end- diastolic 
velocity, and time averaged mean velocity are calculated on three consecutive samples 
during fetal apnea, and the results averaged.  The pulsatility index (PI), a measure of vascular 
resistance in the circulatory bed downstream from the point of  Doppler sampling will be 
calculated according to the equation, PI = (peak systolic velocity – end diastolic velocity)/time 
averaged mean velocity. The ratio of UA-PI to MCA-PI will be designated the U/C-PI ratio. PI 
measurements of the aforementioned vessels will be converted to gestational age-based Z-
scores to allow comparisons between the treatment and control group with previously 
published normal values.  
Fractional Moving Blood Volume 
Ultrasound and power Doppler studies will be performed transabdominally, using a 
Phillips iU 22 (Koninklijke Phillips N.V Eindhoven, The Netherlands) ultrasound system with 
a C-9 MHz linear curved array transducer in the absence of fetal respiratory and body 
movements, and with mother and fetus in suspended respiration.  The following anatomical 
planes of the fetal brain will be included in the pulse Doppler ultrasound color box: anterior 
and complete mid-sagittal, and oblique at the level of the posterior fossa.  During the mid- 
sagittal views, special care will be taken to insonate through the sagittal suture in order to 
avoid the bone barrier and have an uncluttered power Doppler ultrasound recording.  The 
size of box will be maintained as small as possible, in order to cover the different regions 
under investigation and to improve the frame rate.  The ultrasound and power Doppler 
ultrasound settings to be used in all examinations will include: standard gray-scale images 
for obstetrics, medium persistence, high sensitivity, normal image display, pulse repetition 
frequency 610 Hz, medium wall filter, and gain level just below the presence of noise.  The 
mechanical and thermal indices will always be maintained below 1.  A minimum sequence of 
10 consecutive good quality images without flash artifacts would be stored digitally and 
transferred to a personal computer for FMBV analysis in software designed specifically for 
the purpose. (MatLab, MathWorks, Natick, MA, USA).  The regions of interest (ROI) will be 
defined and delimited as follows: 1) anterior cerebral, in an anterior mid sagittal view of the 
fetal head, delimited anteriorly by the internal wall of the skull, posteriorly by an imaginary 
line drawn at 90° at the level of the origin of the anterior cerebral artery and parallel to an 
imaginary line in the front of the face, and inferiorly by the base of the skull, 2) complete mid-
cerebral, in a mid- sagittal plane delimited by the complete internal wall of the fetal skull and 
3) posterior fossa, delimited anteriorly and by the base of the cerebellar hemispheres and 
posteriorly by the fetal skull.  Each image will be rendered and the pixels containing color 
information within the ROI will be selected for analysis. FMBV will be calculated according to 
the methodology described elsewhere.85, 86  

 
  
 
 
 
27
5.2.4 Fetal Brain MRI 
Prenatal MRI examinations will be performed once at 25-28 weeks GA and once at 34-
36 weeks GA.  
The prenatal MRI sequences will include:  
1. T2 weighted images including half-Fourier acquisition single shot turbo spin echo 
T2 images in axial, sagittal, and coronal planes.  
2. Echoplanar images at short and long TE to assess for hemorrhage.  
3. T1 fast low angle shot images (if bleeding or clotting is suspected on other images)  
4. Magnetic resonance spectroscopy.   
5. Diffusion imaging. 
 
Analysis of the fetal Brain MRI examinations will include:  
1. Evaluation for anatomic abnormalities (malformations) and in-utero gross insults.  
2. Size of ventricles.  
3. Assessment of hemorrhage.  
4. Fetal total maturation score (fTMS) evaluation which assesses myelination, cortical 
folding, and the germinal matrix evolution.  
5. Assessment of brain volumetrics. 
6. MR spectroscopic assessment of choline, N-acetylaspartate, lactate, and creatine 
metabolites, including corresponding ratios. 
 
5.2.5 Post-Natal Brain MRI 
Postnatal MRI examinations will include an MRI on the day of surgery and one within 10 
days after surgery.  
The postnatal MRI sequences will include the following:  
1. T1-weighted volumetric imaging.  
2. T2-weighted imaging.  
3. Susceptibility weighted imaging.  
4. Diffusion imaging including diffusion tensor imaging.  
5. Magnetic resonance spectroscopy  
6. Phase contrast flow assessment in the great vessels of the neck and superior 
sagittal sinus. 
 
Analysis of the postnatal MRI will include:  
1. Evaluation for anatomic abnormalities (malformations) and gross insults.  
2. Qualitative and quantitative assessment of overt white matter injury (PVL).  
3. Assessment of hemorrhage.  
4. Postnatal total maturation score (TMS) evaluation which included assessment of 
cortical folding, myelination, germinal matrix evolution, and migrating bands in the 
frontal lobes.  
5. Assessment of whole brain and segmented brain substructure volumetrics 
including volume of myelinated white matter (VMWM).  
6. Assessment of diffusion parameters.  
7. Assessment of flow in the great vessels to and from the brain by phase contrast.  

 
  
 
 
 
28
8. Assessment of choline, N-acetylaspartate, lactate, and creatine metabolites, 
including corresponding ratios in the white matter. 
 
5.2.6 Neurodevelopmental Evaluation 
Contact Information Verification Follow-Up  
After discharge from Visit 1 (post-natal hospitalization) and prior to Visit 2 (18 month 
follow-up), a follow-up phone call/email will be made to verify current contact information at 
the child’s 6 month, 12 month and 15 month birthday.  In addition, brochures from the CHOP 
patient and family education manual about child development and tips for talking will be 
mailed at those intervals.      
The Bayley Scales of Infant Development–III (BSID-III) is an individually administered 
instrument that assesses the developmental functioning of infants and young children 
between 1 month and 42 months of age. 68 It assesses development across five domains: 
cognition, language, motor, social-emotional and adaptive.  The cognitive, language and 
motor composite will be used for this study.  This is the most widely used standardized 
developmental test battery in medical and educational settings.  The composite scores have 
a mean of 100 and a standard deviation of 15.  
The Modified Checklist for Autism in Toddlers (M-CHAT) is a brief screening instrument 
that was designed to detect autism in very young children.  It consists of 23 yes/no items that 
pertain to social relatedness and communication. 69 These items were found to have the best 
discrimination between children diagnosed with and without autism/pervasive developmental 
disorder (PDD). It is an easy questionnaire for parents to complete because of its brevity and 
the clarity of the items.  Those children who reach the cutoff should be referred for further 
evaluation of autism.   
The Infant-Toddler Social and Emotional Assessment (ITSEA) is an empirically validated 
tool for identifying social-emotional problems and competencies that may be areas of concern 
in children 12 months to 35 months 30 days old.87 It yields standard t-scores and percentiles 
in four domains with subscales and item clusters as follows: externalizing-activity/impulsivity, 
aggression/defiance and peer aggression, internalizing – depression/withdrawal, general 
anxiety, separation distress and inhibition to novelty, dysregulation-negative emotionality, 
sleep, eating, sensory sensitivity and Competence-compliance, attention, mastery 
motivation, imitation/play, empathy and prosocial peer relations.  Item clusters include 
maladaptive, social relatedness and atypical.  The ITSEA was standardized and normed 
based on a nationally representative sample that was stratified to match the 2002 US census.   
Medical Follow-up will include obtaining growth measurements and an interim medical 
history followed by performance of a physical and neurological examination by a team of 
developmental pediatricians and a nurse practitioner.    
 
