Comprehensive Echocardiographic Evaluation of Cardiac Function and Pulmonary

Hemodynamics in the Newly Born during Health and Disease

by

Amish Jain

A thesis submitted in conformity with the requirements

For the degree of Doctor of Philosophy

Graduate Department of Physiology

University of Toronto

© Copyright by Amish Jain 2017

ABSTRACT OF THESIS

Comprehensive Echocardiographic Evaluation of Cardiac Function and Pulmonary

Hemodynamics in the Newly Born during Health and Disease

by

Amish Jain

Ph.D., 2017, Department of Physiology, University of Toronto

Very little is known about heart function or pulmonary hemodynamics in newborn infants in

either health or disease states. Animal experiments have shown that the immediate period

following birth is characterized by an increase in pulmonary blood flow, a decrease in pulmonary

vascular resistance and closure of fetal shunts. The specific timing and rate of these events in

human neonates are not well documented. The adaptation of an immature heart during this phase

of physiological ‘pulmonary hypertension’ has also not been studied. Persistent pulmonary

hypertension of newborn (PPHN), a frequent outcome of dysregulation of postnatal adaptation, is

associated with significant mortality and morbidity, however, the role of cardiac dysfunction is

not well known. Clinical appraisal of cardiac performance is difficult in neonates. Two

dimensional echocardiography, the only routinely feasible investigation, has undergone

significant technological advancements, making it suitable to study human neonatal cardiac

physiology. In the studies reported herein, I demonstrated the feasibility and utility of employing

quantitative echocardiography measures for studying cardiopulmonary physiology during the

immediate postnatal period, in both health and diseased states. I prospectively established a

comprehensive set of normative values for various echocardiographic indices of biventricular

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cardiac function and pulmonary hemodynamics during the first two days of life and described

their relative measurement reliability. Subsequently, for the first time, I provided time-sensitive,

quantitative, physiological circulatory data regarding immediate postnatal transition in humans,

starting from < 0.5 hours of life. I demonstrated that in humans, the net decrease in RV afterload

may be slower during the first 10 to 12 hours of life. In addition, I found that in spite of changes

in loading conditions, secondary to changes in heart rate and shunting patterns across ductus

arteriosus and foramen ovale, the right and left ventricular outputs and their ratio remains

relatively constant throughout the first day of life. Further, I demonstrated lower RV function at

< 0.5 hours of age in comparison to measurements performed during the second half of the first

day, suggesting a relative vulnerability of the neonatal right ventricle to high afterload

immediately after birth. Lastly, by employing these methods in neonates with PPHN, I provided

several novel insights into its pathophysiology. I demonstrated that PPHN is characterized by

global dysfunction in both the right and left ventricle, which may be independent of underlying

etiology. In addition, I identified several previously unknown risk factors associated with cardiac

dysfunction in this disorder and showed a linear correlation between functional measures of both

ventricles. I found significant association between RV longitudinal function during the first three

days of life in neonates with PPHN and subsequent occurrence of death or need for

extracorporeal membrane oxygenation, indicating the relevance of RV function in this serious

disorder of postnatal transition.

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ACKNOWLEDGEMENTS

I would like to start by thanking Dr. Robert Jankov and Dr. Patrick McNamara, my thesis

supervisors, for their constant guidance, patience and unfaltering faith in my abilities. I would

also like to sincerely thank Dr. Luc Mertens, my supervisory committee member and

methodology mentor, for always making himself available, providing high quality critiques and

inspiring me to push intellectual boundaries. I would like to thank my other supervisory

committee members, Drs. Brian Kavanagh and Kim Connelly, for their guidance and many

constructive criticisms. In addition, I would like to particularly thank Dr. Prakesh Shah, one of

my senior colleagues, for his guidance, several rewarding academic conversations and insisting

that I “plan before I do” and “do it the right way”. I would like to acknowledge several other

individuals who helped me bring this work to a successful conclusion. I would like to thank Drs.

Adel Mohamed, Afif El-Khuffash and Regan Giesinger for their assistance with various aspects

of this work and Mrs. Michelle Baczynski for her editorial review of my thesis. I would also like

to express my gratitude to the Department of Paediatrics at Mount Sinai Hospital, in particular

Drs. Shoo Lee and Yenge Diambomba, for their support and understanding during this time.

I wish to thank the Clinician Scientist Development Program of Division of Neonatology at

University of Toronto and Clinician Scientist Training Program at the Hospital for Sick Children

Research Institute for provision of a Doctoral award and Queen Elizabeth II/ Heart and Stroke

Foundation of Ontario for providing me with a Graduate Scholarship.

I give my heartfelt thanks to my wife, Dr. Poorva Deshpande, and the pleasure of our lives,

our sons, Rishik and Reyaansh, for sustaining me and enduring my long absences during this

time. Finally, I wish to sincerely thank the infants and their families for their willingness to

participate in the studies included in this work.

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TABLE OF CONTENTS

Page

List of figures …………………………………………………………………………. vi

List of abbreviations …………………………………………………………………. xii

Publication of thesis work ………………………………………………………….... xv

Chapter 1: General introduction ……………………………………………………. 1

Chapter 2: Development of an imaging protocol, rationale and specific aims …… 39

Chapter 3: Methodology ……………………………………………………………... 47

Chapter 4: A comprehensive echocardiographic protocol for assessing neonatal

right ventricular dimensions and function in the transitional Period: normative

data and Z scores …………………………………………………………………......

63

Chapter 5: Left ventricular function in healthy term neonates during the

transitional period …………………………………………………………………….

83

Chapter 6: Cardiopulmonary adaptation during the first day of life in humans ... 97

Chapter 7: Cardiac function and ventricular interactions in persistent

pulmonary hypertension of the newborn ……………………………………………

117

Chapter 8: Project summary and clinical significance …………………………….. 138

Chapter 9: Limitations and future directions ……………………………………… 149

Appendix: Additional figures and data ……………………………………………... 160

References …………………………………………………………………………….. 168

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LIST OF FIGURES

Chapter 1

Fig. 1.1 Relationship between changes in pulmonary vascular resistance and

pulmonary arterial compliance.

p.6

Fig. 1.2 Meta-analysis of clinical trials of inhaled nitric oxide treatment in term

neonates presenting in the perinatal period with severe hypoxic respiratory

failure.

p.11

Fig. 1.3 Hemodynamic effects of exposure of neonatal right ventricle to high

afterload.

p.12

Fig. 1.4 Pulmonary arterial hypertension in preterm neonates with chronic lung

disease.

p.15

Fig. 1.5 Cellular architecture differences between the right and left ventricle and

their relationship with differential contraction patterns.

p.18

Fig. 1.6 Post-mortem diffusion tensor imaging in pigs heart showing the course of

myocardial pathways throughout the left and right ventricle.

p.19

Fig. 1.7 Complex three-dimensional architecture of the right ventricle. p.20

Fig. 1.8 Comparative changes in ejection fraction between the right and ventricle

when exposure to increased afterload.

p.21

Fig. 1.9 Response of the right ventricle when exposed to increased afterload over a

prolonged period of time.

p.22

Fig. 1.10 The net effect of variations in factors that are known to influence right

ventricle’s ability to withstand afterload is unknown in neonates.

p.23

Fig. 1.11 Measurement of myocardial velocities using pulsed tissue Doppler p.32

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imaging.

Fig. 1.12 Assessment of longitudinal deformation from a myocardial wall. p.34

Fig. 1.13 Measurement of fractional area change for the right ventricle from apical

4-chamber view using two-dimensional echocardiography.

p.35

Fig. 1.14 Measurement of tricuspid annular plane systolic excursion as a marker of

right ventricular global longitudinal function using m-mode

echocardiography.

p.37

Fig. 1.15 The majority of established echocardiographic indices of right ventricular

function are limited by the fact that they are derived from a single two

dimensional plane, which may not account for the contractile contribution

of the apex-to-outflow portion of the right ventricle.

p.38

Chapter 2

Fig. 2.1 A comprehensive functional two dimensional echocardiography imaging

protocol designed specifically for use in neonates for the studies involved

in this thesis.

p.41

Fig. 2.2 Right ventricular basal diameter measured from the parasternal long axis

view using echocardiography may corresponds to the posterior depth of the

right ventricular cavity.

p.42

Fig. 2.3 Longitudinal strain of the inferior wall of the right ventricle can be

measured from the new apical 3-chamber view using echocardiography.

p.43

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Chapter 3

Fig. 3.1 Right ventricular linear dimensions measured using two dimensional

echocardiography.

p.50

Fig. 3.2 Quantification of right ventricle’s function using fractional area change,

tricuspid annular plane systolic excursion and tissue Doppler imaging.

p.51

Fig. 3.3 Measurements of tricuspid inflow early and velocities using pulse wave

Doppler.

p.52

Fig. 3.4 Longitudinal strain measured for right ventricular lateral and inferior walls

from apical 4-chamber and 3-chamber views respectively.

p.54

Fig. 3.5 Measurements of left ventricular dimensions using two dimensional

echocardiography.

p.55

Fig. 3.6 Longitudinal strain for left ventricle measured using speckle tracking

echocardiography.

p.58

Fig. 3.7 Schematic representation of measures of pulmonary vascular resistance

obtained from pulse wave Doppler of the main pulmonary artery.

p.60

Chapter 4

Fig. 4.1 A box plot graph of fractional area change measurements obtained from

right ventricular 4-chamber and 3-chamber views on day 1 and day 2 of

life in healthy term neonates.

p.79

Chapter 5

Fig. 5.1 Peak systolic myocardial velocity in left ventricular free wall and p.93

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interventricular septum on day 1 and day 2 of life in healthy term neonates.

Fig. 5.2 Bland Altman plots for inter- and intra-observer measurement variability

for mean longitudinal strain and strain rate measured from left ventricular

4-chamber view in a randomly selected subset of 20 scans obtained in

healthy term neonates.

p.97

Chapter 6

Fig. 6.1 Changes in echocardiographic measures of pulmonary vascular resistance

during the first day of life in healthy human neonates.

p.106

Fig. 6.2 A sequentially obtained pulse wave Doppler trace of the main pulmonary

artery from one of the study infants, showing typical changes observed

during the first 24 hours of age in healthy neonates following successful

postnatal transition.

p.107

Fig. 6.3 Box plot graphs of key echocardiographic measures of right ventricular

systolic function at <0.5 hours and 22-24 hours of life in healthy human

neonates following successful postnatal transition.

p.110

Fig. 6.4 Comparison of peak global longitudinal strain measured using speckle

tracking echocardiography from right ventricular lateral wall (GLS-4C)

and inferior wall (GLS-3C) at < 0.5 hours and 22-24 hours of age in

healthy neonates following uncomplicated postnatal transition.

p.111

Chapter 7

Fig. 7.1 Box plot graph showing comparison of tricuspid annular plane systolic p.126

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excursion (TAPSE) between neonates with persistent pulmonary

hypertension of newborn (PPHN) and healthy controls.

Fig. 7.2 Box plot graphs for overall global peak longitudinal strain for the right and

the left ventricle in neonates with persistent pulmonary hypertension of

newborn (PPHN) and healthy controls.

p.126

Fig. 7.3 Linear correlations between right ventricular (RV) and left ventricular

(LV) overall global peak longitudinal strain (GLS) and RV and LV

myocardial performance index measured using tissue Doppler imaging

(MPI’).

p.128

Fig. 7.4 A box plot graph comparing TAPSE for neonates with persistent

pulmonary hypertension of newborn (PPHN) who survived until discharge

without ECMO vs. those who either died or needed ECMO.

p.130

Chapter 8

Fig. 8.1 A schematic presentation of the long term research strategy developed

during the conduct of this thesis, highlighting research questions for my

ongoing and future work.

p.146

Chapter 9

Fig. 9.1 Future plan to refine diagnostic criteria for chronic pulmonary

hypertension in preterm neonates with chronic lung disease and facilitate

early identification of at-risk neonates.

p.157

Fig. 9.2 A parasternal short axis view obtained using two dimensional p.158

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echocardiography in a preterm neonate with significant chronic pulmonary

hypertension demonstrating severe RV dilatation.

Appendix (additional figures)

Fig. 4.2 Measurement of peak longitudinal strain of right ventricular inferior wall

(GLS-3C) using speckle tracking echocardiography from one of the study

infants.

p.161

Fig. 4.3 Bland-Altman plots showing intra- and inter-observer measurement

variability was lower for biplane-fractional area change (global-FAC) in

comparison to single-plane measurements acquired from RV apical 4

chamber view (FAC-4C) or from RV apical 3 chamber view (FAC-3C).

p.162

Fig. 4.4 Bland-Altman plots showing intra- and inter-observer measurement

variability was relatively lower for global peak longitudinal strain (GLS-

global) obtained by averaging GLS acquired from RV apical 4 chamber

view (GLS-4C) and from RV apical 3 chamber view (GLS-3C) in

comparison to its individual components.

p.163

Fig. 4.5 Bland-Altman plots for inter and intra observer measurement variability

for myocardial performance index calculated using time periods measured

from color flow Doppler (MPI) and tissue Doppler imaging (MPI’).

p.164

Fig. 4.6 Modest linear correlation observed between right ventricular peak systolic

myocardial velocity (s’) measured from tissue Doppler imaging and

speckle racking echocardiography derived peak longitudinal strain of right

ventricular lateral wall.

p.165

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LIST OF ABBREVIATIONS

2D, Two-dimensional

4C, 4-chamber view

3C, 3-chamber view

a’, Late diastolic myocardial velocity measured using TDI

B-4C, Basal diameter in 4C

B-PLAX, Basal diameter in PLAX

BW, Birth weight

BA, Bland-Altman analysis

cPHT, Chronic pulmonary hypertension

CNLD, Chronic neonatal lung disease

CMRI, Cardiac magnetic resonance imaging

COV, Coefficient of variation

ECMO, Extracorporeal membrane oxygenation

ELBW, Extremely low birth weight

EDA, End-diastolic area

ESA, End-systolic area

e’, Early diastolic myocardial velocity measured using TDI

EF, Ejection fraction

FAC, Fractional area change

GLS, Global longitudinal strain

GLS-4C, Global longitudinal strain of RV lateral wall

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GLS-3C, Global longitudinal strain of RV inferior wall

iNO, inhaled nitric oxide

IVRT’, Isovolumic relaxation time measured using TDI

IVRT, Isovolumic relaxation time measured using color flow Doppler

ICC, Intraclass correlation coefficient

IQR, Interquartile range

LV, Left ventricular

LVEDD, Left ventricular end-diastolic diameter

LVESD, Left ventricular end-systolic diameter

LVSV, Left ventricular stroke volume

LVO, Left ventricular output

MPI’, Myocardial performance index measured using TDI

MPI, Myocardial performance index measured using color flow Doppler

MvE, Early mitral diastolic inflow peak velocity

MvA, Late mitral diastolic inflow peak velocity

NICU, Neonatal intensive care unit

PVR, Pulmonary vascular resistance

PAH, Pulmonary arterial hypertension

PPHN, Persistent pulmonary hypertension of the newborn

PHT, Pulmonary hypertension

PLAX, Parasternal long axis view

PAAT, Pulmonary artery acceleration time

PVRI, Pulmonary vascular resistance index

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PDA, Patent ductus arteriosus

PFO, Patent foramen ovale

PAATi, PAAT indexed to total duration of cardiac cycle

RV, Right ventricular

RVET, Right ventricular ejection time measured using color flow Doppler

RVET’ Right ventricular ejection time measured using TDI

RVSP, Peak right ventricular systolic pressure

RVSV, Right ventricular stroke volume

RVO, Right ventricular output

RVETi, RVET indexed to total duration of cardiac cycle

s’, Peak systolic myocardial velocity measured using TDI

STE, Speckle tracking echocardiography

SF, Shortening fraction

SD, Standard deviation

SVR, Systemic vascular resistance

SAX, Parasternal short axis view

TDI, Tissue Doppler imaging

TAPSE, Tricuspid annular plane systolic excursion

TvE, Early tricuspid diastolic inflow peak velocity

TvA, Late tricuspid diastolic inflow peak velocity

VTI, Velocity time integral

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PUBLICATION OF THESIS WORK

1. Amish Jain, Adel Mohamed, Afif El-Khuffash, Kim A. Connelly, Frederic Dallaire, Robert P Jankov, Patrick J McNamara, Luc Mertens. “A Comprehensive Echocardiographic Protocol for Assessing Neonatal Right Ventricular Dimensions and Function in the Transitional Period: Normative Data And Z-scores”. Journal of American Society of Echocardiography 2014; 27(12):1293-3041. Co-author contributions to this work included assistance in patient enrolment (Adel Mohammed), echocardiographic analysis for calculating inter-rater reliability data (Afif El-Khuffash) and calculation of Z-scores (Frederic Dallaire).

2. Amish Jain, Afif F. EL-Khuffash, Bart C.W. Kuipers, Adel Mohamed, Kim A. Connelly, Patrick J McNamara, Robert P. Jankov, Luc Mertens. “Left Ventricular Function in Healthy Term Neonates During the Transitional Period”. Journal of Pediatrics. 2016 Nov 28. Pii: S0022-3476(16)31231-8. Doi: 10.1016/j.jpeds.2016.11.0032. Co-author contribution to this work included assistance in patient enrolment (Adel Mohammed), echocardiographic analysis for calculating inter-rater reliability data (Bart C.W. Kuipers) and teaching and assistance with statistical analysis (Afif El-Khuffash).

3. Amish Jain, Bart C.W. Kuipers, Adel Mohamed, Brian Kavanagh, Prakesh S Shah, Luc Mertens, Robert P. Jankov, Patrick J McNamara. “Cardiopulmonary Adaptation During the First Day after Birth in Humans”. Manuscript in preparation for submission to Circulation. Co-author contribution to this work included assistance in patient enrollment (Adel Mohammed), echocardiographic analysis for calculating inter-rater reliability data (Afif El-Khuffash) and teaching and assistance with statistical analysis (Prakesh Shah).

4. Amish Jain, Afif El-Khuffash, Claar H. van Herpen, Maura Helena, Regan Giesinger, Dany Weisz, Luc Mertens, Robert P. Jankov, Patrick J McNamara. “Cardiac Function and Ventricular Interactions in persistent pulmonary hypertension of the newborn”. Manuscript in preparation for submission to Circulation Imaging. Co-author contribution to this work included assistance in identification of study patients and their echocardiograms (Claar H. van Herpen ) and collection of clinical data (Maura Helena and Regan Giesinger).

xv | P a g e 1Reprinted (Chapter 4) in compliance with Elsevier’s Author User Rights policy.

2Reprinted (Chapter 5) in compliance with Elsevier’s Author User Rights policy.

Chapter 1: General Introduction

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1.1. The clinical relevance of the right ventricle

From the time of William Harvey’s 1628 treatise, De Motu Cordis, correctly describing the

pulmonary circulation, until well into the 20th century, the right ventricle remained largely

ignored, being considered a passive conduit and therefore less important than the systemic

ventricle (1). This notion was further fuelled by experimental observations by Fineberg and

Wiggers in 1936 and Taquini and co-workers in 1960, investigating the effect of an acute rise in

right ventricular (RV) afterload by artificially and variably constricting the pulmonary trunk in

an experimental adult dog model (2, 3). These experiments indicated that up to 50% constriction

of the pulmonary artery resulted in no appreciable change in ventricular pressure or cardiac

output. Additional constriction resulted in an increase in RV systolic pressure but cardiac output

was maintained until almost a 2/3rd occlusion of the pulmonary artery. However, these

experiments were performed with an open pericardium and had the fundamental flaw of failing

to account for the effect of pericardial constraint on inter-ventricular interactions. The belief that

the right ventricle was a relatively dispensable component of the cardiovascular system also

contributed to the development of the Fontan Kreutzer procedure, described separately in 1971

by Dr. Francois Marie Fontan in France and in 1973 by Dr. Guillermo Kreutzer in Argentina, as

a surgical correction for tricuspid atresia (4, 5). Since then, Fontan’s circulation, which involves

creating a pressure passive system for pulmonary blood flow by diverting the deoxygenated

blood returning to the heart via the great veins directly to the pulmonary artery, while the

functioning single ventricle (morphologically the right or left ventricle depending on the

underlying cardiac defect) is used as a pump exclusively for systemic circulation, has become the

model to study and understand the circulatory consequences of an absent right ventricle. Short

and long term follow up of these patients has demonstrated that although sustainable circulation

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is feasible without a functioning right ventricle, it is with significant and often life limiting

consequences, most notably chronic venous congestion and low cardiac output (6). Furthermore,

in the absence of a right ventricle, even a small rise in pulmonary vascular resistance (PVR) is

poorly tolerated and often results in a catastrophic systemic decompensation. Ultimately,

Fontan’s circulation is said to “impact organ system[s] in an indolent and relentless manner, with

progressive decline in functionality likely” (7).

Over the last three decades there has been an increasing recognition of the critical

importance of RV function in health and disease. There is accumulating evidence of a key

prognostic role in several disorders of the cardiovascular system, including congestive heart

failure, left sided heart failure, coronary artery disease, valvular heart disease, post cardiac

surgery and congenital structural heart defects such as Ebstein’s anomaly and tetralogy of Fallot

(8-12). However, in no other disorder is the significance of preserving RV function more

recognized than in pulmonary arterial hypertension (PAH) (13). Although the hallmark feature in

PAH is raised pulmonary arterial pressure, it is now widely regarded as a “right heart failure

syndrome” (14). This is because of the consistent observation across studies, irrespective of

methods used for its assessment, that in this disorder, the severity and progression of symptoms

and clinical outcomes are less related to PVR or mean pulmonary artery pressure than the ability

of the right ventricle to cope with increased afterload while maintaining its size, function and

filling pressures (Table 1.1) (15). Tolerance of the right ventricle to afterload is variable between

individuals and known to depend on factors such as the underlying disorder, gender, age at

presentation and the rate of rise in pulmonary arterial pressure (16, 17).

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Table 1.1(15): Measurements to predict outcome in adults with severe PAH (RV function related measurements shown in bold)

Study (year) No. of patients

Etiology Predictive measurements

Right heart catheterization Fuster (1984) D’Alonzo (1991) Sandoval (1994) McLaughlin (2002) Sitbon (2002) Humbert (2010) Benza (2010)

120 194 61

162 178 354

2716

PPH PPH PPH PPH PPH

IPAH PAH

Mixed venous oxygen saturation (SvO2) RAP, PAP, CO RAP, CO RAP, CO Lack of change in PVR CO RAP, PVR

Echocardiography Eysmann (1989) Raymond (2002) Bustamente (2002) Forfia (2006) Utsunomiya (2009) Ghio (2010) Sachdev (2011) Brierre (2010) Ghio (2011) Haeck (2012) Fine (2013) Ernande (2013) Ameloot (2014) Smith (2014) [24] Yeo (1998) Grunig (2013)

26 81 25 63 50 59 80 79

72

150 575 142 78 97 53

121

PPH PPH PPH

PAH/CTEPH/RD PAH IPAH PAH

PAH/CTEPH/RD

PAH PH PH

PAH/CTEPH PAH/CTEPH PAH/CTEPH

PPH PAH/CTEPH

Pericardial effusion Pericardial effusion, RAS, EI RAS, TR TAPSE Tricuspid E/e’ TAPSE, EI RV strain PAP, pericardial effusion, EI, TAPSE, MPI, IVC RV diameter RV longitudinal strain by speckle tracking RV longitudinal strain by speckle tracking RV isovolumetric contraction velocity TAPSE, dp/dt 3D speckle area strain MPI Stress echo (Δ TR velocity with exercise)

MRI Van Wolferen (2007) Moledina (2013) Yamada (2012) Veerdonk vd (2011) Swift (2014) Freed (2012)

64

100 41

110 80 58

IPAH PAH IPAH PAH IPAH

PH

SV, RVEDV, LVEDV EF, SV RV EDV EF ESV LGE

PPH primary pulmonary hypertension, PAH pulmonary arterial hypertension, IPAH idiopathic PAH, CTEPH chronic thromboembolic pulmonary hypertension, RD respiratory disease, LVD left ventricular disease, RAP right atrial pressure, PAP pulmonary artery pressure, CO cardiac output, RAS right atrial surface area, EI eccentricity index, TR tricuspid regurgitation, TAPSE tricuspid annular plane systolic excursion, RV right ventricular, MPI myocardial performance index, IVC inferior vena cava, SV stroke volume, EDV end-diastolic volume, ESV end-systolic volume, EF ejection fraction, LGE late gadolinium enhancement. Note: Irrespective of the method of assessment, parameters of RV function are more commonly associated with patient outcomes than those reflective of pulmonary vascular disease; indicating the clinical relevance of RV function in PAH. Table adapted from Naeiji R. Curr Hypertens Rep. 2015;17(5):35.

1.2. Right ventricular afterload

One of the reasons for a dichotomy between the severity of PAH and RV dysfunction and

related clinical outcomes is the commonly employed oversimplified practice of using PVR or its

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surrogates as an exclusive measure of RV afterload for quantifying disease severity and

monitoring its progression or response to therapies. PVR is derived by using the measurement of

mean forward flow through the pulmonary vascular bed and is most influenced by changes in the

diameter of small peripheral arteries and arterioles, indicating that it represents the static

component of RV afterload. The blood flow in the pulmonary circulation requires a transmission

of pulsatile waves of pressure and flow down the pulmonary vascular tree, which by virtue of

having bifurcations, is characterized by several upstream deflections of pressure waves; thus

adding a dynamic or pulsatile component to the afterload experienced by the right ventricle. This

additional burden on RV workload is dependent upon the compliance of the pulmonary arterial

circuit (distensibility or pulsatility), determined mainly by the larger pulmonary arteries (up to

the first five branches) (18). Pulmonary arterial compliance has been shown to have an inverse

hyperbolic relationship with PVR (Figure 1.1) (19). Hence, increased resistance is preceded by

reduced compliance in evolving PAH. However, in advanced disease, arterial stiffness has

already reached its peak thereby any further increase in RV afterload is almost entirely secondary

to PVR. The ability of the large pulmonary arteries to maintain a high compliance can

significantly minimize the net afterload experienced by the right ventricle, even in the setting of

rising PVR.

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Figure 2.1: Inverse hyperbolic relationship between changes in PVR and pulmonary arterial compliance in two patients with pulmonary arterial hypertension at different baseline values of PVR (A mild and B severe). Figure from Jan-Willem et at. Eur Heart J. 2008; 29(13):1688-95.

There is an increasing recognition that pulmonary vascular disease is not only contributed

to by sustained pulmonary vasoconstriction (high PVR), but also vascular remodelling and a

resulting reduction in arterial compliance, which can contribute significantly towards increasing

RV workload, particularly early in the disease process (20). However, finding a robust

quantitative measure of net RV afterload has remained an elusive clinical objective. Pulmonary

vascular impedance is said to be such a measure, as it encompasses both the static and dynamic

aspects of RV afterload, however, it can only be measured with right heart catheterization.

Catheterization derived measure of pulmonary vascular impedance in pediatric PAH patients has

recently been shown to correlate better with clinical outcomes in comparison to PVR alone (21).

While we search for the ideal hemodynamic measure of RV afterload, it appears that close

monitoring of RV function itself is the best method for monitoring clinically significant changes

in disease progression, as well as response to commonly used therapies. In fact, several experts

have recently proposed RV function in PAH as the target for drug therapies and an end point for

clinical trials (22, 23). This may be a critical detail in pulmonary hypertensive disorders in

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newborn infants, where due to lack of feasibility of catheterization, our current methods for

detecting pulmonary vascular disease are even more rudimentary.

1.3. Clinical context: Postnatal adaptation and neonatal pulmonary hypertensive disorders

Transition from intra- to extra-uterine life

Birth is a unique physiological event characterized by complex and sudden changes

affecting several organ systems, most notably the respiratory and cardiovascular system (24).

Fetal life is characterized by non-participation of lungs and dependence on placental circulation

for gas exchange, along with its other metabolic functions. The fetal circulation in arranged in

series. The majority of venous return coming from the placenta bypasses the hepatic circulation

via the ductus venosus, reaching the inferior vena cava just before its entry into the right atrium

(25). Enabled by the anatomical location of the inferior vena cava and the high volume of

umbilical venous return, most oxygenated blood jets across to the left side through foramen

ovale (FO), which is kept widely open by the higher right atrial pressure compared to the left. Of

the remaining blood pumped into the pulmonary artery by the fetal right ventricle, a large

proportion joins the systemic circulation via the ductus arteriosus (DA) without passing through

the pulmonary vascular bed; the end result is that only 10-20% of total biventricular cardiac

output enters the lungs during fetal life. This, however, increases to approximately 30% by late

gestation secondary to the increase in reactivity of the fetal pulmonary vasculature during third

trimester (26). In addition to the anatomical features, these circulatory features are made possible

by the high PVR of the fluid filled fetal lungs and low systemic vascular resistance (SVR) in the

fetus secondary to its attachment to the placental circulation. Maintenance of fetal circulation as

well as its rapid adaptation after birth to a parallel circulation, where almost the entire cardiac

output must pass through the lungs for oxygenation, is a result of a cascade of concurrently

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occurring, interconnected but not completely understood mechanical, biochemical and hormonal

factors (27-30).

The sentinel event which triggers dramatic circulatory adaptation at birth is onset of

ventilation (31). A sudden and rhythmic distension of lungs with air enabled by the high negative

pressure of first few breaths taken by the newly born cause displacement of lung fluid from

alveolar to interstitial space. This results in establishment of an air-liquid interface in the

ventilated alveoli and a large initial drop in PVR. Although the specific mechanism(s) by which

ventilation alone reduces PVR are not yet known, the following factors are postulated to play key

role: 1) straightening of airways and untwisting of pulmonary vessels due to alveolar expansion,

2) recruitment of intra-acinar arteries, 3) increased capillary diameter caused by an increase in

the transmural pressure across alveolar-capillary interface secondary to the newly developed

surface tension inside the alveoli and 4) improvement in ventilation-perfusion matching caused

by the vasodilatory effects of increased alveolar oxygen and the production of nitric oxide (28,

30). Coinciding with the fall in PVR secondary to ventilation, is the sudden increase in SVR after

the umbilical cord is cut and the placenta is removed from the systemic circulation. One of the

consequences of these dramatic changes in the PVR: SVR ratio is a change in the directionality

of the flow across DA, resulting in a net left to right shunt (i.e. systemic to pulmonary

circulation). Increased systemic-pulmonary shunting across the DA is thought to be a key

determinant of an initial rapid rise in pulmonary blood flow after birth. The abrupt gush of blood

into the pulmonary vascular bed (both arterial and venous) exposes the endothelium to increased

shearing forces which, in addition to an increase in oxygen tension is thought to induce

production of vasodilatory mediators (e.g. nitric oxide, bradykinin, prostacyclin) and inactivate

production of vasoconstrictor mediators (e.g. thromboxane, endothelin, leukotrienes) (29).

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Contributory changes are also observed in pulmonary vascular smooth muscle cells, which

undergo remodelling and progressive thinning starting shortly after birth.

From a cardiac perspective, an acute rise in pulmonary blood flow causes a significant

increase in left heart preload and a rise in left atrial pressure. This, along with a reduction in

volume and force of venous return from inferior vena cava and lowering of right atrial pressure,

results in closure of the FO. The increase in arterial oxygen concentration, bradykinin production

and reduction in circulating levels of prostaglandins induce constriction of DA soon after birth

followed by functional closure within days. Absence of flow across the ductus venosus following

removal of the placental circulation initially results in constriction of its sphincter followed by

closure. The RV mass at the time of birth is 20% above adult values when indexed to body

surface area, while the mass of the left ventricle is 30% lower (32). Following birth the mass of

the left ventricle increase rapidly over the first two weeks of life but that of the right ventricle

decreases more slowly over months.

Our knowledge of transitional physiology as highlighted above is mostly derived from

animal experiments. The physiologic changes in humans after birth are comparable; however, the

rate and timing of these events is not well understood. Up to 10% of neonates are known to

require resuscitation at birth and dysregulation of postnatal adaptation is a common reason for

need for intensive care treatment. The availability of reliable and clinically relevant physiologic

measures of heart function, pulmonary and systemic hemodynamics in humans may not only

help understand species specific differences but may have direct diagnostic, monitoring and

therapeutic relevance leading to changes in the approach to clinical care. For instance, a recent

physiological study conducted in human fetuses has shown that much less blood bypasses the

liver via the ductus venosus than was previously assumed based on animal experiments; this

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highlights the importance physiology studies in humans (25). In addition, cardiac functional

adaptation in the first day following birth has not been evaluated.

Acute pulmonary arterial hypertension in neonates

Acute PAH in the immediate postnatal period, also known as persistent pulmonary

hypertension of the newborn (PPHN), is an acute cardiopulmonary illness that accounts for up to

10% of all admissions to tertiary neonatal intensive care units (NICUs) (33). Phenotypically, it is

characterized by severe hypoxemic respiratory failure secondary to the failure of a physiological

transition of the pulmonary circulation from a high resistance intra-uterine to a low resistance

extra-uterine circuit. PPHN may arise secondary to other disorders of postnatal transition such as

perinatal asphyxia, meconium aspiration syndrome, respiratory distress syndrome, sepsis or

pulmonary hypoplasia (e.g. congenital diaphragmatic hernia) (33). The incidence of PPHN in

developed countries ranges from 1 to 2 per 1000 live births, with a reported mortality of

approximately 10% (33, 34). Surviving neonates commonly require advanced and prolonged

cardio-respiratory support and are at an increased risk of long-term adverse medical and

neurodevelopmental outcomes (34-37). Although treatment with inhaled nitric oxide (iNO), the

only approved pulmonary vasodilator agent in neonates, has reduced the need for extracorporeal

membrane oxygenation (ECMO), it has failed to impact mortality or long term outcomes (Figure

1.2) (38). A lack of impact of pulmonary vasodilator therapies on long-term outcomes has also

been described in older patients with PAH.

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Figure 1.2: Meta-analysis of clinical trials of inhaled nitric oxide treatment in term neonates presenting in the perinatal period with severe hypoxic respiratory failure. iNO treatment reduced need for extracorporeal membrane oxygenation but not death. Finer NN, Barrington KJ. Cochrane Database Syst Rev. 2006 Oct 18;(4):CD000399.

Acute PAH in neonates may also occur later in postnatal life, after the initial decrease

in PVR from fetal levels, secondary to disorders such as sepsis, pneumonia or aspiration.

Although sepsis- related acute PAH in neonates is associated with a high mortality, it remains a

largely unstudied disorder. Irrespective of the variation in underlying etiology, the hemodynamic

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and clinical sequelae of high PVR are expected to be similar and include a complex but not

completely understood interplay of reduced pulmonary blood flow, perfusion-ventilation

mismatch, hypoxemia, acidosis, persistence of fetal shunts and cardiac dysfunction (Figure 1.3)

(39). How a relatively immature neonatal right ventricle copes with increased afterload, its direct

or indirect effect on the left ventricle and the systemic circulation, as well as its role in eventual

patient outcomes, remains largely unexplored.

Figure 1.3: Hemodynamic effects of exposure of neonatal right ventricle to high afterload may involve a complex, but incompletely understood, interplay between hypoxemia, ventilation-perfusion mismatch and cardiac dysfunction. The severity and relative importance of each alteration may vary between individual patients. Figure from Jain A, McNamara PJ. Semin Fetal Neonatal Med. 2015;20(4):262-71.

There is an urgent need for comprehensive high quality studies examining heart function

in neonatal acute PAH. The few studies that exist on RV function in neonates with PPHN

generally have a small number of patients, include a high proportion of neonates with conditions

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such as congenital diaphragmatic hernia that has a different pathogenesis from PPHN and

employ a few select echocardiographic measures. Nonetheless, these studies have suggested that

RV dysfunction may be a common finding in neonates with PPHN and may be associated with

adverse clinical outcomes; thus emphasizing the importance of comprehensive interrogation of

this problem (Table 1.2) (37, 40-45). The ability to predict which neonates are at risk of

subsequent deterioration will help formulate clinical surveillance policies, reduce the need to act

in response to unforeseen deteriorations and provide opportunities for clinical studies of early

therapeutic interventions.

Table 1.2: Studies on RV function in neonates with PPHN

Study (year)/ Type Population/ No. of patients Measurements tested Conclusion Sernich (2006) Prospective case-control

Congenital diaphragmatic hernia (CDH) with PHT on echocardiography (N=10) Age matched control (N=24)

MPI, PEP/RVET, PEP/PAAT

Early RV dysfunction is present in neonates with CDH and PHT, which improves post-surgical repair as PHT improves.

Peterson (2009) Retrospective cohort

Acute PHT during first 28 days of age (N=63)

RV-EDA, RV-ESA, FAC, LV-EDA, LV-ESA, MV E/A, LVOT-VTI, PDA shunt type

Decreased LV size and output correlates with subsequent need for advanced therapies

Patel (2009) Prospective case-control

Cases: Consecutive NICU patients with PHT on echocardiography (n=15, 11 CDH) Controls: NICU patients with no PHT on echocardiogram (N=28)

RV myocardial velocities measured using tissue Doppler imaging

Quantifying RV function using TDI in neonates with PHT is feasible and demonstrates evidence of systolic and diastolic dysfunction

Moenkemeyer (2014) Retrospective cohort

Cases: CDH (N=20) pulmonary: systemic systolic pressure, RV myocardial velocities measured using tissue Doppler imaging

RV diastolic dysfunction diagnose by lower early diastolic velocity associated with increased duration of respiratory support

Agarwal (2015) Retrospective cohort

Cases: PHT < 1 week age (N=117) Controls: 35 (< 1 week of age)

RV SD/DD measured from tricuspid regurgitant jet, LVO, RVO, MPI, EI

The RV SD/DD ratio is a sensitive prognostic marker for need for ECMO or death

Zakaria (2015) Retrospective case-control

Cases: NICU patients with PHT on echocardiography (N=30, age range 1 to 11 days) Controls: NICU patients without PHT on echocardiography (N=69, age range 5-52 days)

TAPSE, RV s’ measured using tissue Doppler imaging, RV FAC

TAPSE most sensitive measure to distinguish PHT neonates from controls

Malowitz (2015) Cases: PHT on TAPSE, RV/LV end- TAPSE, Global RV strain

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Retrospective cohort echocardiography < 7 days of age (N=86)

diastolic diameter ratio, FAC, RV 4 chamber global strain, PDA shunt type

and right to left PDA shunt associated with progression to death or ECMO

PHT pulmonary hypertension, MPI myocardial performance index, PEP pre-ejection time in main pulmonary artery, RVET right ventricular ejection time, PAAT pulmonary artery acceleration time, RV right ventricular, LV left ventricular, EDA end diastolic area, ESA end systolic area, FAC fraction area change, MV E/A mitral valve early: late inflow velocity ratio, LVOT-VTI left ventricular outflow tract velocity time integral, PDA patent ductus arteriosus, SD systole duration, DD diastole duration, LVO left ventricular output, RVO right ventricular output, MPI myocardial performance index, EI eccentricity index, TAPSE tricuspid annular plane systolic excursion, s’ peak systolic velocity, ECMO extracorporeal membrane oxygenation.

Chronic pulmonary hypertension in neonates

Chronic pulmonary hypertension (cPHT) in neonates is inextricably linked to abnormal

development of the lung, most commonly in chronic neonatal lung disease (CNLD), a frequent

complication of infants born ≤ 1,000 g (known as extremely low birth weight [ELBW] infants)

(46, 47). Advances in neonatal care over the past 25 years have had a major impact on the

survival of ELBW infants. This has also resulted in a greater burden of morbidities. Overall, the

incidence of CNLD in ELBW infants is around 50%, leading to more than 10,000 new cases per

year in the United States alone (48). In Canada, CNLD affects almost 60% of all ELBW infants

(> 800 new cases each year) (47). Pathologically, CNLD is characterized by sustained

pulmonary vasoconstriction and exaggerated vasoreactivity to hypoxemic episodes during early

disease, which after an ill-defined period of time is complicated by characteristic developmental

alterations in the pulmonary vascular bed including vascular hypoplasia and arterial wall

remodeling, as exemplified by smooth muscle hyperplasia and distal extension of smooth muscle

into normally non-muscular arteries; resulting in a relatively ‘fixed’ and often progressive

elevation of PVR (49-51) (Figure 1.4 panel A). Our understanding regarding the prevalence and

relevance of pulmonary vascular disease in CNLD is just beginning to take shape, with the

majority of studies being published in the last few years (52-59). Recent reports have now

confirmed the high prevalence of ‘significant’ cPHT in CNLD (approximately 30-40%) and its

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close link with the severity of underlying lung disease (Figure 1.4 panels B and C) (52).