5.3 
Pharmacokinetic Evaluation 
Not applicable. 
 
5.4 
Safety Evaluation 
Subject safety will be monitored by adverse events, vital signs, physical examinations, 
and laboratory data.  After randomization, participants will be instructed to contact the 
Investigator if any symptoms or side effects develop.  In case of any events, the Investigator 
will initiate the appropriate treatment according to their medical judgment.   Subject diaries 

 
  
 
 
 
29
will also be reviewed at prenatal visits for any symptom or side effects.  Recommendations 
for clinical intervention will be provided in the event of any unanticipated finding on brain MRI. 
Recommendations for clinical intervention will be provided to any family whose child who 
demonstrates a developmental delay or potential genetic abnormality at the 18 month 
Developmental Follow-Up.   
 
6     STATISTICAL CONSIDERATIONS 
6.1   Primary Endpoint 
The primary endpoint is the Motor Scale of the Bayley-III at 18 months of age.  
6.2   Secondary Endpoints 
Secondary endpoints will include the following: 
1. Cognitive and Language Scales of the Bayley-III at 18 months of age 
2. Total maturation score (TMS) 
3. Volumes of whole brain, white matter, gray matter and  myelinated white matter 
(VMWM) 
4. Presence of PVL on early post-natal brain MRI (categorical y/n) 
5. Presence of PVL on early post-operative brain MRI (categorical y/n) 
6. PVL Volume on early post-natal brain MRI 
7. PVL Volume on early post-operative brain MRI 
8. Compliance with progesterone regimen 
9. Compliance with pre- and post-natal MRI’s 
10. Failure to return for 18 month neurodevelopmental evaluation. 
6.3   Statistical Methods 
6.3.1   Baseline Data  
Baseline and demographic characteristics will be summarized by standard descriptive 
summaries (e.g. means or medians and standard deviations or interquartile range for 
continuous variables such as age and percentages for categorical variables such as gender). 
6.3.2   Efficacy Analysis 
The primary analysis will be intent-to-treat and will estimate differences between group 
means and their associated confidence intervals.  In parallel we will test the null hypothesis 
that treatment has no effect on each of three outcomes. Because of its clinical relevance, the 
study is primarily designed to provide an estimate of possible effect sizes for the Bayley-III 
Motor Scale at 18 months. Important information will also be provided on effect sizes for the 
Bayley-III Cognitive and Language Scales at 18 months, as well as total maturation score 
(TMS) and volume of myelinated white matter (VMWM). The analysis will use a two-sided 
two-sample T-interval (estimation) or T-test (hypothesis testing). The confidence level will be 
set at 90% and the Type I error at 10%. Normality will be assessed and a transformation, or 
alternatively a non-parametric test, will be used if needed.  
Based on our earlier work, the occurrence of PVL, both pre- and post-operatively, has a 
complex distribution. At both time points, when occurrence of PVL is considered a categorical 
outcome, there are patients with no evidence of PVL (PVL=0), as well as patients with varying 
amounts of PVL (PVL=1). When occurrence of PVL is considered as a continuous variable 
(volume of PVL), there is a mixture distribution with a peak at zero and the remainder of 

 
  
 
 
 
30
patients demonstrating a continuous highly skewed distribution. Thus it is of interest to know 
whether the prevalence of PVL (categorical outcome) prior to surgery and post-surgery differs 
between treatment groups (intention to treat). It will also be of interest to know if the volume 
of PVL (continuous outcome), among those who have PVL at baseline, differs between 
treatment groups, both at baseline and post-operatively.  
In the secondary analyses, we will use an exact binomial confidence interval to estimate 
differences between groups in the percent of subjects with PVL pre-operatively and the rate 
of increase post-operatively. The null hypothesis that treatment does not reduce the 
frequency of occurrence of PVL pre-operatively and the rate of increase post-operatively will 
be tested using Fisher’s Exact test. The confidence level will be set at 90% and the Type I 
error at 10%. The PVL data can also be modeled in a more unified approach using mixture 
models, where the categorical data for patients with no PVL, and the continuous data for 
patients with evidence of PVL are modeled jointly.88 In addition to the these analyses, we will 
describe compliance, both with treatment and with MRI tests, as well as determine loss to 
follow-up for the 18 month time point. We will secondarily model TMS, VMWM and the 
Bayley-III Cognitive and Language Scales as a function of treatment, as well as other 
potential predictors of fetal brain maturation and development including: gestational age, type 
of CHD, ethnicity, age at surgery, operative management variables, maternal education and 
socioeconomic status. Presence of 22q11 deletions or other identified genetic anomaly and 
apo-lipoprotein E (APOE) genotype will be included as covariates in the analysis of the 
Bayley-III scores. 
6.3.3 Safety Analysis 
All subjects entered into the study at will be included in the safety analysis. The 
frequencies of AE by type, body system, severity and relationship to study drug will be 
summarized for both the mother and the neonate. Serious adverse events (SAE), if any, will 
be described in detail. 
AE incidence will be summarized along with the corresponding exact binomial 95% two-
sided confidence intervals. 
6.4   Sample Size and Power 
Based on the formal statistical considerations described below, we plan to enroll sufficient 
patients to have 80 evaluable patients (40 per treatment group) at the 18 month 
neurodevelopmental testing. Based on known clinical outcomes for this patient population 
and experience with other studies with similar follow-up plans, we estimate a 5% early 
mortality and a further 20% attrition from all causes between hospital discharge and the 18 
month evaluation. There may also be dropout prior to birth for a variety of reasons: fetal 
death, intolerance of therapy, withdrawal from study. We do not have accurate data to 
estimate this loss. We anticipate needing to enroll 110-130 patients to have a sufficient cohort 
at 18 months (40 per treatment group); with 60 to 52-56 patients per treatment group 
available for the post-natal and post-operative brain MRIs respectively.  
Table 1: Expected width of confidence intervals for 
continuous outcomes for several possible sample sizes 
N per 
group 
Distance from Mean Difference to Limits 
of 90% CI based on T-interval 

 
  
 
 
 
31
The primary goal is to 
provide sufficiently precise 
estimates 
of 
the 
mean 
difference in the 18 month 
Bayley-III Motor Scale to 
inform the design of a future 
pivotal trial. Secondarily we 
will provide estimates of 
differences 
in 
the 
TMS, 
VMWM, and the Bayley-III 
Cognitive and Language Scales. All primary analyses will be performed on an intention-to-
treat (ITT) basis. 
 Table 1 shows that for the Bayley-III Motor Scale, assuming 40 evaluable subjects per 
treatment group, and an SD of 15, the ½ width of a two-sided 90% confidence interval on the 
mean difference is expected to be 5.6. Thus, for example, if the mean difference is 6.0 units, 
a clinically relevant value for this population, the 90% two-sided confidence interval is 
expected to be on the order of (0.4, 11.6), suggesting that while only a minor effect cannot 
be ruled out, important positive effects of progesterone appear possible. In contrast, if there 
is no effect of progesterone we expect an interval centered around zero with i.e. (-5.6, 5.6). 
This initial study will thus provide important information about the magnitude of the effect of 
progesterone, and an indication of whether it is worthwhile to proceed with further trials. For 
TMS, results from Licht (personal communication) estimated the mean (median) value of 
TMS in a similar cohort to be 10.1 (10.0) with a standard deviation (SD) of 1.06 and range of 
8.0 to 13.5.  Conservatively assuming an SD that is 25% larger than that in the pilot data 
(SD=1.32), Table 1 shows that, with a sample size of 60 per group, two-sided 90% confidence 
intervals for the mean difference in TMS are anticipated to have a ½ width on the order of 
+0.44. Thus for TMS, if the mean difference is on the order of 2.0, the 90% confidence interval 
would be (1.56, 2.44). Estimated SD’s for VMWM are not yet available.  
 
Next we consider the categorical outcomes. In pilot data (Licht, personal communication), 
19% of subjects had some PVL prior to surgery and 38% of infants increased the level of 
 
SD 
TMS* 
Bayley 
Score** 
40 
+0.372 
+0.49 
+5.6 
50 
+0.332 
+0.40 
+5.0 
*based on a postulated SD=1.32 
**based on a postulated SD=15 for a normalized 
Bayley-III Motor Scale (mean=100) 
Table 2: Expected endpoints of 90% confidence interval (CI) for different scenarios for 
PVL. Calculations are based on Pearson’s Chi-square Method for n=60 (pre-surgical) or, 
conservatively) n=52 (post-surgical) per group. 
Proportion in 
Control Group
Proportion in 
Treated 
Group 
True 
Difference 
in 
Proportions 
Expected Endpoints of 90% CI 
Post-natal PVL (pre-surgical, n=60)  
19.0% 
8.5% 
10.5% 
 (0.3%, 20.7%) 
19.0% 
19.0% 
0% 
(-11.8%, 11.8%) 
Post-operative increase in PVL from baseline (n=52)* 
38.0% 
24.0% 
14% 
 (-0.8%, 28.8%) 
38.0% 
38% 
0% 
(-14.6%, 14.6%) (-15.7%, 15.7%) 
*Conservatively as we expect n=52 to 56 post-operatively 

 
  
 
 
 
32
PVL post-surgery. As part of our secondary aims, the pilot study is intended to estimate the 
differences in these outcomes between groups. To recruit 40 evaluable subjects at 18 months 
we anticipate having 60 patients available pre-surgery (post-natal) and 52 to 56 after surgery.  
Table 2 shows that if PVL at baseline is 19% in the control group, and the true effect is 10.5%, 
then the endpoints of the 90% CI are (0.3%, 20.7%)This would suggest that while we could 
not rule out essentially a null effect of progesterone, the evidence would suggest a possible 
substantial effect. In contrast if there were no effect we expect the confidence interval to be 
centered near zero with endpoints of +11.8%%.  Similarly, if the post-operative increase in 
PVL is 38% in the control group, and the true effect size is 14%, then the 90% confidence 
interval is expected to have endpoints of the 90% CI are (-0.7%, 28.7%). Again, while we 
could not rule out essentially a null effect of progesterone, the evidence would suggest a 
possible substantial effect. In contrast if there were no effect we expect the confidence 
interval to be centered near zero with endpoints of +15.7%%. 
6.5       Interim Analysis 
No interim analysis is planned for this Phase II study. 
7 
STUDY MEDICATION (STUDY DEVICE OR OTHER STUDY INTERVENTION) 
7.1 
Description 
Crinone® (progesterone gel) is a bioadhesive vaginal gel containing micronized 
progesterone in an emulsion system, which is contained in single use, polypropylene vaginal 
applicators. The carrier vehicle is an oil-in-water emulsion containing the water swellable, but 
insoluble polymer, polycarbophil. The progesterone is partially soluble in both the oil and 
water phase of the vehicle, with the majority of the progesterone existing as a suspension. 
Physically, Crinone has the appearance of a soft, white to off-white gel.  
Replens is a hormone-free vaginal moisturizer.  Ingredients include purified water, 
glycerin, mineral oil, polycarbophil, carbomer homopolymer type B, hydrogenated palm oil, 
glyceride, sorbic acid, and sodium hydroxide.  This product will act as the placebo in this 
study. 
7.1.1 Packaging 
Crinone is supplied in a single use, disposable, white polypropylene vaginal applicator 
with a teal twist-off cap. Each applicator delivers 1.125 grams of Crinone gel containing 90 
mg (8% gel) of progesterone in a base containing glycerin, mineral oil, polycarbophil, 
carbomer 934P, hydrogenated palm oil glyceride, sorbic acid, purified water and may contain 
sodium hydroxide. 
Replens Long-Lasting Moisturizer is supplied in pre-filled, sealed and individually 
wrapped applicators. 
Both Crinone 8% and Replens will be obtained from commercial sources and stored 
according to package labeling.  The investigational pharmacy will dispense the study 
medications in a blinded fashion by removing any commercial labeling that contains the 
identity of the product.  The study medications will be dispensed as a 30 +/- 15 day supply.  
Occasionally the timing of prenatal visits to CHOP may require variation in the amount of 
drug supplied to a mother at one time.   
7.1.2 Labeling 
Study drug labeling will be in compliance with applicable local and national regulations 

 
  
 
 