Further, we now know that cPHT in CNLD is independently associated with higher in-hospital

mortality, increased duration of need for respiratory support and longer hospitalization. Severe

cPHT diagnosed late after discharge from initial hospitalization appears to be particularly

malignant, resulting in death of more than half cases from RV failure within two years of

diagnosis (55) (Figure 1.4 panel D).

Figure 1.4: A. Illustration demonstrating characteristic features of arterial wall remodelling seen in chronic neonatal lung disease (muscularization of normally non-muscular distal arteries, intimal fibrosis and medial hypertrophy resulting in narrowing of vascular lumen). B. Overall 30-40% of all extremely low birth weight infants (ELBW) will develop significant chronic pulmonary arterial hypertension (PAH), however this incidence is closely linked to severity of underlying lung disease (panel C.). D. Severe PAH in neonates with CNLD diagnosed after discharge from initial hospitalization is known to be particularly malignant with a high mortality rate secondary to RV failure. Panel A is adapted from Yen-Chun Lai et al. Circ Res. 2014;115:115-130; Panel C is from An HS et al. Korean Circ J 2010; 40:131-136; and D from Khemani E et al. Pediatrics 2007; 120:1260-1269.

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However, the diagnosis of cPHT in these patients is usually made by qualitative and

limited echocardiography criteria that have low sensitivity and are not suitable for early

identification of the disease; the latter is imperative if the focus of care is to maximize the

potential impact of therapeutic interventions and minimize the process of abnormal pulmonary

vascular development. There is a need to develop sensitive, non-invasive, quantitative measures

to enable the study of RV function, given the likelihood that it is a major contributor to adverse

outcomes for this vulnerable and understudied patient population. The field of echocardiographic

evaluation of the right ventricle in extreme preterm neonates is just beginning to develop, with

recent studies demonstrating the feasibility of employing RV-specific indices in this patient

population and describing ‘normal’ values (60-66).

1.4. Differences between the right and the left ventricles

For many years, both ventricles were thought to be similar and the disorders affecting the

right ventricle were commonly managed based on information derived from studies of the left

ventricle (1). However, it has become increasingly clear that the right ventricle is fundamentally

different and needs to be studied and understood independent of left ventricular performance.

Important inter-ventricular differences exist in embryonic origin, cellular architecture,

morphology and functional physiology.

Embryonic origin: The widely accepted view that all structures of the adult heart arise from a

single set of progenitor cells referred to as the ‘primary heart field’, was dispelled as recently as

the year 2001, when three separate groups of researchers, using avian and murine experimental

models, demonstrated the existence of a second contiguous but unrelated region in the

pharyngeal mesoderm contributing cells to the developing outflow tract, referred to as the

‘secondary heart field’ (67-69). Since its discovery, researchers using refined techniques,

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including lineage-tracing experiments, have shown that even though all myocytes of a

developing heart are clonally related, they arise from two distinct lineages: the primary heart

field giving rise to the final left ventricle while the right ventricle, outflow tracts and atria arise

from the secondary heart field (70-72).

Cellular architecture: At a cellular level, the mature myocardium is formed by a complex three-

dimensional meshwork of intermingled myocytes scattered in an extensive fibrous matrix.

Despite the absence of anatomically separate muscular sheets or bands, the myocardial wall can

still be considered as distinct ‘layers’ based on the changing orientation of the myocardial grain

through its depths (73). Here, there are important differences between the two ventricles, which

also explain the differences observed in their respective predominant contraction patterns

(Figure 1.5A). The right ventricular myocardium can be said to have two ‘layers’: superficial

and deep. The myocytes aggregated in the superficial subepicardial layer are oriented

circumferentially, while the deep layer consists of fibers running longitudinally from the base to

the apex. Hence, the right ventricle contracts predominantly in the longitudinal direction, except

for the subpulmonary infundibulum, where the majority of myocytes are oriented in a circular

fashion. In contrast, the LV has an additional middle ‘layer’ of myocytes and a more complex

orientation. The subepicardial layer of the LV is oriented obliquely at an angle of 60 - 80o to the

equatorial plane (called the helical angle), the middle layer has predominantly circular fibers

running parallel to the equator and the deep subendocardial layer has longitudinally oriented

fibers. This complex geometry results in the left ventricle deforming in multiple planes during

each contraction including, longitudinal, circumferential, radial, as well as a twist around its axis

(Figure 1.5B) (74). Interestingly this cellular architectural pattern remains fairly constant from

early fetal to adult life (73).

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Figure 1.5: A. The orientation of myofibers in superficial (subepicardial), middle and deep (subendocardial) components of the right and left ventricle. While the right ventricle has only two orientations – circumferential in superficial and middle and longitudinal in deep layers, the left ventricle demonstrates a more complex orientation with superficial oblique fibres changing to circumferential in middle followed by longitudinal orientation in deep ‘layer’. Due to this orientation, the left ventricle deforms in multiple planes as shown in panel B. The right ventricle on the other hand primarily contracts in longitudinal plane. Panel A is adapted from Siew Yen Ho Eur J Echocardiogr 2009;10:iii3-iii7 and panel B from Wei Yu et al. Eur Heart J Cardiovasc Imaging. 2013 Feb;14(2):175-82.

Using post-mortem diffusion tensor imaging to track myocardial pathways in pig hearts,

Smerup et al have recently demonstrated that each myocardial pathway in the left and the right

ventricle, instead of being a discrete bundle, possessed endocardial, mid wall and epicardial

components, which seamlessly and reproducibly transform into each other, smoothly changing

its angulation as it travels through the thickness of the myocardial wall (75). This study, similar

to previous histological reports, showed a direct continuation of fibers between both ventricles in

the superficial layers (Figure 1.6).

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Figure 1.6: Post-mortem diffusion tensor imaging in pigs heart showing myocardial pathways throughout the left and the right ventricle intruding from the epicardium to the endocardium while seamlessly changing their orientation as they travel through the depth of the myocardium. Panel e clearly shows pathways crossing over from the left to the right ventricle (red color). Figure from Smerup et al. Anat Rec (Hoboken). 2009;292(1):1-11.

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Gross anatomy: Considerable dissimilarities exist in the shape and orientation of the right

ventricle compared to the left. These are important determinants of their varied functional

physiology and are essential to understand to be able to appreciate the challenges involved in RV

imaging, particularly with the use of two-dimensional modalities. Anatomically, each ventricle

can be described in three parts – the inlet which consists of atrioventricular valves and papillary

muscles, the apex consisting of trabeculated myocardium and the infundibular smooth outflow

region (76). The right ventricle is the most anterior chamber of the heart, with its anatomical

regions arranged such as to give it a peculiar crescent shape as it ‘wraps’ around the left ventricle

(Figure 1.7) (77).

Figure 1.7: The right ventricle has a complex three-dimensional architecture. It is the most anterior structure of the heart (A), with its inflow (black dotted line) and outflow tract (black arrow) in different two-dimensional planes with an angle of approximately 37.5o between them. The right ventricle appears triangular in the sagittal plane (B) and crescentic wrapping around the left ventricle in the coronal plane (C). This complex shape necessitates evaluation of RV function from multiple two dimensional planes. Figure adapted from Ho SY and Nihoyannopoulos P. Heart. 2006 Apr;92 Suppl 1:i2-13. RV right ventricle, LV left ventricle, RA right atrium, Ao Aorta, PT pulmonary trunk.

The tricuspid and pulmonary valves are well separated from each other by a muscular

supraventricular crest. This is in sharp contrast to the left ventricle, which has a more circular

profile and has the mitral and aortic valves side by side in fibrous continuity. The wide angle

between the RV inlet and outlet requires the inflow region to start contracting first, which creates

a peristaltic wave of contractile motion to keep the intra-cavity circulation moving in the right

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direction (towards the pulmonary valve) (78). Intra-ventricular dyssynchrony could alter this

balance, compromising the overall efficiency of contraction. In addition, the outlet of the right

ventricle is raised above the ventricular base by a freestanding muscular sleeve, forming the

subpulmonary infundibulum. While this ensures that the RV outflow region remains independent

of the left ventricle, it creates an acute angle of approximately 37.5 degrees between the plane of

the inlet and outlet of the right ventricle (79), necessitating the RV outflow tract to play an

important contributory role in the overall RV contractile function. Unlike the inlet and outlet, the

apex of both ventricles are arranged directly next to each other (80).

1.5. Response of the right ventricle to increase in afterload

Physiologically, a mature adult right ventricle is known to be more tolerant to preload but

sensitive to changes in afterload, presumably due to its thin wall and large surface area. The

slope of decline in ejection fraction in response to rising afterload has been shown to be steeper

for the right ventricle in comparison to the left in adults (Figure 1.8) (9). However, the relative

tolerance of both ventricles to afterload has not been studied in newborns.

Figure 1.8: The slope of regression is steeper for the reduction in ejection fraction determined using angiography in response to rise in afterload for the right ventricle in comparison to the left ventricle. Figure from Nagel E et al. Eur Heart J. 1996;17(6):829-36.

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Clinically, end stage RV failure is characterized by a dilated, poorly contracting right

ventricle, high RV end diastolic pressure, elevated central venous pressure and reduced cardiac

output. Although a normally functioning right ventricle may rapidly progress to RV failure when

exposed to a high afterload, more often this is preceded by homeotropic adaptation (increased

RV contractility and output without dilatation) or heterotropic adaptation (increased contractility

and output accompanied by RV dilatation) (14, 81). Homeotropic adaptation may occur without

any demonstrable change in ventricular morphology or, after a period of time, may be

accompanied by RV hypertrophy (compensatory increase in RV wall thickness maintaining

normal function and output). Heterotropic adaptation on the other hand is likely to lead to high

RV end-diastolic pressure but could be clinically mild in the early phase (RV congestion) before

progressing to frank RV dilatation (RV congestion + grossly dilated RV with preserved systolic

function and cardiac output; Figure 1.9) (82, 83).

Figure 1.9: The right ventricle undergoes progressive dilatation when exposed to increased afterload for prolong period as occurs in pulmonary hypertension. The right ventricle may rapidly become dysfunctional or may undergo adaptive hypertrophy which by compensating for the increased wall stress, allows the right ventricle to pump against high resistance without failing. With increasing duration and/or severity of pulmonary hypertension, the

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compensatory mechanisms fail, resulting in RV dysfunction before culminating in frank RV failure. RV congestion/dilatation is primarily characterized by diastolic dysfunction (i.e. heart failure with normal ejection fraction). This evolution proceeds more rapidly when PHT is diagnosed during neonatal and infancy period in comparison to adults and older children. Preterm neonates may have even lower capacity to adapt. RV right ventricle, LV left ventricle.

The clinical goal in management of PAH is to identify at risk patients by recognizing early

RV compromise before it culminates in end stage RV failure (84, 85). The ability of the right

ventricle to ‘cope’ with rising afterload varies with the severity and rate of progression of PAH,

as well as with the underlying etiology (13, 86, 87). Inter-patient variability may be considerable

(54, 55). Pulmonary stenosis and Eisenmenger’s syndrome are two examples of disorders where

in spite of exposure to a high afterload, RV function remains preserved for a long period, while

in patients with PAH, exposure to high afterload results in RV failure (86, 87).

1.6. Is the neonatal right ventricle more or less tolerant to afterload?

Consistent with developmental and structural differences, the right ventricle also has many

unique physiological properties governing its function in physiological and pathological

conditions (88). The net influence of variations under these conditions may influence a newborn

heart’s tolerance to increases in afterload (Figure 1.10).

Figure 1.10: The net effect of variations in factors that are known to influence right ventricle’s ability to withstand afterload is unknown in neonates.

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The factors which may favourably impact RV tolerance to afterload in the newborn period

include higher RV muscle mass and wall thickness, and lower pericardial constraint. Higher RV

free wall mass (relative hypertrophy) and surface area could be a major advantage during the

newborn period, as it may result in lower wall stress, increased inotropy via Anrep’s effect and

higher ability to increase cardiac output in response to a rise in preload via Frank-Starling’s

mechanism. Wall stress is a measure of effective afterload as sensed by the ventricular wall and

is inversely related to wall thickness. Anrep’s effect refers to the myocardium’s intrinsic

autoregulatory ability to increase its contractility in response to a rise in afterload without any

contribution from extrinsic factors and has been shown in vitro to be higher in the right ventricle

because of its higher surface area (89, 90). The Frank-Starling mechanism represents a

physiological limit for the ventricular wall to increase its surface area and output in response to

increased preload without undergoing dilatation and is related to the muscle mass available for

‘recruitment’ (91).

In contrast, the factors which may adversely impact the newborn right ventricle’s tolerance

to increase workload include, higher oxygen demand due to increased muscle mass, relatively

less coronary blood supply secondary to a higher RV diastolic pressure, a lower duration of

hangout period and a lower contribution from force-frequency relation. The hangout period, a

property unique to the right ventricle, refers to the time period during which blood continues to

be ejected forward into the pulmonary circulation from the right ventricle in spite of a negative

ventriculo-arterial pressure gradient (after completion of RV systole). It is thought to be a result

of the forward momentum of the blood overcoming extremely low pulmonary pressures in

mature hearts and contributes to low RV energy requirements (92). The force-frequency relation

refers to a unique property of the myocardial muscle by which its isometric contractility

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increases in response to a rise in heart rate. The force-frequency curve has been shown to be flat

in in-vitro experiments conducted using ventricular tissue obtained from neonates undergoing

surgical repair for major congenital heart defects (93). In addition, neonatal hearts are known to

be ‘stiffer’ and more dependent on loading conditions for proper functioning, likely secondary to

structural immaturity, as suggested by a higher collagen content, the presence of predominantly

uninucleated cells, a paucity of T tubules and a relative deficiency in calcium handling capacity

(94).

The ability of the neonatal right ventricle to cope with prolonged exposure to high

afterload is not known. The neonatal right ventricle, by virtue of being accustomed to working

against a relatively high resistance during fetal life, could be more tolerant to pulmonary

hypertension (PHT). However, studies have suggested that cPHT secondary to CNLD presenting

in early infancy, is particularly malignant due to an accelerated deterioration in RV function and

a shorter interval between diagnosis and death (95, 96). On the other hand, in patients with

congenital pulmonary stenosis and Eisenmenger’s syndrome, the right ventricle appears to adapt

and tolerate higher afterload for prolonged periods (as much as decades in some cases), at least

in part, by undergoing hypertrophy and re-expression of fetal genes (87). It is therefore likely,

that in line with the pulmonary hypertensive disorders of adults and older children, RV function

plays a key role in determining outcomes in acute and chronic PHT in neonates; therefore early

identification and treatment may favorably alter the clinical course for these patients. This may

be even more important for premature neonates, as their immature right ventricle may have an

even lower capacity to adapt.

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1.7. Assessment of right ventricular function in neonates

Clinical evaluation of RV performance in neonates is challenging: In older patients, RV failure

is considered as a “dyspnea fatigue syndrome” as its primary symptoms, if not in an advanced

stage, are commonly linked to respiratory difficulties and exercise intolerance. Assessment of

RV performance in neonates poses a unique challenge. A large number of at-risk neonates have

associated parenchymal lung disease, which masks the symptoms of pulmonary vascular disease

and RV dysfunction (82, 97). Exercise tolerance in neonates and infants can mainly be assessed

based on changes in feeding ability. The clinical signs (e.g. jugular venous pressure) commonly

utilized in older patients are not easy to elicit in neonates. Physiological monitoring parameters

feasible in neonates in NICUs (blood pressure, heart rate, capillary refill time) can only monitor

the adequacy of systemic circulation and left heart function and that too with limited sensitivity.

In the context of PHT/RV dysfunction, alterations in these indices usually appear late in the

disease process (37, 98, 99). In summary, there are no established clinical parameters, with a

high degree of sensitivity or specificity, which can help in the clinical appraisal of RV function

in neonates. The diagnosis of significant pulmonary vascular disease and RV dysfunction in

neonates is often dependent on clinical awareness and a high index of suspicion.

Two-dimensional (2D) echocardiography is the clinical ‘gold standard’ for neonates: In older

children and adults, right heart catheterization and cardiac magnetic resonance imaging (CMRI)

are often used to confirm the diagnosis of PAH and quantify RV size and function, while

echocardiography is commonly used for screening, frequent monitoring or diagnostic assessment

of sick unstable patients in intensive care units (1, 100-102). Cardiac catheterization and MRI are

generally not feasible in the majority of newborns with PHT for a variety of reasons that include,

small patient size, high risk of complications, clinical instability and need for out-of-facility

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transport. Rarely, when feasible, these investigations can only be performed sporadically and are

not suitable for longitudinal assessments or to monitor response to therapies. Two-dimensional

echocardiography is a safe, relatively quick and generally well-tolerated, non-invasive bedside

investigation that provides clinically relevant information in real time and is ideally suited for

sequential evaluations. It is the investigation of choice for cardiovascular assessment in this

population. However due to a paucity of systematic studies establishing normative data for

quantitative indices, lack of information on reliability testing and knowledge of specific cut-off

values to define clinically significant dysfunction, the evaluation of heart function has for the

most part remained qualitative in neonates (103, 104). With improvements in technology, studies

in this field are beginning to emerge (41, 62, 63, 105-109).

Challenges in right ventricle imaging using 2D echocardiography: The unusual anatomy of the

right ventricle makes it considerably more challenging to assess and judge the adequacy of its

functional performance (77, 110). First, the RV wall lies anteriorly, making it difficult to

visualize due to the relatively inferior near-field resolution of ultrasound. This problem becomes

accentuated in newborns owing to a thin chest wall; hence, the need for higher frequency

ultrasound transducers to ensure reliable imaging. Second, as discussed above, in comparison to

the simple ellipsoidal shape of the left ventricle, the right ventricle has a more complex

geometry. It appears crescentic, wrapping around the left ventricle in the coronal section,

triangular in the sagittal plane and, unlike the left ventricle, has its inflow and outflow in

different planes, making it necessary to employ multiple 2D frames to visualize the entire

chamber (77). Many of these problems can now be overcome with the advent of high frequency

ultrasound transducers (10-12 MHz), high quality image optimization techniques and newer

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imaging modalities such as tissue Doppler imaging (TDI) and deformation imaging, as described

below.

1.8. Ventriculo-ventricular interactions - need for biventricular function assessment:

Various experiments using mature heart models have confirmed the strong and direct

relationship between function and geometry of both ventricles. An artificial non-contractile right

ventricle (created by replacing its free wall with a patch or by electrical isolation), demonstrates

a significant systolic pressure generation secondary to left ventricular (LV) contraction, but the

reverse was not found to be true (111, 112). Preservation of normal RV dimension appears to be

of critical importance. Acute dilatation of the RV not only dampens the ‘right sided’ contribution

of LV contraction but, if severe, can adversely impact LV systolic performance. The individual

contribution of factors which are responsible for these interactions have been poorly isolated but

include: 1) dysfunction of the shared myocardial fibres between both ventricles, 2)

intraventricular septal dysfunction, 3) trans-septal transmission of intra-cavity pressure from one

ventricle to the other, 4) the hemodynamic effects of low RV output resulting in low LV preload

or high LV diastolic pressure causing secondary post-capillary PHT and 5) the mechanical effect

of ventricular dilatation potentiated by pericardial constraint which, by limiting the volume of

heart chambers, results in diastolic dysfunction in the setting of ventricular dilatation. The

adverse effect of pericardial constraint is inversely related to the ventricular wall thickness. In

addition, RV dilatation will cause leftward shift of the interventricular septum, reducing LV

filling capacity (preload), which adversely impacts its contractility and output.