 
33
7.1.3 Dosing 
Crinone 8% is administered vaginally at a dose of 90 mg twice daily. The rounded tip of 
the applicator is inserted into the vagina. After insertion, the plunger is pushed to release the 
gel into the vagina. The applicator is removed. 
Replens Long-Lasting Moisturizer will also be dosed at one applicator intravaginally twice 
daily. 
7.1.4 Treatment Compliance and Adherence 
The Investigational Pharmacy will keep an Investigational Drug Accountability Record for 
the medication and placebo, which keeps track of study drug that is received, dispensed and 
returned. Subjects will be asked to maintain a diary documenting each drug application. 
Compliance with the treatment protocol will be assessed at each visit. The subject diary will 
be reviewed. Patients will be asked to return all unused medication packages. Unused study 
drug will be returned to the Investigational Pharmacy to count the remaining drug to assess 
compliance. Subjects will not be terminated from the study form non-compliance. However 
the compliance protocol will be considered in secondary analyses to determine if there is any 
difference in effect between compliers and non-compliers. 
7.1.5 Drug Accountability 
The Investigational Pharmacy will keep an Investigational Drug Accountability Record for 
the medication and placebo, which keeps track of anything that is received, dispensed and 
returned. 
8 SAFETY MANAGEMENT 
8.1 
Clinical Adverse Events 
Clinical AEs will be monitored throughout the study.  
8.2 
Adverse Event Reporting 
Unanticipated problems related to the research involving risks to subjects or others that 
occur during the course of this study (including SAEs) will be reported to the IRB in 
accordance with CHOP IRB SOP 408: Unanticipated Problems Involving Risks to Subjects. 
AEs that are not serious but that are notable and could involve risks to subjects will be 
summarized in narrative or other format and submitted to the IRB at the time of continuing 
review.  
8.3 
Definition of an Adverse Event 
An adverse event is any untoward medical occurrence in a subject who has received an 
intervention (drug, biologic, or other intervention).  The occurrence does not necessarily have 
to have a causal relationship with the treatment.  An AE can therefore be any unfavorable or 
unintended sign (including an abnormal laboratory finding, for example), symptom, or disease 
temporally associated with the use of a medicinal product, whether or not considered related 
to the medicinal product. 
All AEs (including SAEs) will be noted in the study records and on the case report form 
with a full description including the nature, date and time of onset, determination of non-
serious versus serious, intensity (mild, moderate, severe), duration, causality, and outcome 
of the event. 

 
  
 
 
 
34
8.4 
Definition of a Serious Adverse Event (SAE) 
An SAE is any adverse drug experience occurring at any dose that results in any of the 
following outcomes:  
 death, 
 a life-threatening event (at risk of death at the time of the event),  
 requires inpatient hospitalization or prolongation of existing hospitalization, 
 a persistent or significant disability/incapacity, or 
 a congenital anomaly/birth defect in the offspring of a subject.   
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered a serious adverse drug event when, based upon 
appropriate medical judgment, they may jeopardize the subject and may require medical or 
surgical intervention to prevent one of the outcomes listed in this definition. 
A distinction should be drawn between serious and severe AEs.  A severe AE is a major 
event of its type.  A severe AE does not necessarily need to be considered serious.  For 
example, nausea which persists for several hours may be considered severe nausea, but 
would not be an SAE.  On the other hand, a stroke that results in only a limited degree of 
disability may be considered a mild stroke, but would be an SAE.  
The Common Terminology Criteria for Adverse Events (CTCAE) Version 4.0 
(http://ctep.cancer.gov) provides a grading system that is used to categorize the severity of 
adverse events, as follows: 
Grade 
Severity 
Definition 
1 
Mild 
Asymptomatic or mild symptoms; clinical or diagnostic 
observations only; intervention not indicated. 
2 
Moderate 
Minimal, local or noninvasive intervention indicated; limiting 
age-appropriate instrumental activities of daily living (ADL). 
3 
Severe 
Severe or medically significant but not immediately life-
threatening; hospitalization or prolongation of 
hospitalization indicated; disabling; limiting self-care ADL. 
4 
Life-
threatening 
Life-threatening consequences; urgent intervention 
indicated. 
5 
Death 
Death related to AE 
A SAE, as defined above, encompasses CTCAE grades 4 and 5, and any Grade 3 event 
that requires or prolongs hospitalization, or that substantially disrupts the ability of the subject 
to conduct normal life functions.   
Expectedness  
The purpose of reporting is to provide new, important information on serious reactions or 
events previously unobserved or undocumented.  Therefore, all AEs will be evaluated as to 
whether their occurrence was unexpected, using the following definitions:  
Unexpected: An unexpected AE or adverse reaction is one for which the nature or 
severity is not consistent with information in the protocol, consent form, or product brochure. 
An AE or adverse reaction also may be categorized as unexpected if the event has not 
previously been observed at the same specificity and/or severity.    

 
  
 
 
 
35
 
Expected: An event is considered expected if it is known to be associated with the study 
drug(s) and/or the disease state.  
Pregnant subjects: The standard definition for SAE will be used (Section 7.4).   
However, the following adverse events will not be classified an SAE for the purposes of 
this study as they are expected events associated with pregnancy and delivery: 
a) Hospitalizations for treatment planned prior to randomization 
b) Hospitalizations for elective treatment of a pre-existing condition  
c) Miscarriage 
d) Preterm labor / suspected preterm labor 
e) Premature rupture of membranes (PROM) / suspected PROM 
f) Preterm delivery  
g) Preterm delivery in maternal interest 
h) Preterm delivery in fetal interest 
i) 
Hospitalization for pregnancy induced hypertension 
j) Hospitalization for “maternal discomfort”  
k) Hospitalization for “rest”  
l) 
Hospitalization for “observation” or “monitoring” for which the women are 
admitted for a period of less than 12 hours.   
Newborn subjects: It is important to note at baseline these newborns are medically 
fragile pediatric patients requiring high-risk neonatal cardiothoracic surgery.  This puts them 
at risk for many expected adverse events and serious adverse events without any additional 
interventions.  In response to a surgical randomized clinical trial (RCT) run by the Pediatric 
Heart Network at the NIH, a novel adverse event reporting system was developed for this 
medically fragile population.89  The system is based on the concept of the identification of 
“sentinel” SAEs.  For this study, these events will be  
a. death 
b. listing for cardiac transplantation 
c. endocarditis 
d. thromboembolic event 
e. need for surgical or catheter-based intervention 
f. heart block  
g. hemolysis.   
The remaining adverse events will be organized in a code list by body system.  We have 
used this system of sentinel SAE reporting with another RCT study Protocol No 10-007750.   
8.4.1 Relationship of SAE to Study Drug 
The relationship of each SAE to the study intervention should be characterized using one 
of the following terms in accordance with CHOP IRB Guidelines: definitely, probably, 
possibly, unlikely or unrelated. 
Type of Unanticipated 
Problem 
Initial Notification  
(Phone, Email, Fax) 
Written Report 
Internal (on-site) SAEs 
Death or Life Threatening  
24 hours 
Within 2 calendar days 
Internal (on-site) SAEs 
All other SAEs 
7 days 
Within 7 business days 

 
  
 
 
 
36
8.5 
IRB Notification of SAEs and Other Unanticipated Problems 
The Investigator will promptly notify the IRB of all on-site unanticipated, serious Adverse 
Events that are related to the research activity. Other unanticipated problems related to the 
research involving risk to subjects or others will also be reported promptly. Written reports 
will be filed using the eIRB system and in accordance with the timeline below. External SAEs 
that are both unexpected and related to the study intervention will be reported promptly after 
the investigator receives the report. 
8.5.1 Follow-up report 
If an SAE has not resolved at the time of the initial report and new information arises that 
changes the investigator’s assessment of the event, a follow-up report including all relevant 
new or reassessed information (e.g., concomitant medication, medical history) should be 
submitted to the IRB. The investigator is responsible for ensuring that all SAE are followed 
until either resolved or stable.  
8.6 
Investigator Reporting of a Serious Adverse Event to Sponsor 
Not applicable. 
8.7 
Medical Emergencies 
These subjects are either pregnant mothers or newborns undergoing cardiac surgery 
requiring postoperative cardiac intensive care.  Maternal Fetal Medicine professionals in the 
Center for Diagnosis and Treatment will be responsible for the care of pregnant subjects in 
the event of medical emergencies.  Cardiac surgical, medical, anesthesia, and nursing 
professionals in the Cardiac Center will be responsible for the care and treatment of these 
infants in the event of medical emergencies.  
9 STUDY ADMINISTRATION 
9.1 
Treatment Assignment Methods 
9.1.1Randomization 
The study biostatistician, Dr. Putt, will generate a block randomization schedule based 
on stratification by fetal CHD diagnosis: TGA, HLHS, and other CHD. The randomization 
schedule will be based on equal allocation of subjects to the progesterone and placebo arms 
within each block. The schedule will be generated using variable block sizes. Upon 
confirmation of eligibility in the DMS, the randomization module will be programmed to 
generate a randomization number that corresponds to a unique kit number of progesterone 
or placebo treatment. This kit number will be used to distribute treatment. 
8.1.2    Blinding 
The study biostatistician, Dr. Putt, will generate a block randomization schedule based 
on stratification by fetal CHD diagnosis: TGA, HLHS, and other CHD. The randomization 
schedule will be based on equal allocation of subjects to the progesterone and placebo arms 
within each block. The schedule will be designed so that the block size is not fixed. Upon 
Unanticipated Problems 
Related to Research 
7 days  
Within 7 business days 
All other AEs 
N/A 
Brief 
Summary 
of 
important 
AEs 
may 
be 
reported 
at 
time 
of 
continuing review 

 
  
 
 
 
37
confirmation of eligibility in the DMS, the randomization module will be programmed to 
generate a randomization number that corresponds to a unique kit number of progesterone 
or placebo treatment. This kit number will be used to distribute treatment. 
9.1.3 Unblinding 
Unblinding can occur if the PI instructs the Investigational Pharmacy to reveal this 
information.  A direct request from the PI to Investigational Pharmacy must be documented. 
9.2 
Data Collection and Management 
The following non-research data sources will be queried for demographic and medical data 
etc.: ChartMax, CardioIMS, EPIC, CardioAccess, CompuRecord, PC4 and other applicable 
clinical databases. 
1. Confidentiality: The following methods will be utilized to ensure confidentiality of 
research study data [including source documents, case report forms, & data in 
computerized systems] 
a. Access to identifiable study data, databases containing identifiers, and 
systems will be limited to study personnel who have valid CHOP enterprise 
password and login, who have been designated by the PI and approved by 
the IRB, and who are using a computer that is connected to the internal CHOP 
network.   
b. Password-protected data base will be utilized  
c. Data files copied to removable disk drives will be encrypted  
2.  Security: Data will be entered, cleaned and maintained in a password-protected 
research database in the located in the Cardiothoracic Surgery suite.  All data 
management applications, databases, and data are hosted on secure servers in the 
offsite CHOP data center, hosted by a commercial provider. Physical access to the 
data center is monitored, logged, controlled and limited to approved CHOP personnel. 
System administration and support is provided by CHOP Research Information 
Systems. Servers and the Case Report Application are actively monitored 24x7x365 
and replicated between two geographically separate fully redundant data centers. 
Server backup at the file system level is performed multiple times per day, and a multi-
day real-time accessible rolling archive will be maintained. After seven days, data will 
be compressed and moved to an online archive that may be accessed upon 
documented request to Research Information Systems. System and data recovery 
are accomplished through documented standard operating procedures.  
3. Anonymization, de-identification or destruction: Identifiers will be kept in conjunction 
with the study identification number for the purposes of the18 month follow-up and 
future investigation. 
9.3 
Confidentiality 
All data and records generated during this study will be kept confidential in accordance with 
Institutional policies and HIPAA on subject privacy and that the Investigator and other site 
personnel will not use such data and records for any purpose other than conducting the study. 
Safeguards are described under Data Collection and Management (9.2). 

 
  
 
 
 
38
9.4 
Regulatory and Ethical Considerations 
9.4.1 Data and Safety Monitoring Plan 
The purpose of this Data and Safety Monitoring Plan is to ensure the safety of research 
participants and the integrity of the research data. 
Safety of Research Participant:  
Protection of the rights and welfare of subjects and their families will be maintained during 
the recruitment, consenting process, and study participation.  Dr. Gaynor and his study team 
have over a 15-year experience in recruiting and enrolling patients for research studies in the 
Cardiac Center.  Subject privacy and confidentiality will be maintained as described in Section 
8.3.  All research responsibilities delegated by the principal investigator to study team 
members will be carried out in accordance with the protocol, federal regulations, federal, state 
and local laws and institutional policies and procedures.  The study team has two research 
nurses, one of which is a Certified Clinical Research Coordinator (CCRC).  If any the results 
of research procedures appear to be important for immediate clinical care, these results will 
be provided to the Principal Investigator (Dr. Gaynor) of the study who will forward the 
information to the subject’s surgeon, cardiologist and/or primary physician.  
The Principal Investigator (Dr. Gaynor) and Co-Investigators (Drs. Rychik, Szwast, 
Johnson, Gebb, and Licht,) will be responsible for reviewing subject enrollment data, 
maternal events, and clinical event data as described in Section 7.4 every 2 weeks.  Dr. 
Gaynor is a pediatric cardiothoracic surgeon. Dr. Licht is a pediatric neurologist. Drs. Rychik 
and Szwast are fetal cardiologists. Drs. Johnson and Gebb are experts in Maternal-Fetal 
Medicine. Data will be reviewed for adverse events and actions appropriate to the event will 
be initiated. 
Data Safety Monitoring Board (DSMB): 
An independent Data Safety Monitoring Board has been established. The members have 
expertise in maternal-fetal medicine, pediatric neurology, pediatric critical care, peri-operative 
care of the neonate with CHD, and neurodevelopmental outcomes for patients with CHD. 
The DSMB will review the protocol with respect to ethical and safety standards and making 
recommendations as necessary. This committee will monitor the emerging results for safety 
and efficacy. The DSMB will also review the performance of the trial in terms of recruitment, 
protocol adherence and data quality. The interval and timing of DSMB meetings after the 
initial review of the protocol will be at the discretion of the committee itself. 
DSMB Membership: 
Jane Newburger, Pediatric Cardiology, Boston Children’s Hospital (Chair) 
Dean Andropoulos, Pediatric Cardiac Anesthesia, Texas Children’s Hospital 
Richard Ittenbach, Biostatistician, Cincinnati Children’s Medical Center 
Patrick McQuillen, Pediatric Cardiac Critical Care Medicine, University of California, San 
Francisco 
Steven Miller, Pediatric Neurology, Hospital for Sick Kids, Toronto 
David Stamilo, Maternal-Fetal Medicine, Wake Forest Baptist Health 
9.4.2 Risk Assessment 
Progesterone Therapy 