1.9. Echocardiography methods for heart function assessment in neonates

A number of 2D echocardiography methods have been developed for assessment of

ventricular size and function. Detailed guidelines have been published describing standard

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methodology, normal values and clinically relevant interpretations for these variables in adults

and older children (113-115). No such guidelines exist for neonates or infants, presumably due to

lack of information regarding reliability testing, normative data and clinical utilization. With

technological advancements in echocardiography equipment and increased availability of newer

imaging modalities, many of the established techniques are now being employed in neonates and

preliminary reports describing the feasibility and normal values for related indices are beginning

to emerge. However, high quality normative datasets are still missing for the majority of indices

(Table 1.3) (104, 116). In the studies described in subsequent chapters, I utilized a wide array of

functional indices derived using a comprehensive imaging protocol developed based upon, but

not restricted to, the guidelines published by American Society of Echocardiography (ASE).

While the standard commonly used techniques are defined in the methods chapter, some specific

newer methods are described below.

Table 1.3: Echocardiographic indices of ventricular dimensions and function and availability of normal data for neonates Echo window Adult data Neonatal data Description RV Dimensions Basal diameter Apical 4C Available NA Anterior cavity Mid-cavity diameter Apical 4C Available NA Anterior cavity Base-apex length Apical 4C Available NA Anterior cavity RV areas Apical 4C Available NA Anterior cavity RV Functional indices FAC Apical 4C Available NA Systolic function (anterior cavity) TAPSE Apical 4C Available Available Longitudinal systolic function

(base-apex) TDI (peak systolic) Apical 4C Available Available Longitudinal systolic/diastolic

function 2D Strain (lateral wall) Apical 4C Available Limited Systolic function (base-apex) MPI Apical 4C Available Available Global function LV Dimensions Basal diameter Apical 4C Available NA Anterior cavity Base-apex length Apical 4C Available Available Anterior cavity LV volume Apical 4C Available Limited Anterior cavity LV Functional indices Fractional shortening Parasternal

short axis Available Available Systolic function (dependent on

septal motion) Ejection fraction (Simpson’s)

Apical 4C Available Limited Systolic function

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TDI (peak systolic) Apical 4C Available Available Longitudinal systolic/diastolic function

2D Strain Apical 4, 3 and 2 chamber views

Available NA (only apical 4C described in few neonates)

Systolic function (base-apex)

MPI Apical 4C Available Available Global function FAC fractional area change, TAPSE tricuspid annular plane systolic excursion, TDI tissue Doppler imaging, 2D two dimensional, MPI myocardial performance index, 4C 4 chamber view, NA: not available

Myocardial Tissue Doppler Imaging (TDI):

Traditionally, spectral pulsed Doppler echocardiography, due to its property of detecting

high velocity low amplitude motion, has been used in routine clinical practice to evaluate the

velocity of blood flow. Tissue Doppler imaging (TDI), is a robust technique, relatively recently

introduced to the field of neonatology, which enables detection of velocities directly from the

tissue (117-121). Contrary to spectral Doppler echocardiography, TDI detects low velocity, high

amplitude motion, ideally suited for studying the movement of the myocardial wall, while

avoiding artefacts from movement of blood (122, 123). Unlike conventional methods of

functional assessment, which mainly assess changes in cavity dimension and blood flow

velocities, this modality directly assesses muscle wall characteristics. TDI captures information

using high frame rates; the resulting high temporal resolution facilitates reliable and reproducible

measurements of the velocities of the myocardial wall during various phases of cardiac cycle, as

well as the measurements of the duration of these phases (systolic and diastolic times /

isovolumic contraction and relaxation times). Currently, there are two methods available to

derive these measurements - pulsed TDI and colour TDI (124, 125). Of these, pulsed TDI is the

preferred approach due to its advantages of high temporal resolution, ease and speed of

measurement and high inter-rater reliability. I used pulsed TDI method in studies described in

chapters 4 to 7. As the name suggests, it involves measuring TDI velocities by spectral analysis

using pulse wave Doppler technique. By convention, use of this method is restricted for

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assessment of RV and LV free wall and septal motion at the base, which is then considered as a

marker of global longitudinal function of the respective myocardial wall.

Images are usually obtained from an apical four-chamber view. The sector width of the

field of view is usually narrowed to only include the wall of interest. This ensures that the

temporal resolution is enhanced and a frame rate of over 200 frames per second is obtained. A

pulsed wave Doppler sample is then placed at the base of the respective wall while minimizing

the angle of insonation, less than 20 degrees in all cases to avoid underestimation of velocities

(Figure 1.11A). The sample gate is narrowed to only capture the velocity of the area of interest

(usually 1 – 2 mm) and peak velocities are recorded in systole (s’), early (e’) and late (a’)

diastole, as well as during isovolumic contraction and relaxation (126). The duration of these

phases can also be easily measured (Figure 1.11B). Using the values of event timings,

myocardial performance index (MPI’), a simple hemodynamic variable which represents global

ventricular function, can be calculated using a simple formula; MPI’ = [isovolumic contraction

time (IVCT) + isovolumic relaxation time (IVRT)]/ejection time (107, 127-129).

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Figure 1.11: Measurement of myocardial velocities using pulsed tissue Doppler imaging. By convention measurements are performed at the basal segment of the RV free wall, inter-ventricular septum and LV free wall (A). Peak velocities in each phase of cardiac cycle as well as event timings can then be easily measured (B). For the right ventricular, peak systolic myocardial velocity (s’) has been shown to increase with gestational age; Koestenberger et al. Neonatology. 2013;103(4):281-6. Panel A courtesy of Dr. Afif El-Khuffash.

The main disadvantages of this technique are low spatial resolution, only registering

motion parallel to the line of interrogation at the base, and inability to differentiate a tethering

effect i.e., movement of a non-contractile wall segment due to contraction of an adjacent

segment. While pulsed TDI measures peak myocardial velocities, color TDI is an angle

independent offline method, which allows measurements of mean myocardial velocities at

various segments. However, it has poor inter-rater reliability and relatively lower temporal

resolution. Normative data for pulsed TDI velocities for preterm and term neonates is now well

established (63, 106, 121, 125, 130). Previous studies have demonstrated a positive linear

relationship between gestational age and RV s’ (Figure 1.11 C) (63). In older patients, RV s’

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correlates well with MRI obtained RV ejection fraction, in the absence of regional dysfunction

(131).

Deformational imaging – 2D speckle tracking echocardiography (STE):

Deformation refers to the change in shape of the myocardium from its baseline state at end-

diastole to its deformed state at end-systole (Figure 1.12A). This occurs as a result of sarcomere

shortening during contraction and leads to a reduction in cavity size and ejection of blood from

the ventricle (132). The direction and type of deformation is dependent on the orientation of the

myocytes and as described above, is thought to be predominantly longitudinal for the right

ventricle (and circumferential to a lesser extent), while the left ventricle undergoes longitudinal,

circumferential as well as radial (thickening) deformation (74). Myocardial strain is the term

used to define this deformation occurring in systole relative to the original length at end diastole

and is expressed in a percentage (%) (133). Longitudinal and circumferential deformations are

assigned a negative strain to indicate shortening, and radial deformation a positive strain to

indicate thickening. Strain rate refers to the speed at which myocardial deformation occurs and is

expressed in 1/s. One major advantage of strain and strain rate imaging is the ability to quantify

both global as well as regional wall function (134-136).

Two-dimensional speckle tracking echocardiography is an imaging technique that

measures deformation by tracking the movement of speckles within the myocardial wall.

Speckles are acoustic backscatter generated by the ultrasound beam and form a unique pattern in

each wall segment (137). Those speckles can be tracked frame-by-frame using offline software,

which then directly measures strain and calculates the strain rate for various segments of the

ventricular wall to generate a strain curve (Figure 1.12B). The values obtained from various

segments can then be averaged to obtain an overall global value for the respective wall. This

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technique has been validated by ultrasonomicrometry in the longitudinal direction and it has

been widely applied to the left ventricle and most recently to the right ventricle in older patients

(74, 132, 138-145) and children (138, 142, 146-148). A major advantage of STE over color TDI,

the aforementioned method of measuring myocardial deformation, is that it is angle independent

i.e. not dependent on the alignment of the wall relative to the ultrasound beam (141).

Furthermore, it possesses a relatively improved signal to noise ratio. The main disadvantage of

STE is a limitation of the software to track speckles when image quality is suboptimal; as a

result, dropouts and reverberations can negatively affect tracking. In neonates, a frame rate to

heart rate ratio of 0.7 to 0.9 has been suggested to minimize inter-rater measurement variability

(105). This is important due to the higher heart rates in neonates. In term neonates, the normal

data for STE derived deformation parameters has not been fully established. Few small studies

have suggested its feasibility, described normal LV values derived exclusively from the apical 4-

chamber (apical-4C) view (instead of the standard 4, 3 and 2 chamber views) and have

highlighted the potential utility of strain and strain rate for evaluation of pathological states in

preterm and term neonates (109, 127, 149-157).

Figure 1.12: A. Longitudinal deformation refers to the change in length of a segment from its baseline shape in diastole to its deformed shape in systole. Strain refers to the degree of change in shape relative to the baseline and is

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expressed in (%). B. Strain peaks during end systole at aortic valve closure (AVC) and returns to baseline during diastole at mitral valve closure (MVC). On the other hand strain rate peaks in mid systole and returns to baseline at AVC when no deformation occurs. SRe: early diastolic strain rate; SRa: late diastolic strain rate (during atrial contraction). Figure courtesy of Dr. Afif El-Khuffash.

Additional 2D echocardiography derived RV specific functional measures:

Fractional area change (FAC): Fractional area change is the relative change in the area of the

RV cavity in end-systole compared to end-diastole expressed as a percentage and by convention

is calculated from measurements of the RV cavity areas as visualized in the RV-focused apical-

4C view (Figure 1.13). FAC is the RV equivalent of ejection fraction and represents global

pump function. In older patients, FAC has been shown to have a good correlation with MRI

measured RV ejection fraction (158). A major disadvantage of this measure is the dependence on

a high quality 2D image, as manual tracing of RV endocardial borders is required. This may be

more challenging in neonates given the increased trabeculations in the right ventricle. A recent

study has demonstrated the feasibility of using this index in preterm neonates and reported higher

RV areas and lower FAC in patients with CNLD compared to controls (61).

Figure 1.13: Fractional area change is calculated for the right ventricle from apical-4C view by measuring RV areas in end-diastole (EDA) and end-systole (ESA) and then applying the simple formula of FAC (%)= [(EDA-

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ESA)EDA] x100. FAC shows good correlation with MRI derived ejection fraction in adults. Anavekar et al. Echocardiography. 2007;24(5):452-6. Figure courtesy of Dr. Luc Mertens.

Tricuspid annular plane systolic excursion (TAPSE): TAPSE is a linear measurement of the

maximum displacement of the tricuspid annulus during each contraction, as obtained from the

RV-focused apical-4C view using M mode echocardiography by placing the cursor through the

tricuspid valve annulus (159) (Figure 1.14). In adults with PAH, TAPSE has been shown to be a

sensitive marker of clinical prognosis and global RV longitudinal function (base to apex). In

addition it has been shown to be reflective of the extent of RV remodelling and disproportion in

the sizes of the right and left heart and severity of PVR (159-162). Preliminary reports in term

neonates with PPHN have suggested TAPSE to be a potentially useful index for measurement of

RV function and prognosis, however its relevance in comparison to other markers has not been

tested (43, 44). Similar to RV s’, TAPSE in preterm neonates has been shown to increase with

gestational age (62). TAPSE has several advantages such as simplicity of measurement,

immediate bedside availability of results and high inter-rater reliability. Its disadvantage is that it

only indicates longitudinal function of the RV free wall, does not account for the function of the

outflow tract and may be falsely elevated by LV motion (163).

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Figure 1.14: Tricuspid annular plane systolic excursion (TAPSE) is a simple measure of global longitudinal function of the right ventricle and is shown in preterm and term neonates to have a linear positive correlation with birth weight and gestational age. Koestenberger et al. Neonatology. 2011;100(1):85-92.

1.10. Application of echocardiographic indices during the newborn period

A number of factors need to be considered for application of published imaging guidelines

in the newborn population, particularly during the first few days of life, normally characterized

by significant circulatory changes - a fall in PVR, an increase in pulmonary blood flow and

closure of fetal shunts. First, given that the majority of neonates with acute PHT present during

the early postnatal period, it is crucial to know the influence of normal physiological transition, if

any, on the normal values of echocardiography-derived parameters. Second, by convention, most

of the established RV functional parameters are derived exclusively from a single 2D view (RV-

focused apical-4C view), which only visualizes the anterior RV cavity and thus may not account

for the significant contribution of the RV infundibulum (Figure 1.15) (8). Third, the RV cavity

dimensions as measured in the apical-4C view are influenced by interventricular septal motion,

which is physiologically ‘abnormal’ during the first few days of life in neonates (164). There is a

clinical need to develop and test comprehensive imaging protocols for quantifying cardiac

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function in neonates. Such protocols should ideally make use of multiple modalities as available

and include parameters derived from 2D views other than the apical-4C view. To prevent

misinterpretation of the results obtained in disease states, information is required regarding the

effect of physiological changes occurring during normal newborn transition on various

functional parameters.

Figure 1.15: The majority of established echocardiographic indices of RV function are derived from one 2D plane (apical 4C view), which only visualizes the anterior RV cavity (yellow shaded area) and may not account for the contractile contribution of a significant portion (infundibulum; green shaded area). Schematic adapted from Sheehan F and Redington A. Heart. 2008;94(11):1510-5.

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Chapter 2: Development of an imaging protocol, rationale and

specific aims

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2.1 Development of an imaging protocol and novel indices for quantifying RV function

The development of the comprehensive functional imaging protocol used in subsequent

studies, as summarized below, was undertaken during my clinical fellowship prior to

commencement of my graduate studies in the Department of Physiology. As summarized in

chapter one, a number of 2D echocardiography techniques suitable for studying ventricular

function in neonates are now available. These methods have been extensively used in older

patients to study the left ventricle, and more recently the right ventricle in PAH (165). While

sporadic use of some of these indices in neonates have emerged, clinical assessment of heart

function has remained qualitative for the most part, presumably due to lack of quality normative

datasets for the majority of available indices, as well as their relative reliability testing. This is

particularly true for the immediate postnatal period, which is marked by important changes in

biventricular loading conditions and is also the period during which the majority of cases of

neonatal acute PHT are encountered. These considerations coupled with my own experience in

neonatal echocardiography and the American Society of Echocardiography’s latest guidelines led

us to define a comprehensive functional two-dimensional echocardiography imaging protocol

suitable for neonates (Figure 2.1) (113-115). This protocol integrated various available

modalities and enabled quantitative estimation of right and left ventricular size, systolic and

diastolic function using multiple 2D planes, as well as quantified pulmonary hemodynamics

using standard measures previously employed in neonates.

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Figure 3.1: A comprehensive functional two dimensional echocardiography imaging protocol designed specifically for use in neonates for the studies involved in this thesis.

In addition, as also highlighted in the introductory chapter, currently established indices of

RV function have the limitation of being exclusively derived from a single 2D view (the apical-

4C view), which only allows visualization of the RV lateral wall and a part of its crescentic

cavity (base to apex). Hence, these measures may not account for the contribution of a

significant part of the right ventricle (apex to outflow) and may be influenced by the shape and

movement of the interventricular septum, which is particularly important in the immediate

postnatal period. Under these considerations, I incorporated the following additional imaging

sequences and developed related functional indices to allow a more comprehensive RV

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functional assessment in neonates, independent of the effect of the interventricular septum

(technical details provided with methods in chapter 3).

Basal diameter -PLAX: This linear dimension was developed to allow measurement of the

previously unmonitored ‘posterior depth’ of the RV cavity. From the parasternal long axis view

(PLAX) of the RV inflow, the antero-inferior linear dimension of the RV cavity, the RV basal

diameter (B-PLAX), can be measured (Figure 2.2).

Figure 2.2: In addition to the four standard linear dimensions measured from the apical 4C view, RV basal diameter was also measured from the parasternal long axis view of the RV inflow. Anatomically this corresponded to the posterior depth of the RV cavity. Schematic adapted from Sheehan F and Redington A. Heart. 2008;94(11):1510-5.

RV apical 3 chamber view (3C): The rationale of developing indices using this view, was to

allow quantitative estimation of the functional contribution of the ‘wrap around’ infundibular

portion of the RV cavity (Figure 2.3A). From the standard apical imaging window, a short axis

view of the right ventricle can be obtained, visualizing both its inflow and outflow portions

within the same 2D view along with the RV inferior wall (Figure 2.3B). This view was then

used to measure the end-systolic (ESA-3C) and end-diastolic (EDA-3C) areas of the visualized

RV cavity and calculate the related fractional area change (FAC-3C). Further, using 2D STE, the

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inferior wall of the right ventricle could be tracked and the peak global longitudinal strain of the

inferior wall (GLS-3C) obtained (Figure 2.3C).

Figure 2.3: A. The green shaded area highlights the infundibular ‘wrap around’ portion of the right ventricle which is usually not included in RV functional assessment. B. A short axis view of the right ventricle obtained from the standard apical acoustic window. This view allow simultaneous visualization of the inflow and outflow portion of the right ventricle, its posterior cavity and inferior wall. C. The RV inferior wall seen in this view can be tracked using speckle tracking echocardiography and its longitudinal strain can then be measured. Schematic in panel A adapted from Sheehan F and Redington A. Heart. 2008;94(11):1510-5. Panel B reproduced from Jain et al. J Am Soc Echocardiogr. 2014;27(12):1293-304.

2.2 Rationale and aims

Cardiovascular decision-making in critically ill neonates is complicated by a lack of

accurate and reliable measures of the adequacy of ventricular performance, and limited

understanding of the interplay between cardiovascular physiology in neonatal disease and

outcomes. Assessment of cardiovascular well-being in the immediate period after birth is even

more challenging due to physiological changes in fetal shunts and vascular resistance. Unlike

older patients, the clinical symptoms and signs of cardiovascular compromise in neonates, in

particular RV dysfunction, are non-specific and more definitive investigations, such as cardiac

catheterization and CMRI, are generally not feasible. With relatively recent technical

advancements, 2D echocardiography has become more suitable for cardiac functional imaging in

neonates. It offers the potential of a timely and longitudinal evaluation of myocardial

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performance and/or pulmonary hemodynamics in sick neonates, however a robust

comprehensive normative dataset and information on relative measurement reliability is lacking.

Further, availability of these methods, if reliable, also offer an opportunity to study and

understand the cardio-pulmonary adaptive changes and their specific timing in relation to normal

physiological transition from an intra-uterine to an extra-uterine environment in humans. To

date, our knowledge of these events is mostly based on animal models and limited to circulatory

changes. It is important to understand the effect of postnatal circulatory changes on

echocardiography-derived indices of cardiac function, as the majority of sick newborns present

clinically within the first 48 hours of life. Defining normative data and normal physiologic

adaptation is an essential pre-requisite of determining whether deviations seen in any

echocardiography measurement is reflective of a disease process. The overall long term objective

of this work is to understand the incidence, progression and short and long term clinical

significance of cardiac dysfunction in neonatal pulmonary hypertensive disorders and to

facilitate early identification of at-risk neonates. Currently, there are major gaps in our

understanding of the factors governing the high rates of adverse outcomes observed in neonatal

PHT. Right heart function, a likely key prognostic factor, has been grossly understudied in

neonates because of technological lag and a lack of appreciation of its probable critical role in

clinical outcomes. High quality, comprehensive and systematic clinical studies are critical to

understand these factors and to establish methods for early identification of infants at highest

risk. The specific aims for the studies conducted for this thesis are as follows:

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Chapter 4 (RV function) and Chapter 5 (LV function):

• To test the feasibility and reliability of a comprehensive echocardiographic protocol

including novel indices specifically developed for use in neonates, for quantifying RV

and LV dimensions and function in healthy term human neonates.

• To establish normative data for indices derived using 2D echocardiography, TDI and 2D

STE.

• To investigate the effect of early transitional changes on these measurements by

comparing results obtained on days 1 and 2 of life.

Chapter 6:

• To delineate the natural history of pulmonary vascular resistance, right and left

ventricular outputs and transitional shunts during the first 24 hours of life in humans.

• To study the cardiac functional adaptation associated with the immediate postnatal

period.

• To investigate the reproducibility of echocardiographic measures of pulmonary vascular

resistance and cardiac outputs in another larger cohort of healthy neonates.

Chapter 7:

• To identify echocardiographic correlates of biventricular size, function, and

hemodynamics, including shunt patterns, in neonates presenting with persistent

pulmonary hypertension of the newborn (PPHN) during the first 3 days of life by

employing a comprehensive imaging protocol as described in Chapters 4 and 5.

• To investigate associations between key echocardiographic findings and subsequent

adverse clinical outcomes in neonates with PPHN.

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• To identify risk factors associated with cardiac dysfunction in neonates with PPHN.

• To examine inter-ventricular functional correlations in PPHN during the immediate

postnatal period.

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Chapter 3: Methodology

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3.1 Study design and sites

The work encompassed in this thesis included performing and analyzing echocardiograms

for two prospective observational studies conducted in healthy term human newborns (Chapter

4-6) and one retrospective cohort study including analysis of echocardiograms for newborns

admitted to the NICU with a diagnosis of PPHN (Chapter 7). The studies in Chapters 4 to 6 were

carried out in the well Mother and Baby Unit and Labour ward of Mount Sinai Hospital, and the

study described in chapter 7 was performed in the NICU of the Hospital for Sick Children; both

sites are teaching hospitals affiliated with the University of Toronto, Ontario, Canada.

3.2 Ethics

All studies received approval from the institutional Research Ethics Board. For prospective

studies, informed consent was obtained from the parents of recruited infants. A consent waiver

for the retrospective study was obtained from the Research Ethics Board.

3.3 Echocardiography material and personnel

I performed all scans using a Vivid 7 ultrasound scanner with a 10 MHz transducer or a

Vivid E9 ultrasound scanner with a 12 MHz transducer (GE Medical Systems, Milwaukee, WI).

Studies were digitally stored as DICOM raw data and analyzed off-line, in a random order, at

completion of recruitment for each study using a dedicated workstation (EchoPAC, version

BT10, GE Medical Systems). Wherever applicable, only the raw measurements were recorded

initially, while the calculations to generate the final results were carried out after completion of

the analysis of echocardiograms. For work described in chapter 7, to ensure blinding, clinical

data was collected separately and was merged with echocardiography data prior to statistical

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analysis. All scans were analyzed as per published guidelines and the indices described below

were collected (113, 114).

3.4 Right ventricular parameters

RV linear dimensions:

The majority of linear dimensions were measured from the RV-focused apical-4C view

(Figure 3.1A). Tricuspid valve annular diameter (TVA) was defined as a straight line joining the

hinge points of the anterior and septal valve leaflets. The basal diameter (RV B-4C) was

measured at the basal third of the RV cavity as the maximal distance from the RV lateral wall to

the septum while maintaining a parallel orientation to the TVA. A straight line joining the mid-

point of the TVA to the RV apex constituted the RV length (RVL). The mid-cavity diameter (MC-

4C) was defined by a straight distance between the RV lateral wall and the septum running

parallel to the TVA but passing through the mid-point of RVL. In addition, as previously

described, with the aim of quantifying the antero-inferior dimension of the RV cavity, I

measured the B-PLAX using the PLAX RV-inflow view (Figure 3.1B). This view was obtained

by anteriorly tilting the ultrasound probe from the standard PLAX view until the interventricular

septum was no longer in view and was replaced on screen by the RV anterior wall and attached

tricuspid valve leaflet. The measurement was then performed as the maximal diameter at the

basal one-third of the RV cavity from its anterior to inferior walls, while maintaining a parallel

orientation with the TVA. All dimensions were measured in end-diastole.

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Figure 4.1: A. Anterior RV cavity dimensions measured in apical 4C view: 1. Tricuspid valve annular diameter (TVA), 2. Basal diameter (B-4C), 3. RV length (RVL), 4. Mid cavity diameter (MC-4C), B. RV basal diameter measured from parasternal long axis view (B-PLAX), corresponding to the posterior depth of the RV cavity. Figure from Jain et al. J Am Soc Echocardiogr. 2014;27(12):1293-304.

Functional measurements obtained from the apical-4C view:

Fractional area change (FAC-4C): The RV areas at end-diastole (EDA4C) and end-systole

(ESA4C) were calculated by manually tracing the RV endocardial borders including the RV

trabeculations within the area (Figure 3.2A). FAC-4C (expressed as a %) was calculated using

the formula [(EDA4C – ESA4C)/EDA4C] x 100.

Tricuspid annular plane systolic excursion (TAPSE) was measured using M-mode

echocardiography with the line of interrogation passing through the lateral aspect of tricuspid

annulus while maintaining vertical alignment with the apex (Figure 3.2B).

Myocardial velocities: Pulsed TDI of the tricuspid annulus was obtained by placing a pulse

wave sample (gate 2 mm) on the basal segment of the RV free wall just below the lateral

tricuspid annulus. Peak systolic (s’), early diastolic (e’), late diastolic (a’), as well as peak

isovolumetric contraction (IVCV’) velocities were measured. From the Doppler tracings, the

time intervals including the closing to opening of the tricuspid valve (TcOt’), isovolumic

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relaxation time (IVRT’), duration of systole (systole’ = beginning to the end of s’= RVET’) and

diastole (diastole’= beginning of e’ to end of a’) were obtained (Figures 3.2C & 3.2D). e’:a’,

TvE:e’, systole’:diastole’ and the TDI-based myocardial performance index [MPI’= (TcOt’-

RVET’)/ RVET’] were then calculated.

Figure 3.2: A. Fractional area change measured from the apical-4C view (FAC-4C), B. Tricuspid annular plane systolic excursion (TAPSE) is the downward vertical distance the tricuspid annulus moves (yellow arrow) during systole and is measured using m-mode echocardiography C. and D. Using pulsed tissue Doppler imaging, various RV myocardial velocities (cm/s) and time periods (msec) are measured at the basal segment of the RV free wall, just below the lateral tricuspid annulus. Time from closing to opening of tricuspid valve (TcOt’)= from the end of a’ to beginning of next e’; right ventricle ejection time (RVET’)= start to end of s’; isovolumetric relaxation time (IVRT’)= end of s’ to beginning of e’. s’/e’/a’/IVCV’ systolic/early diastolic/late diastolic/isovolumetric contraction velocities. Figure from Jain et al. J Am Soc Echocardiogr. 2014;27(12):1293-304.

Tricuspid valve inflow was assessed by placing a pulsed wave sample gate of 2 mm at the tip of

the tricuspid valve leaflets during diastole with the Doppler beam parallel to the inflow as

visualized using color Doppler echocardiography. Early (TvE) and late (TvA) inflow velocities

and their ratio (TvE:TvA) were measured. The area under the curve for the tricuspid inflow was

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estimated as velocity time integral (VTI) measured by tracing the borders of the inflow Doppler

(Figure 3.3A). Conventional myocardial performance index (MPI) was also calculated by

measuring the time between end of TvA and beginning of next TvE wave (TcOt), along with the

right ventricular ejection time measured from pulse wave Doppler of right ventricular outflow

tract (RVET) (Figure 3.3B).

Figure 3.3: Measurements of tricuspid inflow early (TvE) and late (TvA) velocity as well as velocity time integral (VTI). Myocardial performance index (MPI) can also be calculated from measurement of duration between closing and opening of tricuspid valve (TcOt) from tricuspid inflow Doppler and right ventricular ejection time (RVET) from pulmonary artery Doppler.

Peak longitudinal strain of the RV lateral wall: The grey scale images recorded at a frame rate of

80-100 frames/sec were analyzed off-line using 2D speckle-tracking analysis software

(EchoPAC, version 10, GE Medical Systems). The RV lateral wall endocardial border was

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manually traced at end-systole from the lateral basal attachment of the tricuspid valve to the apex

(133). The width of the region of interest was reduced to the smallest allowed by the software.

The software’s automated tracking was visually inspected before accepting the results. The

tracing points were manually re-positioned in cases of inadequate tracking. A maximum of 10

minutes was allowed to obtain adequate tracking before the image was deemed ‘non-analyzable’.

Overall peak GLS of the RV lateral wall (GLS-4C) was calculated by averaging peak GLS of the

basal, mid and apical segments (Figure 3.4A).

The RV-3 chamber view:

From the apical window, a ‘RV-focused apical-3-chamber view’ (RV-3C) was acquired by

rotating the transducer counter clockwise from the standard RV-4C view while maintaining a

slight rightward tilt to keep the RV in view. The probe was rotated until the left heart was

completely out of the view, the ascending aorta was in the center of the image and simultaneous

visualization of RV inflow, outflow and the inferior wall was achieved. Precaution was taken to

avoid visualizing the anterior wall of the right ventricle. Our aim was to ‘capture’ the maximum

RV cavity while keeping these anatomical landmarks in view (Figure 2.3B). Using this view, we

measured FAC (FAC-3C) and peak GLS for the inferior wall (GLS-3C) using similar methods as

described above. Briefly, FAC-3C (%) = [(EDA3C – ESA3C)/EDA3C] x 100, where EDA3C

and ESA3C are RV areas at end diastole and systole obtained by manually tracing the

endocardial borders (Figure 3.4B). For GLS-3C, the inferior wall’s endocardial border was

manually traced in end-systole from the lateral basal attachment of the tricuspid valve to just

before the pulmonary valve attachment at the infundibulum. The values for all segments were

averaged to obtain an overall GLS-3C for the inferior wall (Figure 3.4C). Similar to GLS-4C,

the image was deemed ‘non-analyzable’ if adequate tracking was not achieved in 10 minutes.

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The results of RV-4C and RV-3C views were then averaged to calculate global FAC and global

GLS.

Figure 3.4: A. Calculation of longitudinal strain of RV lateral wall (pLS-4C) using speckle tracking echocardiography. The pLS value of each segment (represented graphically by color-coded lines) is averaged to generate an overall pLS-4C. B. Measurement of fractional area change from RV-3C view (FAC-3C). Manual tracing of the endocardial borders was performed to generate respective RV areas at end-diastole (EDA-3C) and end-systole (ESA-3C). FAC-3C was then calculated by formula (EDA-3C – ESA-3C)/EDA-3C). C. Calculation of longitudinal strain of RV inferior wall (pLS-3C) using speckle tracking echocardiography. Figures from Jain et al. J Am Soc Echocardiogr. 2014;27(12):1293-304.

3.5 Left ventricular parameters

LV Dimensions:

LV dimensions were obtained at end-diastole from the apical 4-C view, the LV PLAX view and

the parasternal short axis view taken at the level of papillary muscle. From the apical 4-C view,

mitral valve annulus diameter (MVA) was measured by joining a straight line between the two

leaflet hinge points; the basal diameter (LV B-4C) was measured by drawing a straight line at the

basal segment of the LV cavity, parallel to the MVA to get the maximal distance between LV

free wall and septum; LV length was determined by a straight line joining the midpoint of the

MV annulus and LV apex (Figure 3.5A). Left ventricular end diastolic diameter (LVEDD) was

determined using M-mode of the long and short axis parasternal views. Care was taken to ensure

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that the line of interrogation was perpendicular to the interventricular septum and ‘cuts’ the LV

cavity at the level of the mitral valve leaflet tips (Figure 3.5B). Finally, left ventricular end

diastolic circumference (LVEDC) was determined in 2D by manually tracing the endocardial

borders of the LV cavity in the short axis parasternal views at the level of the papillary muscles

just below the mitral valve chordae (Figure 3.5C).

Figure 3.5: Measurement of Left Ventricular dimensions using the apical 4C view (A), M-mode of the long axis parasternal view (B), and the short axis parasternal view at the level of the papillary muscles (C). MV: mitral valve; LV: left ventricle; LVEDD: left ventricle end diastolic diameter; LVEDC: left ventricle end diastolic circumference.

LV functional indices:

Shortening fraction (SF): Using the same M mode echocardiography image of the left ventricle

used to measure LVEDD from the LV parasternal long axis view, left ventricular end systolic

diameter (LVESD) was also measured. Shortening fraction was then calculated using the

formula SF(%) = [(LVEDD – LVESD)/LVEDD] x 100 (166).

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Ejection fraction (EF) using Simpson’s biplane method: From the standard apical 4- and 2-

chamber views of the left ventricle, and using the analysis software’s built-in calculation

package, the endocardial borders of the LV cavity were manually traced at end-diastole and end-

systole to generate respective volumes in millilitres for each view. Biplane EF is then calculated

and provided as a percentage by the software (167).

Mean velocity of circumferential fibre shortening (mVCFc): Mean velocity of circumferential

fibre shortening is considered as a preload independent marker of LV systolic function which is

also adjusted for a given afterload. It is calculated using LV end-diastolic and end-systolic

circumference measured by manually tracing the endocardial borders of the LV cavity at

respective phase in the cardiac cycle in the parasternal short axis view at the level of papillary

muscle. It is then calculated using the formula mVCFc (circumference/sec) = (LVEDC –

LVESC)/LVEDC x ETc); where LVEDC and LVESC are left ventricular circumference in end-

diastole and end-systole respectively and ETc is the LV ejection time corrected for heart rate to

60 beats/seconds by dividing the measured ejection time from the pulse wave Doppler of the LV

outflow by square root of R-R interval measured from the simultaneously recorded

electrocardiographic tracings (168).

Mitral valve inflow was assessed by placing a pulse wave sample gate of 2 mm at the tip of the

mitral valve leaflets during diastole with the Doppler beam parallel to the inflow as visualized

using color Doppler echocardiography. Early (MvE), late (MvA) inflow velocities, their ratio

(MvE:MvA) and VTI were measured as for tricuspid valve inflow. In addition IVRT was

measured as the time lag between cessation of mitral valve inflow and start of flow out of the LV

outflow tract.

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Myocardial velocities: Similar to the right ventricle, pulsed TDI was obtained by placing a pulse

wave sample gate of 2 mm on the basal segment of the LV free wall just below the lateral mitral

annulus. Peak systolic (s’), early diastolic (e’) and late diastolic (a’) velocities were then

measured. From the Doppler tracings, the time intervals including closing to opening of the

mitral valve (McOt’), IVRT’, duration of systole (systole’= beginning to the end of s’= LVET’)

and diastole (diastole’= beginning of e’ to end of a’) were obtained. As for the right ventricle, e’:

a’, MvE: e’, systole’: diastole’ and MPI’ [= (McOt’-LVET’): LVET’] were calculated. In

addition, s’, a’ and e’ were also measured using pulsed TDI for the interventricular septum by

placing the sample gate at its basal segment.

Speckle Tracking Echocardiography: Longitudinal strain analysis was carried out by obtaining

grey scale images of the left ventricle in three views (the LV apical four- two- and three-chamber

views) at a frame rate of 80 - 100 frames/second. As for the right ventricle, images were

optimized to visualize the myocardial walls and analysis was performed using the 2D STE

software (EchoPAC, version 11, GE Medical Systems). Briefly, the endocardial border was

manually traced at end-systole and the region of interest was narrowed to only include the LV

myocardium. Tracking was automatically performed, and the analysis was accepted after visual

inspection of all the segments and software confirmation of adequate tracking. If tracking was

suboptimal, the endocardial border was retraced. If satisfactory tracking was not accomplished

within 10 min, the non-tracking segments were excluded from analysis. Global peak longitudinal

strain (GLS) was then calculated by averaging all values of the segmental peak longitudinal

strain obtained from the 17 segments in three views (Figure 3.6) (133). Peak strain was defined

as peak value during the entire cardiac cycle as identified by the software (peak G values) (137).

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For both the left and right ventricle, we chose to measure peak strain instead of end systolic

strain (obtained by manual tracing of each strain curve to the point of aortic valve closure) with

the premise that it would make measurements more user friendly by significantly reducing post

processing time and may minimize inter-rater variability by removing the need for manual

tracing of each strain curve. Further, for a random subset of 100 segments, we recorded and

compared end systolic strain values to the automated peak G values provided by the software and

found them to be not significantly different. The frame rate: heart rate ratio for all STE images

ranged from 0.65 to 0.90.

Figure 3.6: Speckle tracking echocardiography. Strain is measured in the three planes: four chamber, two chamber, and three chamber, and displayed in a bulls eye view with the three individual values and the global average. Figure adapted from Jain et al. J Pediatr. 2016 Nov 28. pii: S0022-3476(16)31231-8. doi: 10.1016/j.jpeds.2016.11.003.

3.6 Hemodynamic measurements

Changes in PVR were estimated by measuring pulmonary artery acceleration time (PAAT)

and pulmonary vascular resistance index (PVRI =RVET: PAAT); both of which have previously

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been validated in adults and older children and are commonly used parameters in neonatal and

animal studies (169-172). PAAT and RVET were measured from the pulse wave Doppler tracing

of the main pulmonary artery obtained from the parasternal long axis view of the right

ventricular outflow tract with a 2-mm wide Doppler gate placed in the middle of the main

pulmonary artery at the level of the pulmonary valve (Figure 3.7). Further, as a surrogate

qualitative marker of RV systolic pressure, the curvature of the interventricular septum motion at

end-systole was assessed by visual inspection from a 2D parasternal short axis view acquired at

the level of the mitral valve (164). The septal motion was considered as ‘flat’ if there was

complete absence of concavity towards the left ventricle, and as per routine clinical practise, was

taken as an indication for the RV systolic pressure to be between 50% to 100% systemic systolic

pressure. Presence of concavity in the septal motion in end-systole towards the left ventricle

indicated RV systolic pressure to be below 50% systemic systolic pressure, while presence of

concavity towards the right ventricle indicated RV systolic pressure to be above systemic

systolic pressure (102).

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Figure 3.7: A schematic representation showing measurements of pulmonary artery acceleration time (PAAT) and right ventricular ejection time (RVET) from pulse wave Doppler tracing of the main pulmonary artery obtained at the level of the pulmonary valve. Pulmonary vascular resistance index (PVRI) can then be calculated as shown.

Non-invasive calculation of peak right ventricular systolic pressure (RVSP) were made

whenever feasible, by measuring the peak velocity of tricuspid regurgitant jet using continuous

wave Doppler. The RVSP was then calculated using simplified modified Bernoulli’s equation as

RVSP = 4 x v2 + RAP; where v is the peak velocity of tricuspid regurgitant jet and RAP is right

atrial pressure which in these studies was always assumed to be 5 mmHg (173, 174).

Changes in cardiac outputs were assessed by measuring right ventricular stroke volume

(RVSV) and left ventricular stroke volume (LVSV) respectively using the standard formula [π x

(respective outflow tract diameter/2)2 x velocity time integral] (175). For RVSV, diameter was

measured at the level of pulmonary valve at peak systole from the parasternal long axis view of

the right ventricular outflow tract and VTI was obtained by tracing the pulse wave Doppler trace

obtained at the same level. For LVSV, measurements were performed just proximal to the level

of the aortic valve with diameter being measured from LV PLAX view in 2D image and VTI

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from the pulse wave Doppler trace obtained from the standard apical-5-chamber view (166, 176).

Stroke volumes were multiplied by the observed heart rate to calculate right ventricular output

(RVO) and left ventricular output (LVO), which were indexed to body weight and expressed as

ml/min/kg. All flow related measurements were measured and averaged over at least 3

consecutive cardiac cycles.

3.7 Assessment of transitional shunts

The shunt at the level of patent ductus arteriosus (PDA) and patent formamen ovale (PFO)

was visualized using 2D and spectral color flow Doppler. When present, the shunt pattern was

characterized using pulse wave Doppler from the standard views - high parasternal view for

PDAs and subcostal view for PFOs. The shunt pattern was classified as shunting from systemic

to pulmonary circulation (i.e. left to right), pulmonary to systemic circulation (i.e. right to left) or

bidirectional. In cases of bidirectional shunts, the duration of right to left shunt was measured

and represented as a percentage of total duration of the cardiac cycle.

3.8 Patient selection and exposure

The inclusion and exclusion criteria as well as experimental exposure related on each study

are outlined in detail in specific Chapters.