 
  
 
 
 
39
The existing evidence suggests that progesterone therapy is associated with potential 
benefits to the mother and fetus in singleton pregnancies, with greater than minimal risk.  
Maternal Risks 
Multiple reviews have found that there do not appear to be any significant maternal or 
fetal risks associated with progestin therapy. , ,40, 42, 43, 45 The lower blood levels after vaginal 
administration have been associated with a lower prevalence of systemic side effects.3, 5 The 
most common side effect of vaginal progesterone administration is local irritation. A recent 
randomized trial of Crinone vaginal gel vs. intramuscular progesterone during ART showed 
that patient satisfaction in terms of ease of administration, convenience of administration and 
discomfort was significantly greater for patients receiving Crinone.90 Additional supportive 
evidence of safety and efficacy is found in the 2011 FDA Advisory Committee Briefing Book. 
(Attached to application) 
 
Stopping Rule 
In previous studies, the vaginal progesterone gel has been well tolerated with few adverse 
effects. The most common effect is vaginal irritation which will be treated with sitz baths; and 
for severe cases a low potency topical steroid cream (hydrocortisone 1 % cream). If a woman 
goes into preterm labor, this will be treated according to standard clinical protocols. We 
anticipate that need to terminate study drug prior to 39 weeks or delivery (if earlier) will be 
infrequent. 
Risks to Fetus and Newborn 
The effects of progesterone on perinatal outcomes and the neonate are of special 
interest. A recent meta-analysis attempted to quantify the effect of progesterone therapy for 
the prevention of preterm birth on perinatal outcomes.42 The authors concluded that 
progesterone administration in singleton pregnancies at risk for preterm birth improves 
perinatal outcomes, but may actually have adverse effects in multiple pregnancies.42 Other 
reviews have also found that there do not appear to be any significant maternal or fetal risks 
associated with progestin therapy.45 Longer term outcomes have also been evaluated.36 
Surviving children from a trial of 17-OHP-C vs. placebo to prevent preterm birth underwent 
physical examination and developmental screening at 48 months of age. Progesterone 
therapy was not associated with any adverse physical or neurodevelopmental sequelae.36 
Risk Assessment for Pre-Natal Evaluations 
Fetal Magnetic Resonance Imaging:  
All of the fetal brain MRI examinations performed in this study will be during the 25th week 
GA or beyond. No MRI will be performed in the first half of pregnancy or embryonic period. 
None of the studies will be performed with gadolinium contrast administration. None of the 
studies will be done with sedation or general anesthesia. General MRI safety guidelines for 
metallic objects in the MRI environment will be monitored as part of standard practice by the 
MRI technologists and radiology nursing staff. At the Children’s Hospital of Philadelphia, 
clinical fetal MRI studies are routinely and safely performed at 16 weeks GA and beyond.  To 
avoid conflict of interest concerning decisions about termination or continuation of a study, 
the anesthesiologist for the research MRIs will not be a member of the study team. 
There are no verified or scientifically proven additional side effects of clinical MRI on 
human fetuses within the safety limits of performing MRI studies. The FDA has specific limits 
on the amount of energy deposition by MRI which is built in as a fail-safe warning mechanism 

 
  
 
 
 
40
on all of the MRI systems at CHOP, which used for all patients. There is little direct scientific 
evidence of harmful effects with short-term exposure to high static magnetic fields.91 Two to 
three decades of practice generally supports this widely-held belief. 78, 79 FDA guidelines now 
classify MRI devices as “nonsignificant risk” when neonates (infants less than one month of 
age) are scanned at a static magnetic field of 4T or less. As a result, it has become 
widespread routine clinical practice to scan even the youngest premature infants who are 
stable for transport. Myers and coworkers reported no significant reduction in fetal growth in 
74 volunteer subjects exposed in utero to MRI vs. matched controls.92 Also the recent practice 
parameter from the American College of Radiology and the Society for Pediatric Radiology 
for fetal MRI states, “At this stage, the preponderance of research studies have failed to 
discover any reproducible harmful effects of exposure of the mother or developing fetus to 
the 3T or weaker magnetic fields used in the routine clinical MR imaging process.”93   Some 
MRI sequences are for research purposes and are not FDA approved. However, all the 
sequences will be below the FDA energy deposition limits and no gadolinium contrast will be 
used. 
A “potential” side effect of the MRI environment is exposure to high sound levels.  Baker 
and associates reported no demonstrable increase in disease, disability, or hearing loss in 
20 children examined in utero using echoplanar MRI for suspected fetal compromise.94 In a 
2010 study, Reeves and colleagues found no evidence that exposure of the fetus to 1.5T MR 
imaging during the second and third trimesters is associated with an increased risk of 
substantial neonatal hearing impairment. 95   
Risk Assessment for Post-Natal Evaluations 
Transport:  
This is a high-risk group of subjects being transported from the cardiac intensive care unit 
(CICU) to the MRI suite. The risks of transport include exposure to cooling temperatures, 
dislodging arterial and venous lines and accidental extubation. To accomplish safe transport 
of subjects, all subject transports will be performed by a team of cardiac anesthesiologists 
including an attending, a fellow and a respiratory therapist.  This team of physicians has a 
great deal of experience both in transporting high-risk neonates (n>200) to the MRI suite for 
preoperative and post-operative studies. 
Magnetic Resonance Imaging:  
MRI is an electromagnetic imaging technique that does not use ionizing radiation such as 
x-rays to obtain the images; as such there are minimal risks involved with this technique.  The 
Food and Drug Administration (FDA) guideline for average specific absorption ratio (SAR) is 
3W/kg over the head for 10min. The SAR level of this CASL sequence, calculated using 
quasi-static approximation (spherical head model), is 1W/kg on an average head size with 
radius of 10cm. The SAR level is also automatically calculated and monitored by integrated 
product program during MR scanning according to the legal requirement value (IEC 60601-
2-33, accepted by the US FDA). A mean SAR level (percent of maximal allowable SAR) of 
54.5±15.6% (40-80%) was observed on 13 healthy adult subjects (radiology in press paper). 
The SAR level actually is lower in children and infants than in adults because it is roughly 
proportional to the square of the head size (radius).96 The 3T CASL method is therefore very 
safe for pediatric imaging.  
 
Sedation for the Pre-Operative MRI:  

 
  
 
 
 
41
The pre-operative MRI will be performed during the same anesthetic period as the 
planned surgery. On the morning of heart surgery, the patient will be induced for anesthesia 
using published protocols.66, 97 Immediately prior to surgery, the patient will be brought to the 
MRI scanner located between the CICU and the operating rooms. Upon completion of the 
pre-operative MRI protocol, the infant will be transported to the operating room where the 
surgery will take place. During transport and the MRI procedure, the patient will be monitored 
by a team of cardiac anesthesiologist. To avoid conflict of interest concerning decisions about 
termination or continuation of a study, the anesthesiologist for the research MRIs will not be 
a member of the study team. 
Sedation for the Post-Operative MRI:  
For the post-operative MRI procedure, primary consideration will be given toward the 
possibility of a non-sedated, “sated sleep” protocol. All infants will be transported to and from 
the MRI scanner by a team of pediatric cardiac anesthesiologists. For a sated-sleep MRI the 
infant would be held NPO for 3 or 4 hours prior to the study. The subject would be transported 
awake to the scanner, ear protectors and ECG leads would be placed and the subject 
swaddled with warm blankets. The infant would then be offered formula/breast milk ad-lib or 
as recommended by the ICU protocol. When postprandial sleep is achieved the subject is 
placed in the scanner. The MRI sequences will be run in order of relative importance to the 
study, with T1 MPR and DWI taking highest priority. 
The decision to use the sated sleep protocol will be made by either Dr. Susan Nicolson 
or Dr. Lisa Montenegro and it will be done on a patient-by-patient basis. There are some 
concerns that may make this procedure untenable: first, the patient must tolerate oral feeds 
to be eligible for such a protocol; second, agitation in a post-operative infant may cause 
unsafe increase in blood pressure. The advantages are clear and include primarily decreased 
risk of respiratory compromise when compared to sedation. 
If a “sated sleep” MRI procedure is not an option (if the infant is not tolerating oral feeds, 
for instance), an individualized sedation protocol will be administered by Cardiac Anesthesia 
for the post-operative MRI scan. The sedation will follow protocols that have established 
safety and have been used successfully in the past. The medications used for sedation will 
be determined by the anesthesiologist responsible for the case but will likely include the use 
of pentobarbital. All subjects will have heart rate, blood pressure and oxygen saturations 
monitored throughout the study. Any deviation in oxygen saturations of more than 10% would 
necessitate the discontinuation of the study and immediate medical evaluation of the subject.   
Stopping Rule 
Our previous experiences with pre-operative and post-operative MRI suggest that these 
are safe procedures with our research team. In this study we expect similar results. This 
notwithstanding, enrollment for this study will be halted temporarily if two subjects experience 
vital sign fluctuations that require termination of their MRI. Enrollment will not resume until 
independent review of procedures determines the safety for continuation. All adverse events 
will promptly be reported to the IRB. All SAEs will be reported to the board within 24 hours of 
occurrence 
Review of Medical Record  
Collecting data from the medical record may increase the risk of breach of privacy and 
confidentiality. All data and records generated during this study will be kept confidential in 
accordance with institutional policies and HIPAA on subject privacy and the Investigator and 
other site personnel will not use such data and records for any purpose other than conducting 
the study.   

 
  
 
 
 
42
Blood Work for Mothers/Fathers 
Taking blood may cause some pain, bleeding or bruising at the spot where the needle 
enters your body. Rarely, taking blood may cause fainting or infection.   
Blood Work for Infant 
The sample will be obtained from an existing central venous or arterial line, which is 
associated with minimal risk of pain or complication. 
18-month Developmental and Medical Follow-Up 
There are no physical risks but toddler-age children may become uncooperative.  The 
Neonatal follow-up team has over 10 years of experience in developmental assessments of 
infants and children with CHD. Parents and child will remain together throughout this 
assessment in order to provide support and comfort to the child.  Because the early 
assessment measures for children from birth to 3 are based on play, typically children enjoy 
these sessions. 
9.4.3 Potential Benefits of Trial Participation 
There is a significant risk of preterm birth for women carrying a fetus with CHD. A potential 
benefit to the mother and child is prolongation of the pregnancy with a reduction in perinatal 
mortality and morbidity. 
There is a significant risk of brain injury and subsequent neurobehavioral disability for the 
newborn undergoing cardiac surgery. A potential benefit of the study is a reduction in prenatal 
and perioperative brain injury resulting in improved neurobehavioral outcomes. 
The possibility of collateral benefit from participation in the post-operative MRI has been 
demonstrated in a related study. Over the course of this study, entitled “Hemodynamic 
Neuroimaging” (IRB # 4357), 7.4% of participants (2/27) from the same target population 
have benefitted directly from a post-operative brain MRI; including one patient whose MRI 
revealed an incidental finding of a pre-symptomatic hyperacute non-occlusive sino-venous 
thrombus of both transverse sinuses, also involving the torcula. This condition is life-
threatening, and the post-operative MRI allowed physicians to intervene early with both 
primary and secondary prevention. The other patient’s MRI revealed an arterial ischemic 
stroke, which allowed for early secondary prevention. For all subjects in the present study, 
Dr. Licht will meet with the parents to review all the imaging data. The significance of injury, 
if present, will be explained to the parents in layman language and interventional therapies 
will be put in place if they are deemed necessary. 
Current data demonstrates that some degree of neurobehavioral disability is present on 
over 50% of survivors of cardiac surgery in the newborn period. Indeed, neurodevelopmental 
disability is now recognized as the most common complication of critical CHD (i.e. those 
patients requiring cardiac surgery in infancy) and has the most negative impact on quality of 
life, academic performance and opportunity for independence as an adult. Regardless of their 
origin, neurodevelopmental problems adversely affect learning and the attainment of 
academic, social, and vocational skills. Ultimately they undermine mental health and 
employment opportunities in adulthood. There is a wealth of disturbing data documenting the 
impact of neurodevelopmental deficits among children and adults affected by CHD. The need 
for special and rehabilitative services results in significant costs to the patients’ families and 
to society. A potential benefit of the study is a reduced incidence of neurobehavioral disability 
leading to lower health care costs for society. 