3.9 Statistics

All data are presented as frequency (proportion), mean (± standard deviation, SD) or

median (interquartile range, IQR) unless otherwise stated. For chapter 4 and 5, results from D2

scans were compared with D1 using paired student’s t-tests for continuous variables and chi

square test or Fisher’s exact test for categorical variables as appropriate. For chapter 6, data from

4 sequential echocardiograms was analyzed using one way repeat measures ANOVA; while

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heart function indices only measured on the 1st and 4th scans were compared using paired student

t-test. For chapter 7, comparison of cases with controls was performed using t-test for continuous

variables with parametric distribution or Mann Whitney U test for continuous variables with

non-parametric distribution, and chi square test or Fisher’s exact test for categorical variables as

appropriate. These being observational physiological studies, minimizing type 2 error rate was

considered more important than type 1 error; hence, Bonferroni correction for multiple

comparisons was not used and a p value < 0.05 was considered as significant.

All inter-observer measurement reliabilities were assessed using 20 randomly selected

studies. For intra-observer variability, one investigator performed two off-line analyses 12 weeks

apart to avoid recall bias while inter-observer variability was assessed by a second investigator

who was blinded to the measurements of the first investigator. Inter-observer agreement was

tested using intraclass correlation presented as intraclass correlation coefficient (ICC) and 95%

confidence interval, Bland-Altman (BA) analysis presented as 95% limits of agreement and

coefficient of variation (COV). COV was calculated for each parameter using the formula COV

(expressed as %) = (SD of absolute differences between repeated measurements/ arithmetic mean

of all repeated measurements) x 100. This was done to facilitate a more intuitive comparison

between reproducibility of different parameters. The lower the COV is, the lower the inter-

observer variability. We accepted a p value of < 0.05 as significant. The strength of linear

dependency between indices was examined by estimating Pearson or Spearman’s correlation

coefficient as and when appropriate. Only relationships with p values < 0.05 are shown. SPSS

statistics software version 21 was used to perform the analyses.

Additional study-specific details of statistical methods are provided in individual chapters.

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Chapter 4: A Comprehensive echocardiographic protocol for assessing

neonatal right ventricular dimensions and function in the transitional Period:

normative data and Z scores*

*Reproduced in compliance with Elsevier’s Author User Rights policy as published in Journal of American Society

of Echocardiography (J Am Soc Echocardiogr. 2014;27(12):1293-304)

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4.1 Introduction:

Right ventricular dysfunction is a strong predictor of outcomes in many adult and pediatric

diseases (1). It is recognized, in infants with PHT and various congenital heart defects involving

the right ventricle, that an RV functional assessment is an important aspect of care. Despite its

importance, the appraisal of RV function in routine clinical practice has largely remained

qualitative. The American Society of Echocardiography (ASE) has recently published separate

comprehensive guidelines for assessment of the right heart in adults (114), and in children (115).

The applicability of these guidelines to the newborn population is hampered by the limited

availability of normative data and the potential influence of the transitional circulation on these

measurements. Transition from fetal to postnatal life is characterized by major circulatory

changes, which includes a decrease in PVR, an increase in pulmonary blood flow and closure of

fetal shunts - PDA and PFO. Although these changes take place over the first 4-6 weeks after

birth, the most rapid alterations occur within the first few days of life. While research regarding

the use of echocardiography to quantify neonatal RV function is growing, there remains a

paucity of comprehensively acquired data, with even fewer studies looking at the effect of the

transitional circulation during the first 48 hours of age (62, 63, 104, 177). This is crucial as the

majority of critically ill newborns present during this period. Further, the majority of RV

functional parameters established for use in older children and adults are derived almost

exclusively from apical 4C view and hence can be influenced by interventricular septal motion,

which is often restrictive and variable during the first few days of life.

The primary objective of this study was to test the feasibility and reliability of a

comprehensive echocardiographic protocol, including novel indices independent of septal

motion, for quantifying RV dimensions and function in healthy term human neonates. This

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protocol was used to establish normative data and calculate Z-scores for 2D measurements, TDI

and 2D STE parameters. Finally, we investigated the effect of early transitional changes on these

measurements by comparing results between days 1 and 2 of life. We hypothesized that in

healthy neonates, using a comprehensive approach, it was possible to image the neonatal right

ventricle from multiple views and reliably quantify RV dimensions and function. We further

hypothesized that the novel parameters established for this study would allow quantification of

size and function of the neonatal right ventricle independent of septal motion.

4.2 Additional methods

Inclusion and exclusion criteria: Healthy, term (gestational age 37 to 42 weeks), singleton

newborns with normal birth weight (BW) (between 10th to 90th percentiles for gestational age at

birth), born following an uncomplicated low risk pregnancy were considered eligible for

recruitment. Exclusion criteria were conditions which may alter the transitional circulation

and/or effect neonatal cardiac function during this period and included maternal diseases

(diabetes mellitus; preeclampsia; chorioamnionitis; antenatal diagnosis of placental dysfunction

defined by absent or reversed end-diastolic flow in umbilical arteries on fetal ultrasound; or the

prenatal use of antidepressant medications) and any newborn disease (evidence of a perinatal

depression defined for this study as umbilical cord pH < 7.0 and/or Apgar score of < 5 at 5

minutes of age; the need for active resuscitation at birth; admission to the neonatal intensive care

unit; any congenital malformation; documented episode of neonatal hypoglycemia or clinical

examination suggestive of genetic or cardiovascular abnormalities). We also intended to exclude

neonates with an incidental finding of a congenital heart defect on echocardiograms, except those

with a PFO, small PDA or mild peripheral pulmonary artery stenosis as these are considered

‘normal’ during the transitional period.

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Exposure: The health records of infants receiving routine postnatal care in the Mother and Baby

Unit at Mount Sinai Hospital were screened and eligible families were approached for consent

within 12 hours of birth. A total of 50 healthy eligible neonates (pre-defined sample size for

convenience) were recruited between December 2011 and September 2012. Each infant

underwent two serial comprehensive evaluations - the first between 12 to 20 hours (D1) and a

second between 30 to 40 hours (D2) of life with a minimum interval of 18 hours between scans.

Each echocardiogram was performed using the study imaging protocol to enable measuring all

functional indices as described above. Blood pressure was recorded using a non-invasive

oscillometric method (DINAMAPR Pro 100, GE healthcare, Tampa, FL).

Additional statistical considerations:

Estimation of reference values and Z-scores: All echocardiographic measurements were assessed

for dependency to BW. Associations with infant length or body surface area were not explored

because of the concerns regarding the accuracy of length measurement in the newborns.

Regression models were empirically tested to optimize the goodness-of-fit between

echocardiographic measurements and BW. Linear (y = ax + b), allometric (y = axb), 2nd order

polynomial (y = ax2 + bx + c), and 3rd order polynomial (y = ax3 + bx2 + cx + d) models were

tested. Selection of the most adequate model for each echocardiographic measurement was based

on goodness-of-fit, on visual inspection of residual values, on fit diagnostic aid plots, and on fit

plots of the residual values over the independent variable by linear regression and polynomial

regression. Mathematical transformation of the dependent variable was only considered if the

distribution of the normalized echocardiographic measurement suggested significant departure

from the normal distribution. Preliminary analysis showed that the variance of the residual values

was not always homogenous across the range of BW (i.e. heteroscedasticity). However, weighted

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regression approaches yielded clearly over-adjusted results (not shown). Therefore, weighted

regressions were not further explored.

For each echocardiographic measurement, the selected regression model was used to calculate

the predicted mean value according to BW. Residual values (observed measurement minus

predicted mean) were calculated and the SD of the residual values was computed. Z-scores were

then calculated as follows:

Z-Score = (Observed value − Predicted mean) / SD of residual values.

Finally, newly computed Z-scores were tested to ensure that residual association with BW (by

linear regression and polynomial regression) and significant departure from the standard normal

distribution (distribution histograms, box plots, and normal probability plots, and Shapiro-Wilk

Statistic) were not present.

4.3 Results:

Study population:

Fifty neonates (17 male) with a mean gestational age of 39.7 (± 1.2) weeks were included.

Twelve neonates were delivered by caesarian section. The mean cord pH was 7.23 (± 0.07) and

median Apgar score at 5 minutes was 9 (range 8 – 9). Two infants were noted to have premature

atrial contractions during the echocardiogram that were confirmed by a 12-lead

electrocardiogram. The mean time interval between the two scans was 19.9 ± 1.8 hours. Table

4.1 summarizes clinical and echocardiographic characteristics of the included neonates. A small

PDA was present in 44% of subjects on the first day of age which closed spontaneously in the

majority by the second day. An increase in PAAT suggested a decrease in PVR on day 2 vs. day

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1 of age; which was also consistent with the decreased frequency of flat interventricular septal

motion in systole on day 2.

Table 4.1: Clinical and baseline echocardiographic characteristics during the first two days of life

Characteristic Day 1 Day 2 P

Weight (kg) 3.49 ± 0.44 3.30 ± 0.43 <0.01

Age at scan (hrs) 15 ± 2 35 ± 2 -

Systolic blood pressure (mmHg) 67 ± 8 68 ± 6 0.75

Diastolic blood pressure (mmHg) 39 ± 9 41 ± 7 0.32

Mean blood pressure (mmHg) 48 ± 8 51 ± 8 0.18

Heart rate (beats/min) 118 (108-128) 121 (110-140) 0.18

Patent ductus arteriosus, n (%) 22 (44) 2 (4) <0.001

Patent foramen ovale, n (%) 41 (82) 37 (74) 0.47

Right ventricular ejection time (ms) 216 ± 24 216 ± 23 0.89

Pulmonary artery acceleration time (ms) 49 ± 17 59 ± 15 <0.001

Interventricular septum flat at end systole, n (%) 16 (32) 6 (12) 0.03

Results presented as mean (± SD) or median (IQR).

Feasibility and reliability of RV measurements:

All RV dimensions were measurable from each of the 100 scans analyzed. STE-derived

GLS-4C and GLS-3C measurements were feasible in 94% of the scans. The RV lateral wall

could not be tracked in 2 subjects on D1 and in 4 on D2. The RV inferior wall was traceable in

all but 1 infant on D1 and 5 on D2. At least one wall was analyzable in 98% of scans while a

global GLS calculation was feasible in 90%. Measurements for tricuspid inflow velocities were

not taken in cases of complete fusion of TvE and TvA (10 scans on D1 and 9 on D2). Similarly,

myocardial diastolic velocity measurements were considered non-feasible when e’ and a’ were

indistinguishable (4 cases on D1 and 7 on D2). Remaining functional indices could successfully

be measured from all studies. Majority of parameters demonstrated low intra- and inter-observer

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measurement variability (Table 4.2). The variability in measuring RV ESA in both views was

relatively higher than end-diastolic area. The biplane-FAC (global-FAC) calculations were less

variable than single-plane FAC-4C and FAC-3C. Tissue Doppler peak velocities were highly

reproducible while MPI’ was more variable.

Table 4.2: Intra- and inter-observer variability of echocardiographic RV measurements

Intra-observer Inter-observer

ICC*

(95% CI)

BA

95% LOA COV (%)

ICC*

(95% CI)

BA

95% LOA COV (%)

RV linear dimensions

B-4C (cm) 0.92 (0.80, 0.97) -0.21, 0.18 5.4 0.86 (0.69, 0.94) -0.26, 0.18 6.4

MC-4C (cm) 0.90 (0.76, 0.96) -0.26, 0.18 6.3 0.83 (0.62, 0.93) -0.21, 0.29 7.0

RVL (cm) 0.89 (0.75, 0.96) -0.25, 0.22 3.8 0.58 (0.21, 0.81) -0.32, 0.49 6.8

B-PLAX (cm) 0.87 (0.71, 0.95) -0.20, 0.22 4.5 0.66 (0.27, 0.86) -0.41, 0.22 6.7

RV cavity areas

EDA-4C (cm2) 0.93 (0.84, 0.97) -0.67, 0.55 6.7 0.86 (0.67, 0.94) -0.76, 0.82 8.7

ESA-4C (cm2) 0.95 (0.89, 0.98) -0.30, 0.35 4.9 0.74 (0.45, 0.89) -0.73, 0.74 10.5

EDA-3C (cm2) 0.87 (0.69, 0.95) -1.07, 1.02 7.8 0.85 (0.67, 0.94) -1.08, 0.85 7.3

ESA-3C (cm2) 0.81 (0.58, 0.92) -0.86, 0.86 10.2 0.80 (0.56, 0.91) -0.97, 0.87 11.0

RV functional indices

FAC-4C 0.82 (0.61, 0.92) -0.09, 0.07 16.7 0.51 (0.09, 0.77) -0.12, 0.13 25.0

FAC-3C 0.67 (0.33, 0.86) -0.10, 0.09 13.9 0.65 (0.29, 0.84) -0.13, 0.12 16.7

Biplane FAC 0.72 (-0.42, 0.88) -0.08, 0.06 13.3 0.76 (0.49, 0.90) -0.06, 0.06 10.0

TAPSE (cm) 0.97 (0.93, 0.99) -0.11, 0.11 3.4 0.52 (0.11, 0.78) -0.16, 0.22 10.9

s’ (cm/s) 0.96 (0.89, 0.98) -0.36, 0.55 3.7 0.94 (0.86, 0.98) -0.53, 0.67 4.9

e’ (cm/s) 0.97 (0.93, 0.99) -0.65, 0.60 3.8 0.52 (0.00, 0.85) -0.99, 0.69 5.0

a’ (cm/s) 0.99 (0.97, 1.00) -0.34, 0.48 2.7 0.77 (0.22, 0.93) -1.20, 0.91 7.0

IVCV’ (cm/s) 0.99 (0.97, 1.00) -0.25, 0.41 3.6 0.98 (0.96, 0.99) -0.48, 0.51 5.2

MPI’ 0.97 (0.92, 0.98) -0.08, 0.06 9.5 0.80 (0.56, 0.92) -0.13, 0.21 20.9

GLS-4C (%) 0.93 (0.78, 0.97) -4.42, 2.37 9.5 0.96 (0.89, 0.98) -3.65, 2.61 9.2

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GLS-3C (%) 0.77 (0.50, 0.90) -5.11, 5.61 12.5 0.84 (0.63, 0.93) -5.16, 5.10 12.0

Global GLS (%) 0.89 (0.74, 0.96) -3.17, 2.30 7.0 0.87 (0.70, 0.95) -3.28, 2.90 8.0

SD standard deviation; ICC intraclass correlation coefficient; CI confidence interval; BA Bland-Altman; LOA limits of agreement; COV coefficient of variability; 4C apical 4-chamber view; 3C apical 3-chamber view; TVA tricuspid annulus; B/MC basal/mid-cavity diameter; RVL RV length; B-PLAX basal diameter in parasternal long axis view; EDA end diastolic area; ESA end systolic area; FAC fractional area change; TAPSE tricuspid annular plane systolic excursion; s’/e’/a’/IVCV’ peak systolic/early diastolic/late diastolic/isovolumetric contraction tricuspid annular velocities; MPI’ myocardial performance index; GLS peak longitudinal strain of lateral and inferior wall in 4C and 3C respectively; Global FAC = (FAC-4C + FAC-3C)/2; Global GLS = (GLS-4C + GLS-3C)/2; *p<0.05 for all parameters.

Echocardiographic RV dimensions in the transitional period:

Results obtained for RV dimensions parameters for each day are outlined in Table 4.3.

The mean basal antero-inferior diameter (B-PLAX) was larger than the mean B-4C and mean

EDA-3C was larger than the mean EDA-4C (p<0.001). We noted a small but significant increase

in some RV dimensions on D2 (MC-4C, B-PLAX and EDA-4C).

There was a modest but significant association between most RV dimension

measurements and BW; a simple linear regression was sufficient to describe their relationship.

The use of more complex models, e.g. allometric, polynomial, had a tendency to produce

noticeably over-adjusted regression curves. We therefore elected to use a linear model for all RV

dimension measurements. Z-scores can be computed with the beta estimate and the SD of

residual values presented in table 3 using the following equation:

𝑍𝑍 =𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝑀𝑀𝑑𝑑𝑑𝑑𝑀𝑀𝑑𝑑𝑀𝑀𝑑𝑑𝑑𝑑𝑑𝑑 − [𝑑𝑑𝑑𝑑𝑖𝑖𝑀𝑀𝑀𝑀𝑖𝑖𝑀𝑀𝑖𝑖𝑖𝑖 + (𝑏𝑏𝑀𝑀𝑖𝑖𝑀𝑀 𝑀𝑀𝑀𝑀𝑖𝑖𝑑𝑑𝑑𝑑𝑀𝑀𝑖𝑖𝑀𝑀 × 𝐵𝐵𝐵𝐵)]

𝑆𝑆𝑆𝑆 𝑑𝑑𝑜𝑜 𝑀𝑀𝑀𝑀𝑀𝑀𝑑𝑑𝑀𝑀𝑀𝑀𝑀𝑀𝑟𝑟 𝑣𝑣𝑀𝑀𝑟𝑟𝑀𝑀𝑀𝑀𝑀𝑀

Z-scores were adequately distributed with little or no residual association with BW.

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Table 4.3: Right ventricular dimensions and area measurements during the transitional period

Measurements

Day 1 Day 2

P-value for day 1 vs. day 2

Mean SD Intercept Beta estimate for BW

slope

SD of residual values

Mean SD Intercept* Beta estimate for BW slope*

SD of residual values*

RV linear dimensions

TVA (cm) 1.29 0.15 1.14 0.044 0.151 1.31 0.15 0.54

B-4C (cm) 1.71 0.17 1.20 0.146 0.158 1.74 0.21 0.31

MC-4C (cm) 1.55 0.19 0.86 0.197 0.167 1.71 0.20 1.12 0.168 0.183 <0.01

RVL (cm) 3.06 0.23 2.52 0.154 0.219 3.05 0.23 0.82

B-PLAX (cm) 2.06 0.24 1.89 0.048 0.243 2.24 0.29 1.70 0.153 0.279 <0.01

RV cavity areas

EDA-4C (cm2) 4.11 0.69 1.16 0.841 0.581 4.32 0.64 1.16 0.841 0.581 0.04

ESA-4C (cm2) 3.02 0.57 -0.11 0.893 0.418 3.15 0.54 0.12

EDA-3C (cm2) 6.44 1.18 1.62 1.373 1.014 6.64 0.86 0.29

ESA-3C (cm2) 3.91 0.68 1.58 0.667 0.607 4.10 0.70 0.10

SD standard deviation; BW birth weight; 4C RV-focused apical 4-chamber view; TVA tricuspid valve annulus; B/MC basal/mid-cavity diameter; RVL RV length; B-PLAX basal diameter in parasternal long axis view; EDA end diastolic area; ESA end systolic area. *Only presented if mean values differ significantly from Day 1.

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Echocardiographic RV functional measurements in the transitional period:

Results obtained for RV functional parameters for each day are outlined in Tables 4.4 and 4.5.

FAC-3C values were higher than FAC-4C on both days (Figure 4.1). The mean difference

between two FAC measurements was 12.2 ± 9.3% on day 1 and 11.9 ± 10.9 on day 2.

Measurement of GLS was similar for both lateral and inferior walls. Apart from a very small but

significant drop in e’, no changes in RV functional parameters were observed between day 1 and

day 2.

Unlike RV dimension measurements, most functional measurements were not

significantly associated with BW, with the exception of FAC. Z-scores for RV functional

measurements which did not show an association with BW, are estimated with the mean and SD

presented in Table 4.5 using the following equation:

𝑍𝑍 =𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝑖𝑖𝑀𝑀𝑀𝑀𝑀𝑀𝑑𝑑𝑀𝑀𝑖𝑖𝑀𝑀𝑀𝑀 − 𝑑𝑑𝑀𝑀𝑀𝑀𝑑𝑑

𝑆𝑆𝑆𝑆

Most RV functional measurements had normal or near normal distribution with the

exception of systole’: diastole’ and MPI’. systole’: diastole’ ratio adopted a log-normal

distribution. Consequently, a log transformation was necessary to obtain a normally distributed

Z-score, which can be estimated with the mean and SD of the log-transformed value in Table 5

using the following equation:

𝑍𝑍 =Ln(𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝑖𝑖𝑀𝑀𝑀𝑀𝑀𝑀𝑑𝑑𝑀𝑀𝑖𝑖𝑀𝑀𝑀𝑀) −𝑑𝑑𝑀𝑀𝑀𝑀𝑑𝑑

𝑆𝑆𝑆𝑆

MPI’ distribution was very different on D1 compared to D2; such departure from the normal

distribution prevented the computation of a valid Z-score.