 
  
 
 
 
43
9.4.4 Risk-Benefit Assessment 
Risk-Benefit Ratio: 
There are significant potential benefits to the mother and fetus including prolongation of 
pregnancy, and improved brain development, with a reduction in peri-operative white injury 
leading to better long-term neurodevelopmental outcomes. Previous studies indicate that 
maternal progesterone therapy during the third trimester is associated with only a minor 
increase above minimal risk.  
As outlined above, there is a possibility of collateral benefit from participation in the MRI 
assessments, however because of the potential need for sedation, this is a greater than 
minimal risk study with prospect for collateral benefit (Subpart D category §46.406 or §50.53).  
9.5 
Recruitment Strategy (or Case Ascertainment) 
Subjects will be recruited from the Cardiac Center at CHOP. Eligible mothers and with a 
fetus with a prenatal diagnosis of CHD, who are followed by the Fetal Heart Program, will be 
recruited in person during a prenatal visit. 
We plan to enroll sufficient patients to have 80 evaluable patients (40 per treatment group) 
at the 18 month neurodevelopmental testing. Based on known clinical outcomes for this 
patient population and experience with other studies with similar follow-up plans, we estimate 
a 5% early mortality and a further 20% attrition from all causes between hospital discharge 
and the 18 month evaluation. There may also be dropout prior to birth for a variety of reasons: 
fetal death, intolerance of therapy, withdrawal from study. We do not have accurate data to 
estimate this loss. We anticipate needing to enroll 110-130 patients to have a sufficient cohort 
at 18 months (40 per treatment group). We will use the IRB approved Cardiac Center 
Research Intro Sheet. This sheet is given to families in CICU and families followed by the 
Fetal Heart program at CHOP and lists all the studies in the Cardiac Center that recruit pre-
operatively. [See current version attached in appendix section].  In addition, we plan to 
develop a study specific brochure similar to that used in the OPPTIMUM trial (attached in 
appendix), which we will submit for IRB approval.     
9.6 
Informed Consent/Assent and HIPAA Authorization 
Dr. Gaynor and/or one of the research nurses will approach eligible families in person 
during a prenatal visit or upon admission to CHOP and ask their permission to review the 
research study.  If families are willing, the following informed consent process will be initiated 
in order to assure subjects comprehend the purpose of the study, the study procedures, and 
the risk-benefit profile.   
1. The research protocol will be reviewed verbally. 
2. The consent form document will be reviewed. 
3. The HIPAA authorization form will be reviewed. 
4. Time will be provided for questions and decision making. There will be ongoing 
discussion with the parents to give them adequate time to consider participation.    
Dr. Gaynor and/or one of the research nurses will approach eligible families in person during 
an outpatient visit or upon admission to CHOP and ask their permission to review the consent 
addendum. If families are willing, the following informed consent process will be initiated in 
order to assure subjects comprehend the purpose of the consent addendum, the additional 
study procedures, and the risk-benefit profile. 
1. The research protocol will be reviewed verbally. 

 
  
 
 
 
44
2. The consent addendum form document will be reviewed. 
3. Time will be provided for questions and decision making. 
 
9.7 
Payment to Subjects/Families 
9.7.1 Reimbursement for travel, parking and meals 
At the time of both fetal brain MRIs, mothers will be reimbursed for travel, parking, need 
for babysitting, and meals. This will average between $130 (local)-$900 (outside the tristate 
needing hotel and airfare) based on where mothers reside during study period.   
At the 18-month follow-up families will be reimbursed for travel, parking, need for 
babysitting, and meals.  We have budgeted $425-500 on average per family based on a 
range of $190 for families coming by car with no hotel stay (70% referral base), $600 for 
families coming by car with one night hotel stay or the need for round trip limo service (13% 
of referral base) to $1300 for families coming by air with one-two hotel night stay (17% of our 
referral base).   
9.7.2 Payments to parent for time and inconvenience (i.e. compensation) 
Upon completion of each fetal brain MRI each mother will be given a $50.00 gift card to 
compensate for time and inconvenience.     
At the 18 month follow-up the parent will receive a $50.00 gift card to compensate for 
time and inconvenience.   
9.7.3 Gifts 
At the 18-month follow-up the child will receive a book or small toy as a token of 
appreciation.  
10 PUBLICATION 
Any publication or presentation of the data will be done in a de-identified manner and under 
the guidance of J. William Gaynor, Principle Investigator. 
 
 

 
  
 
 
 
45
 
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2004;128:841-849 
 
 
 
 

 
  
 
 
 
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APPENDIX 
Appendix 1  Data Elements from Medical Record Review from Section 5 
 
Section 5.1.1 Pre-Natal Medical Record Review  
Date of Birth 
Race& Ethnicity 
Marital Status 
Occupation 
Demographic: address, phone number(s), email address(s), alternate contacts 
Physical Examination 
Vital Signs: Blood pressure/ pulse (at each visit) 
Growth Parameters  
Concomitant medications  
Allergies 
Last Menstrual Period 
Gestational Age 
Estimated Date of Confinement 
IVF fertilization date or embryo transfer 
Pre-pregnancy weight 
Body Mass Index  
Weights and weight gain 
OB History: Gravida, Para, Term, Preterm, TAB, SAB, Ect, Mult Living 
Antepartum, intrapartum, postpartum complications 
History of STD's 
Abnormal Pap Smears 
Genitourinary Abnormalities 
Family medical history 

 
  
 
 
 
53
Medical and surgical history 
Psychiatric history 
Smoking 
Drugs 
Alcohol 
Exposure to Teratogens 
Stress 
Mental Status 
Social Support 
History of past or current physical abuse 
Pregnancy Readiness Planned/Unplanned 
Housing 
Finances 
Clinically indicated laboratory evaluations obtained by referring physician or CHOP’s CFDT 
(Section 5.1.5) 
 
Section 5.1.2 Post-Natal Medical Record Review 
 
BIRTH  
Date of Birth 
Gender 
Location of Birth 
Gestational Age 
Mode of Delivery  
Apgar Scores 
Weight at Birth 
Length at Birth 
Head Circumference at Birth 
Adverse Events at delivery [Seizures, arrest, intubation, PGE] 

 
  
 
 
 
54
 
 
INPATIENT ADMISSION(s)  
Date of Admission 
Medical Record Number 
Diagnosis: CHD 
Genetic Anomaly  
Extra-Cardiac Anomaly 
Data from clinical genetic consult(s)  
Daily Weight 
Daily Feeding Status 
Daily Lowest Diastolic Blood Pressure 
Daily Arterial Blood Gas [lowest PO2, lowest PCO2, lowest pH, and highest pH] 
Type and duration of mechanical ventilation 
Type and duration of intrathoracic catheters 
Type and duratioin of intracardiac lines 
Daily Events [ NeuroCardiac Research Program database daily event code list] 
Date of Surgery 
Weight at Surgery 
Surgical Procedure 
Surgeon 
Number of CPB Runs 
CPB Time(s) 
DHCA Time(s) 
            Cross Clamp Time(s) 
Cooling Time(s) 
Rewarming Time(s) 
MUF 

 
  
 
 
 
55
NP Temperature 
Baseline Hematocrit 
Hematocrit on CPB 
Hematocrit following modified ultrafiltration 
Hematocrit on admission to CICU 
Anesthesiologist 
Arterial Blood Gas 5 minutes after Commencement of Bypass 
Blood Product Usage 
Medication Usage 
Cardiac Arrest Events 
Reoperations 
ECMO/VAD 
Delayed Sternal Closure 
Death  
Date of Death 
Date of Discharge 
Cardiologist 
 
OUTPATIENT  
Events including procedures between discharge from neonatal surgery and 
18month follow up