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Table 4.4: Right ventricular fractional area change during transitional period

Measurements

Day 1 Day 2

P-value for day 1 vs. day 2

Mean SD Intercept* Beta estimate for BW slope*

SD of residual values*

Mean SD

FAC-4C (%) 0.26 0.072 0.48 -0.063 0.0667 0.27 0.081 0.68

FAC-3C (%) 0.39 0.068 0.30 0.025 0.0670 0.38 0.058 0.74

Global-FAC (%)

0.33 0.053 0.40 -0.021 0.0520 0.33 0.042 0.89

SD standard deviation; BW birth weight; 4C apical 4-chamber view; 3C apical 3-chamber view; FAC fractional area change; Global FAC=(FAC-4C + FAC-3C)/2. *Only presented for Day 1 as mean values were similar on both days.

Figure 4.1: A box plot graph of fractional area change (FAC) measurements obtained from RV-4C and RV-3C view on day 1 and 2 of age. FAC-3C was significantly greater than FAC-4C on both days.

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Table 4.5: Right ventricular functional measurements in the transitional period

Measurements

Day 1 Day 2

p value Mean SD Mean SD

Systolic function

TAPSE (cm) 0.92 0.14 0.91 0.13 0.69

s’ (cm/s) 6.55 1.16 6.54 1.09 0.95

IVCV’ (cm/s) 4.88 1.52 4.84 1.39 0.95

GLS-4C (%) 21.17 5.25 21.27 5.35 0.79

GLS-3C (%) 21.38 4.36 20.68 4.12 0.40

Global GLS (%) 21.21 3.93 21.16 4.21 0.72

Diastolic function

TvE (cm/s) 45.60 9.48 44.02 7.96 0.16

TvA (cm/s) 52.09 10.49 53.49 8.64 0.36

TvE: TvA 0.89 0.17 0.83 0.13 0.07

e’ (cm/s) 7.99 1.57 7.33 1.26 <0.01

a’ (cm/s) 8.27 1.57 8.01 1.28 0.45

e’/a’ 0.99 0.23 0.93 0.21 0.09

TvE: e’ 5.85 1.34 6.19 1.36 0.24

IVRT’(ms) 41.38 17.09 43.32 15.55 0.51

Global function

MPI’ 0.42 0.15 0.42 0.12 0.89

systole’: diastole’ 1.08 0.26 1.11 0.31 0.29

Log [systole’: diastole’]

0.05 0.22 0.07 0.25 -

SD standard deviation; 4C apical 4-chamber view; 3C apical 3-chamber view; TAPSE tricuspid annular plane systolic excursion; s’/e’/a’/IVCV’ systolic/early diastolic/late diastolic/isovolumetric contraction tricuspid annular velocities; GLS peak longitudinal strain of lateral and inferior wall in 4C and 3C respectively; TvE: TvA early/late

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tricuspid inflow velocities; IVRT’ isovolumetric relaxation time; MPI’ myocardial performance index; systole’: diastole’ ratio of duration of systole to diastole; Global GLS = (GLS-4C + GLS-3C)/2.

Systolic functional parameters measured using the RV-4C view demonstrated weak to

modest positive linear correlations (Table 4.6), with the correlation coefficient of 0.45 being the

highest between s’ and GLS-4C. Comparing indices acquired from the RV-3C view with those

measured from the RV-4C view revealed similar results for GLS-3C with GLS-4C (r=0.45,

p<0.001) and GLS-3C with TAPSE (r=0.23, p=0.03). FAC measurements did not correlate

linearly with each other or with any other functional parameters.

Table 4.6: Linear correlations examined for right ventricular functional indices derived from apical 4C view

TAPSE s’ GLS-4C FAC-4C

TAPSE - 0.29 (0.004) 0.29 (0.005) -0.01 (0.88)

s’ - - 0.43 (<0.001) -0.06 (0.53)

GLS-4C - - - 0.07 (0.48)

Data presented as Pearson’s correlation coefficient (p value). RV right ventricle; TAPSE tricuspid annular plane systolic excursion; s’ systolic tricuspid annular velocity; GLS-4C peak longitudinal strain for RV lateral wall; FAC-4C fractional area change in apical 4-C view.

4.4 Discussion:

Clinical significance: In this study we described a comprehensive echocardiographic protocol for

imaging the right ventricle in neonates and established normal values for various RV dimensions

and functional parameters. Preservation of RV function is recognized as a critical prognostic

factor in the management of cardio-pulmonary disorders of adults, in particular PAH (8, 84,

178). Cardio-pulmonary disorders are not uncommon during the newborn period. Acute PHT has

been reported as a primary diagnosis in up to 4% of admissions in tertiary neonatal intensive care

units with a reported mortality ranging from 5-20% (33, 34). Many chronic neonatal respiratory

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illnesses such as chronic lung disease or congenital diaphragmatic hernia are complicated by

cPHT and high rates of adverse patient outcomes (41, 128, 179). RV function may also be of

critical importance in various congenital heart defects. It is plausible that early recognition of RV

compromise may modify the clinical outcomes for this vulnerable patient population. Clinical

appraisal of RV health in neonates is challenging, as clinical symptoms and signs are non-

specific and investigations such as cardiac catheterization and CMRI are generally not feasible.

2D echocardiography is considered to be the gold standard for the assessment of heart function

in this population, with research emphasis beginning to shift towards RV performance. The

establishment of a comprehensive normative dataset in this study, attained in a systematic

manner, is an important and timely initial step towards examining the relevance of RV

physiology in neonatal disease states.

Although recent reports have highlighted the feasibility of quantifying RV function in

healthy neonates, the majority of these studies used either TAPSE or TDI-derived myocardial

velocities (60, 62, 63, 106, 108, 109, 153, 154). We demonstrate the practicality of employing a

more integrated and comprehensive approach using different 2D echocardiographic views and

emerging techniques for RV assessment in neonates. This approach allowed us to provide

comparative reliability data as well as normative ranges for various measures of RV dimensions

and function. Further, we demonstrated the feasibility of acquiring the RV 3-chamber view and

related novel functional measurements - FAC-3C and GLS-3C.

Rationale for using ‘newer’ indices: The majority of previously established echocardiographic

RV indices for adults have been derived from the RV-4C view, which provides a coronal cut

through the RV. Both 4C-derived FAC and RV linear dimensions are likely to be heavily

influenced by interventricular septal motion, which is often restricted and variable during the

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early neonatal period (164). TAPSE, TDI-derived tricuspid annular velocities and lateral wall

GLS are measured from the same region of the RV and may not always reflect global RV

performance. Adding additional views may help to overcome some of these limitations. We

hypothesized that, as B-PLAX diameter is independent of septal motion, it may be more

reflective of changes in RV dimensions in newborns. We also observed that FAC values

obtained using RV-3C view were significantly higher than those obtained from 4C view for both

days 1 and 2 of age. As we hypothesized, the RV-3C view may be more reflective of ‘true’ RV

systolic function as it is measured independent of the septum. Another possible explanation

could be that FAC-3C represents systolic function of a different RV region, the RV

infundibulum. The finding that GLS measurements obtained from both RV walls (GLS-4C and

GLS-3C) were not different suggests lack of interference from septal motion to be the primary

reason for higher FAC-3C values. In addition, it was our qualitative experience that the RV

cavity visualized in the RV-3C view had less trabeculations than the RV-4C, which may explain

the superior reliability observed for FAC-3C when compared to FAC-4C in our study. Recent

data have shown that the RV infundibulum is inherently different from the other regions of the

right ventricle (8, 84). Further, we measured and generated normative data for IVCV as it has

recently been described to correlate with invasive measurements of RV contractility in adults

(180). Although it is tempting to hypothesize that use of these indices may provide for a more

global functional appraisal and may facilitate investigations into differential impact of

pathologies and treatments on different regions of the right ventricle, additional testing is

required in a variety of disease populations.

Reproducibility testing: In this study we used three different methods to investigate the

reproducibility of these measurements – intraclass correlation coefficient (ICC), Bland Altman

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analysis (BA) and coefficient of variability (COV). This was done to ensure a comprehensive

analysis as each method has its strengths and weaknesses. Though ICC is easier to interpret, it

can occasionally be misleading as it compares the variability in repeated measurements with the

overall variability in the study sample. In cases of measurements where the overall variability in

study population is low, ICC may falsely show poor reproducibility and vice versa. This is amply

demonstrated in the results for e’ and MPI’. The ICC for e’, which had low sample variation,

demonstrated poor reproducibility but the reproducibility was excellent when assessed using

COV/BA. On the other hand, MPI’, which varied widely between study patients appeared to

have a good ICC but, showed poor reproducibility on BA/COV. Though calculating limits of

agreement using BA analysis provides the most comprehensive information, its interpretation

and inter parameter comparison is not always intuitive. This is because the results are usually

expressed in units specific to the measurement being evaluated. In contrast, COV allows easy

comparison between different parameters as results are expressed in percentage and are

independent of specific units.

Many previous studies have used pooled images from the right ventricle and the left

ventricle for estimation of inter-observer reliability of certain measurements. This could be

misleading, as the right ventricle, owing to its complex shape and anterior location, is

considerably more challenging to image. The ASE guidelines for targeted neonatal

echocardiography has recommended the use of higher frequency ultrasound transducers (8-12

MHz), to help with the known limitation of inferior near-field resolution of ultrasound, which is

expected to be accentuated in newborns, owing to a thin chest wall (181). Being the most

anterior cardiac structure, this may be especially relevant for imaging the right ventricle. Only

two studies have reported RV-specific measurement reproducibility in neonates (60, 182). The

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superior reproducibility for myocardial velocities in our study in comparison to Joshi et al (COV

ranging from 2.7-7% vs. 15-24%) could in part be explained by our use of higher resolution

transducers and by the fact that half of the patients in their study were preterms who, as

described earlier, are more difficult to scan. Recently, Levy and coworkers reported a lower

COV than reported in our study for measuring STE-derived RV lateral wall GLS in preterm

infants (COVs of 2.7% and 3.9% vs. 9.5% and 9.2% for intra- and inter-observer respectively)

(60). This may be explained by the rigorous criteria applied in their study for assessment of

image quality, resulting in the exclusion of 16% of scans. We attempted tracking on all acquired

scans and were able to perform GLS-4C measurements in 94% with a COV of <10%. Contrary

to all other parameters investigated, we found that the inter-observer measurement bias was

significant for FAC-4C and MPI’ measurements. This could be due to inherent subjectivity

associated with performing these measurements; specifically, delineating the endocardial borders

in end-systole in a heavily trabeculated neonatal right ventricle and identifying the edges of

tissue Doppler waves. Interestingly, calculation of global-FAC resulted in a noticeable

improvement in inter-observer reproducibility. Conversely, MPI’ measurements showed wide

variation between days with no clear pattern, making calculation of Z-scores impractical and

suggesting against the use of MPI’ for RV assessment in neonates.

Among the indices reported in this study, TVA, trans-tricuspid flow profile, TDI-derived

myocardial velocities and TAPSE, are the only functional indices for which good quality

normative data exists for neonates (62, 63, 130, 183). Our data for these indices were comparable

to previously published results, thus supporting the validity of our study population.

Furthermore, we found that the majority of indices of RV systolic function measured in this

study, demonstrated statistically significant linear correlations. Although these correlations were

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weak to modest and of uncertain clinical significance, it provided circumstantial evidence

supporting the appropriateness of our methods in eliciting these ‘newer’ indices. Interestingly,

FAC estimated from either views did not correlate with each other or with other indices. This

could suggest the unreliability of FAC measurements, or perhaps, FAC represents a ‘different’

aspect of RV systolic function as FAC is derived from cavity dimensions while all other

parameters are derived from the myocardium. Nonetheless, FAC measurements showed

relatively wide standard deviations and high inter-observer bias, which may limit its clinical

utility in this population.

Deformation measurements: Although they are not inter-changeable, two methods currently exist

for measuring myocardial wall strain. Contrary to previous neonatal studies, we chose not to

utilize TDI-derived strain measurements, but rather employ STE to measure GLS for the RV

walls. Measurements obtained using STE are independent of the angle of the ultrasound beam

and of the shape of the myocardial wall (133). To avoid errors from dividing a short neonatal

myocardial wall into segments, we calculated an overall GLS for the lateral and inferior walls, to

generate a ‘global’ RV-GLS. We chose not to measure strain rate in this study. STE, in contrast

to TDI, uses relatively low frame rates and though this does not significantly influence strain

measurements, because the software determines strain rate by measuring the change in speckle

position frame by frame, this could lead to under sampling and therefore an underestimate of

peak strain rate. This problem is likely to be accentuated in neonates given their higher heart

rates. Further, it’s been shown that while TDI and STE derived longitudinal strain measurements

are comparable in children; the strain rate measurements derived using STE are much lower than

TDI and have poor inter-rater reliability (184). The relative benefits of using STE-derived strain

and strain rate in neonates need further investigation.

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Effect of postnatal transition: Another consideration while designing this study was to

investigate the effect of physiological circulatory alternations associated with the transition to

postnatal life. This was important for the clinical applicability of our data. Contrary to

expectations, we found no increase in RV GLS from day 1 to day 2 of life, in spite of a reduction

in PVR. There could be several explanations for this finding. First, the decrease in PVR under

physiological conditions may not be large enough to affect STE-derived GLS. Second, the

relatively immature contractile properties of the neonatal myocardium may be less affected by

changes in loading conditions. Lastly, it is plausible that the reduction in preload secondary to

physiological diuresis expected to occur in the first few days of life in healthy neonates negated

the effect of changes in PVR on RV deformational indices. Converse to the effect of changes in

afterload, a drop in preload is expected to lower RV GLS. Indeed, as expected, neonates enrolled

in this study demonstrated weight loss on day 2 of life ranging from 0.6% to 9.3% [mean (±SD)

= 5.6 (± 2.1) %]. We confirm that in healthy neonates, ‘normal’ changes in RV loading

conditions during early postnatal life had no appreciable effect on quantitative echocardiographic

measures of RV function. Mori et al also reported no differences when they compared TDI-

derived myocardial velocities between healthy neonates < 24 hours old (n = 130) with those

between 1-7 days of life (n = 135) (108). We did observe a small increase in some RV

dimensions on day 2 of life. We speculate that this could have been explained by an increase in

left to right shunting of blood across the PFO associated with the reduction in right-sided

pressures on day 2 of life. Although quantitative echocardiographic measurement of shunt

volume was not feasible to test this hypothesis, persistence of a PFO in most infants is highly

suggestive. Nevertheless, as the magnitude of the observed change was small, this finding is

unlikely to be clinically significant.

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In conclusion, a comprehensive, quantitative RV appraisal using 2D echocardiography is

feasible in neonates and should include measurements of its dimensions and function using

multiple 2D views. We propose that a practical echocardiographic protocol should include

measurement of posterior basal diameter in addition to the conventional apical RV-4C view

derived linear dimensions. Further, RV functional assessment should include parameters which

demonstrated the least inter-observer measurement bias – TAPSE, myocardial velocities, GLS of

the lateral and inferior walls and global GLS. To reduce measurement bias, FAC, when used,

should be measured from both the RV-3C and RV-4C views to generate a global FAC. RV

posterior basal diameter and parameters obtained from RV-3C view, may provide novel indices

for neonatal RV assessment which are independent of septal motion. The normative data

established in this study may provide a reference for future comprehensive investigations into

RV physiology in neonates.

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Chapter 5: Left ventricular function in healthy term neonates during the

transitional period*

*Reproduced in compliance with Elsevier’s Author User Rights policy as published in the Journal of Pediatrics (J

Pediatr. 2016 Nov 28. pii: S0022-3476(16)31231-8).

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5.1 Introduction

Impaired LV function is a known association of many disorders of the early neonatal

period, including infants of diabetic mothers, birth asphyxia, PPHN, sepsis, and intrauterine

growth restriction (155, 157, 185-187). Two-dimensional echocardiography is considered the

clinical reference investigation to evaluate LV size and function in neonates. Traditionally, the

most common echocardiographic methods employed to objectively assess LV systolic function

are, M-mode derived shortening fraction (SF) and B-mode derived ejection fraction (EF) using

Simpson’s bi-plane method, both of which are derived from LV cavity dimensions. Although

these methods provide a simple index of LV function and size, their utility in the first few days

of age is hampered by physiological changes in interventricular septal motion and LV geometry

(164). This is due to the progressive increase in SVR, decrease in PVR and decrease in LV

preload with closure of the PDA during this period. Further, SF measures change in LV cavity

dimension at a single point in one plane, which makes it highly operator dependent and not

always reflective of global LV function.

TDI and STE are parameters which assess regional and global LV function by directly

measuring muscle wall characteristics rather than cavity dimensions. TDI is used to measure the

velocity of muscle movement in systole and diastole of the LV free wall and septum, as well as

the timing of cardiac events in a cardiac cycle (188). STE is used to measure LV longitudinal

deformation which is expressed as percentage change in muscle length in systole compared to

end diastole (strain), as well as provide a measure of the speed with which this deformation

occurs (strain rate) (189). Although individual quantitative measure of LV function have been

shown to be feasible in recent neonatal studies (106, 153-155, 157), LV evaluation remains

largely qualitative in neonatal practice. This may reflect a paucity of data regarding normal

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values and relative measurement reliability of conventional and newer echocardiographic

methods. Further, it is not known whether normal values for these indices change over the first

few days of life (147).

We aimed to characterize LV function with a comprehensive echocardiographic protocol

that incorporates conventional, TDI and STE in order to compare measurement reliability,

establish reference values, and determine their maturational patterns during the early neonatal

transitional period.

5.2 Additional methods

The inclusion and exclusion criteria as well as exposure was similar to that of Chapter 4,

and for ease of reference is reproduced below.

Inclusion and exclusion criteria: Healthy, term (gestational age 37 to 42 weeks), singleton

newborns with normal birth weight (BW) (between 10th to 90th percentiles for gestational age at

birth), born following an uncomplicated low risk pregnancy were considered eligible for

recruitment. Exclusion criteria were conditions which may alter the transitional circulation

and/or effect neonatal cardiac function during this period and included maternal diseases

(diabetes mellitus; preeclampsia; chorioamnionitis; antenatal diagnosis of placental dysfunction

defined by absent or reversed end-diastolic flow in umbilical arteries on fetal ultrasound; or the

prenatal use of antidepressant medications) and any newborn disease (evidence of a perinatal

depression defined for this study as umbilical cord pH < 7.0 and/or Apgar score of < 5 at 5

minutes of age; the need for active resuscitation at birth; admission to the neonatal intensive care

unit; any congenital malformation; documented episode of neonatal hypoglycemia or clinical

examination suggestive of genetic or cardiovascular abnormalities). We also intended to exclude

neonates with an incidental finding of a congenital heart defect on echocardiograms, except those

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with a PFO, small PDA or mild peripheral pulmonary artery stenosis as these are considered

‘normal’ during the transitional period.

Exposure: The health records of infants receiving routine postnatal care in the Mother and Baby

Unit at Mount Sinai Hospital were screened and eligible families were approached for consent

within 12 hours of birth. A total of 50 healthy eligible neonates (pre-defined sample size for

convenience) were recruited between December 2011 and September 2012. Each infant

underwent two serial comprehensive evaluations - the first between 12 to 20 hours (D1) and a

second between 30 to 40 hours (D2) of life with a minimum interval of 18 hours between scans.

Each echocardiogram was performed using the study imaging protocol to enable measuring all

functional indices as described above. Blood pressure was recorded using a non-invasive

oscillometric method (DINAMAPR Pro 100, GE healthcare, Tampa, FL).

Additional statistical considerations: The strength of linear dependency between infant’s

gestational age at birth and weight on the day of the scan and LV dimensions and functional

indices was examined by estimating Pearson or Spearman correlation coefficients as appropriate.

5.3 Results

The study population’s baseline demographic characteristics were as listed in Chapter 4.

In brief, the study involved 50 healthy term neonates receiving routine postnatal care in the well

Mother and Baby Unit. The mean ± SD gestation and BW was 39.3 ± 1.2 weeks and 3.5 ± 0.44

kg respectively. Sequential scans were performed at 15 ± 2 hours [range 12 – 19] (D1) and 35 ±

2 hours [range 30 – 40] (D2) of age; with a mean time interval of 19.9 ± 1.8 hours. Weight on D2

was significantly lower (3.3 ± 0.4 Kg, p<0.001) with a mean weight loss of 5.6 ± 2.1%. There

was no difference in heart rate [118 (108 – 128) vs. 121 (110 – 140); p=0.20)], systolic blood

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pressure [(67 ± 8 vs. 68 ± 6); p=0.75] or diastolic blood pressure [(39 ± 9 vs. 41 ± 7); p=0.32]

between two time points. Small restrictive PDAs were present in 22 (44%) of subjects on the

first day of age, remaining patent in only 2 infants (4%) by the second day (p<0.001), while

patent foramen ovale was present in 41 (82%) and 37 (74%) neonates on D1 and D2 respectively

(p=0.47).

LV Dimensions during the Transitional Period

LV dimensions were measurable from all scans (Table 5.1). With the exception of a

slight reduction in LV basal diameter on D2, there was no difference in any of the measurements

between the two time points. A mild to moderate linear correlation was observed between birth

weight and the majority of LV dimensions. There was no difference in dimensions between

males vs. females and scans with PDA open vs. closed (data not shown).

Table 5.1: LV dimensions on days 1 and 2 of age in healthy term neonates

Correlation with weight

Correlation with weight

D2 vs. D1

Variable D1 r p D2 r p p

MV annulus 0.99 ± 0.09 0.29 0.04 0.99 ± 0.08 0.10 0.50 0.73

LV basal diameter 1.91 ± 0.13 0.46 <0.01 1.84 ± 0.14 0.44 <0.01 <0.01

LV length (4C) 3.08 ± 0.26 0.30 0.04 3.07 ± 0.26 0.24 0.10 0.77

Aortic Diameter 0.69 ± 0.06 0.30 0.04 0.69 ± 0.05 0.44 <0.01 0.77

LVEDD (PLAX) 1.79

(1.64 – 1.92)

0.44 <0.01 1.74

(1.65 – 1.87)

0.42 <0.01 0.17

LVEDD (SAX) 1.73 ± 0.23 0.42 <0.01 1.72 ± 0.26 0.49 <0.01 0.91

LVEDC (SAX) 7.61 ± 0.73 0.49 <0.01 7.59±0.67 0.55 <0.01 0.79

Values are presented as mean ± SD or medians (IQR). All units are in centimetres. MV: Mitral Valve; LV: left ventricle; 4C: 4 Chamber; LVEDD: left ventricular end diastolic diameter; PLAX: parasternal long axis view; SAX: parasternal short axis view; LVEDC: left ventricular end diastolic circumference.

LV Function during the Transitional Period

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All measurements were feasible from each scan except STE in 10% images on D1 (15

out of 150 images; 4 in the four-chamber, 6 in the two chamber and 5 in the three chamber) and

21% images on D2 (32 out of 150 images; 10 in the four-chamber, 9 in the two-chamber and 13

in the three-chamber). With the exception of s’ (Figure 5.1) and mitral valve VTI, all measured

functional indices remained unchanged on D2 (Table 5.2, 5.3). All myocardial velocities were

lower in the septum than respective values for the LV free wall on both days (p < 0.01). There

was no linear correlation between gestation and birth weight with any of the functional

parameters. There was no difference observed between males vs. females, or between scans with

PDA vs. no PDA (data not shown).

Figure 5.1: Peak systolic myocardial velocity in LV free wall and interventricular septum on day 1 and day 2 of life in healthy term neonates.

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Table 5.2: LV systolic function parameters on days 1 and 2 of age in healthy term neonates

Variable D1 D2 p

Shortening Fraction (%) 39 ± 7 39 ± 8 0.91

mVCF (circ/sec) 1.79 (1.55 – 2.10) 1.89 (1.55 – 2.08) 0.34

Ejection Time (ms) 217 ± 22 212 ± 18 0.15

Systolic Time (ms) 188 (172 – 203) 184 (172 – 195) 0.74

Ejection Fraction (%) – Simpson’s

Two-Chamber 56 ± 9 54 ± 8 0.24

Four Chamber 54 ± 9 53 ± 9 0.38

Biplane 55 ± 7 53 ± 6 0.26

Tissue Doppler s` (cm/s)

Lateral Wall 4.9 ± 0.8 4.6 ± 0.6 <0.01

Septal 3.6 ± 0.6 3.5 ± 0.5 0.04

Peak Longitudinal Strain (%)

Four Chamber - 21.2 ± 2.6 - 21.1 ± 2.2 0.91

Two Chamber - 22.5 ± 2.9 - 22.1 ± 3.0 0.75

Three Chamber - 21.5 ± 2.7 - 20.4 ± 2.5 0.14

Global - 21.7 ± 1.9 - 21.2 ± 1.8 0.23

Peak Longitudinal Strain Rate (1/s)

Four Chamber 2.10 ± 0.44 1.98 ± 0.34 0.15

Two Chamber 2.19 ± 0.39 2.32 ± 0.52 0.09

Three Chamber 2.12 ± 0.43 2.20 ± 0.48 0.53

Global 2.05 (1.90 – 2.28) 2.17 (1.93 – 2.43) 0.27

mVCF: Mean velocity of circumferential fibre shortening; Ejection time was measured using pulsed wave Doppler. Systolic time was measured as the duration of the s` wave using tissue Doppler imaging. Longitudinal strain and strain rate were measured using 2D speckle tracking echocardiography.

Table 5.3: LV diastolic and global function parameters on days 1 and 2 of age in healthy term neonates

Variable D1 D2 p

Pulsed Wave Doppler

Mitral Valve E wave (cm/s) 55.9 ± 8.3 55.7 ± 8.9 0.84

Mitral Valve A wave (cm/s) 50.6 ± 8.2 49.7 ± 9.2 0.45

E:A ratio 1.1 (1.0 – 1.2) 1.1 (0.9 – 1.3) 0.99

Velocity Time Index 9.1 ± 1.5 8.6 ± 1.7 0.02

Isovolumic Relaxation Time (ms) 54 (44 – 68) 55 (46 – 65) 0.77

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Tissue Doppler - Lateral Wall

e’ (cm/s) 6.7 ± 1.4 6.5 ± 1.2 0.29

a’ (cm/s) 5.5 ± 1.3 5.4 ± 1.3 0.62

e’:a’ 1.3 ± 0.4 1.3 ± 0.4 0.98

Isovolumic Relaxation Time (ms) 53 ± 12 52 ± 10 0.57

Diastolic time (ms) 199 ± 42 195 ± 44 0.66

E:e`ratio 8.7 ± 2.5 8.9 ± 2.8 0.59

Tissue Doppler - Septum

e’ (cm/s) 4.7 ± 1.1 4.8 ± 0.9 0.32

a’ (cm/s) 4.2 ± 0.8 4.4 ± 0.8 0.32

e’:a’ 1.1 (0.9 – 1.3) 1.1 (0.9 – 1.3) 0.85

Longitudinal Strain Rate (1/s)

Two Chamber Early SR 2.92 ± 0.69 2.96 ± 0.63 0.67

Two Chamber Late SR 2.09 ± 0.59 2.22 ±0.60 0.22

Three Chamber Early SR 3.21 ± 0.64 3.06 ± 0.63 0.16

Three Chamber Late SR 2.03 ± 0.66 2.12 ± 0.69 0.70

Four Chamber Early SR 3.12 ± 0.79 3.09 ± 0.53 0.53

Four Chamber Late SR 2.12 ± 0.75 2.14 ± 0.56 0.94

Global Function Parameters

Left ventricular output (mls/min/kg) 128 ± 28 137 ± 28 0.11

Systole: diastole (Pulsed Wave) 0.79 (0.69-0.90) 0.82 (0.70 – 0.98) 0.17

Systole’: diastole’ (Tissue Doppler) 0.98 ± 0.24 0.98 ± 0.19 0.90

Myocardial Performance Index 0.61 (0.53 – 0.70) 0.61 (0.54 – 0.70) 0.80

Measurement Reliability of LV measurements

Overall, all measured indices demonstrated acceptable intra- and inter-observer

measurement reliability (Table 5.4), with longitudinal strain faring better than strain rate (Figure

5.2) as well as conventional measures of FS and EF.

Table 5.4: Intra- and inter-observer variability of left ventricular size and functional measurements

Variable Intra-observer Variability Inter-observer variability ICC*

(95% CI) Mean Bias (95%CI)

COV (%)

ICC* (95% CI)

Mean Bias (95%CI)

COV (%)

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LV Dimensions MV Annulus 0.97

(0.92 – 0.99) -0.01 (-0.09 – 0.07) 4 0.86

(0.44 – 0.95) -0.05 (-0.18 – 0.09) 7

Basal diameter 0.86 (0.68 – 0.95)

0.01 (-0.16 – 0.18) 5 0.71 (-0.19 – 0.92)

0.14 (-0.10 – 0.38) 7

Length 0.89 (0.73 – 0.96)

0.01 (-0.24 – 0.25) 4 0.88 (0.70 – 0.95)

-0.02 (-0.33 – 0.29) 5

LVEDD 0.97 (0.93 – 0.99)

0.00 (-0.08 – 0.08) 2 0.97 (0.93 – 0.99)

-0.01 (-0.13 – 0.10) 3

Ao diameter 0.93 (0.81 – 0.97)

0.01 (-0.03 – 0.06) 4 0.87 (0.67 – 0.95)

-0.01 (-0.07 – 0.05) 5

LV Functional Indices – Conventional SF 0.92

(0.80 – 0.97) -1 (-8 – 7) 10 0.93

(0.83 – 0.97) 2 (-5 – 8) 9

EF 0.87 (0.67 – 0.95)

-1 (-9 – 7) 6 0.71 (0.24 – 0.89)

4 (-7 – 15) 9

MvE 0.94 (0.84 – 0.98)

-1.41 (-7.24 – 4.43) 6 0.94 (0.82 – 0.98)

2.80 (-3.52 – 9.13) 6

MvA 0.98 (0.96 – 0.99)

-0.80 (-5.36 – 3.76) 5 0.97 (0.92 – 0.99)

1.12 (-4.57 – 6.81) 6

Aortic VTI 0.97 (0.93 – 0.99)

0.02 (-0.71 – 0.75) 4 0.98 (0.92 – 0.99)

0.15 (-0.64 – 0.93) 4

LV Functional Indices – TDI s’ 0.99

(0.97 – 1.00) -0.04 (-0.44 – 0.36) 4 0.99

(0.97 – 1.00) 0.00 (-0.41 – 0.42) 4

e’ 0.94 (0.87 – 0.98)

-0.07 (-0.82 – 0.68) 5 0.92 (0.81 – 0.97)

0.14 (-1.1. – 1.38) 9

a’ 0.97 (0.92 – 0.99)

-0.01 (-0.83 – 0.81) 7 0.98 (0.92 – 0.99)

0.18 (-0.63 – 1.00) 7

IVRT’ 0.94 (0.86 – 0.98)

-0.63 (-6.17 – 4.91) 5 0.95 (0.87 – 0.98)

-0.01 (-5.69 – 5.68) 5

MPI’ 0.95 (0.88 – 0.98)

-0.01 (-0.09 – 0.07) 6 0.89 (0.73 – 0.89)

0.00 (-0.10 – 0.11) 9

Peak longitudinal Strain (GLS) 2 Chamber 0.92

(0.82 – 0.97) 0.05 (-0.20 – 2.30) 7 0.86

(0.66 – 0.94) -0.63 (-3.50 – 2.25) 8

3 Chamber 0.93 (0.84 – 0.97)

0.14 (-2.40 – 2.68) 7 0.90 (0.76 – 0.96)

-0.08 (-3.15 – 2.98) 9

4 Chamber 0.87 (0.71 – 0.94)

-0.06 (-2.95 – 2.84) 8 0.80 (0.54 – 0.92)

0.81 (-2.68 – 4.30) 10

Peak longitudinal Systolic Strain Rate (pLSSR) 2 Chamber 0.81

(0.57 – 0.92) -0.01 (-0.36 – 0.33) 10 0.71

(0.03 – 0.91) -0.19 (-0.51 – 0.14) 9

3 Chamber 0.80 (0.57 – 0.92)

0.04 (-0.38 – 0.46) 13 0.79 (0.54 – 0.91)

-0.02 (-0.47 – 0.43) 14

4 Chamber 0.93 (0.82 – 0.97)

0.00 (-0.27 – 0.28) 8 0.84 (0.65 – 0.94)

0.03 (-0.34 – 0.41) 11

ICC intraclass correlation coefficient; CI confidence interval; BA Bland-Altman; LOA limits of agreement; COV coefficient of variability; 4C apical 4-chamber view; 3C apical 3-chamber view; 2C apical 2-chamber view; * p<0.001 for all ICC;

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Figure 5.2: Bland Altman plots for inter- and intra-observer measurement variability for mean strain and strain rate measured from left ventricular 4 chamber view in a randomly selected subset of 20 scans. Bias is presented as an absolute difference between two values.

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5.4 Discussion

In this study, we utilized a comprehensive echocardiographic protocol that included

conventional imaging, TDI, and emerging STE to quantify LV size and function in healthy

newborns during the first two days of age, and provide the first comprehensive set of normal

values for this population and relative measurement reproducibility for these indices. We found

that LV dimensions, but not functional parameters, had a mild to moderate positive linear

correlation with birth weight. Further, we confirmed the validity of our data as normal values in

term neonates after the initial 12 hours of age, by demonstrating no significant change in

association with expected physiological changes in loading conditions between day 1 and day 2

of age.

Although there is an increasing desire to move from a qualitative to quantitative appraisal

of cardiac function, the field of quantitative assessment of LV function in neonates remains

grossly understudied, both for conventional and newer echocardiographic techniques. The

existing data, including inter-rater reliability and normative values, is patchy at best and needs to

be systematically evaluated, especially now that high-resolution ultrasound equipment and newer

imaging modalities are routinely available in NICUs. With the growth of neonatal

echocardiography programs, functional cardiac ultrasound is increasingly being utilized in daily

clinical practice by neonatologists. In this study, we demonstrated that among the tested

variables, measurement bias was least for tissue Doppler derived parameters. STE-derived

longitudinal strain fared slightly better than strain rate and conventional parameters (SF and EF).

This is likely due to the inherent limitation in these measures of the need for the operator to

manually identify and/or trace myocardial borders and that in STE, contrary to TDI, the strain is

directly measured by the software while the strain rate is calculated. In addition, we found that

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LV related hemodynamic variables commonly employed in targeted neonatal echocardiograms

(mitral valve inflow, aortic diameter, VTI) demonstrated excellent reproducibility. We believe

the results of this study will be particularly valuable to the increasing number of intensivists with

echocardiography skills and will serve to inform day-to-day echocardiographic practices in

NICUs.

This study should be viewed through the prism of introducing a new routine bedside

application to the field of neonatal cardiology. This pilot study in healthy term infants serves as a

derivation cohort that now needs to be followed up with similar comprehensive and systematic

efforts to validate these markers by relating them to patient outcomes in different disease states.

Although it may be premature at this stage to provide categorical recommendations based on a

pre-defined cut-off, it may be feasible to incorporate some, if not all of these parameters, along

with qualitative assessment, for longitudinal monitoring. However, it should be recognized that

similar to other techniques, the measurement bias and clinical utilization of these methods is

likely to vary with image quality and operator experience. The specific threshold of imaging

experience for functional echocardiographic measurements is beyond the scope of this study, but

we recommend that prior to using these techniques in clinical practice, individual practitioners

should, review the emerging image acquisition and data analysis protocols, become aware of the

measurement bias for different indices in their own practice, and discuss the proper ways to

incorporate this knowledge while interpreting results. Overall, we report that the majority of LV

functional indices had acceptable reproducibility with ICC > 0.85, relatively narrow LOA on BA

analysis, and COV < 15%. These results were comparable to those of older children, as well as

that of the right ventricle previously reported by our group in the same population (190, 191).

Future studies should evaluate the role of these techniques, in early identification of pathological

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deviations to obtain novel insights into the disease mechanism and progression, and to measure

the impact of common therapies, such as inotropes.

Interest in the use of TDI and STE for the assessment of regional and global LV function in

neonates is increasing (192). A recent review of left ventricular systolic strain and strain rate

measurement in infants and children has highlighted the lack of normative data in term neonates,

particularly during the first few days of age (147). Nevertheless, strain measurements have now

been applied to evaluate LV function in few neonatal conditions (150, 151, 157). Smaller studies

have reported that in comparison to healthy controls, LV GLS is lower in term small for

gestational age infants (-15.9% ± 2.1 vs. -21.3% ± 2.8; n=20 in each group) (151), in infants

undergoing total body hypothermia treatment for severe perinatal asphyxia (-11.01% ± 2.48 vs -

21.45% ± 2.74; n=24 in each group) (150), and in infants born after being exposed to pre-

gestational (-10.4 ± 3.2; n=20) and gestational diabetes (-13.1 ± 4.7; n=25) (157). Strain has also

shown to be negatively correlated with serum troponin levels in cases of severe perinatal

asphyxia (r2=0.64) (150). Although these studies highlight the potential utility of STE in

neonates, systematic studies with an a priori aim of establishing a comprehensive normative

dataset in this specific population is a necessary prerequisite to subsequent higher impact studies

in disease states. The control group data of STE-derived LV GLS presented in above studies

(150, 151, 157), as well as TDI-derived strain reported in previous studies which included

neonates on day 1 of age (153, 154), closely match the values obtained in our study.

In this study, we found that the majority of functional parameters when measured after 12

hours of age remained unchanged over the next two days; with the exception of small reductions

in LV lateral wall and septal s`, mitral valve inflow VTI and LV basal diameter. Although it is

not clear whether one or a combination of expected physiological changes associated with

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postnatal transition were responsible for these changes, these could be explained by a relative

decrease in LV preload on day 2 of age. There are a number of factors which could explain this,

such as an expected increase in left to right shunt across the PFO secondary to decreasing right

sided pressures; the closure of the PDA on day 2 (observed in the majority of neonates), which

though restrictive on day 1, were contributing to LV preload by virtue of the left to right shunt

pattern (observed in 44% of neonates); and normal postnatal diuresis, as evident by the observed

weight loss on day 2. Tissue Doppler systolic velocities have previously been reported to be

influenced by changes in preload (193), while diastolic function assessment using TDI is

considered to be relatively less load dependent, which was also observed in this study (194).

In conclusion, we demonstrated the feasibility and describe the relative reliability and

normal values for LV functional parameters in heathy neonates during the first two days of life.

We also confirm the validity of data generated after 12 hours of age, by confirming the lack of

significant impact of further physiological transitional circulatory changes on these

measurements. We propose that LV function assessment in neonates should be based on a

comprehensive protocol which, in addition to traditionally employed parameters of FS and EF,

should also include TDI, STE derived longitudinal strain and flow derived hemodynamic

variables. Our results suggest that TDI derived myocardial systolic velocity (relatively higher

dependency on preload) and STE derived strain rate (high variability and low reliability), should

be used with caution in neonates during the first few days of age. Clinicians involved in

performing functional echocardiograms in neonates should focus on consolidating their

experience in measuring quantitative indices, be aware of the measurement bias for various

indices in their own practice and carefully consider such bias while interpreting longitudinal

changes over time or response to treatments.

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Chapter 6: Cardiopulmonary adaptation during the first day of life in

humans*

*Draft of manuscript to be submitted to Circulation.

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6.1 Introduction

The transition from intrauterine to extrauterine life represents a critical phase of

physiological adaptation, which impacts many organ systems, most notably the heart and the

lungs. The majority of neonates complete this transition without complications; however,

dysregulation of normal postnatal adaptation may lead to acute cardiopulmonary instability

necessitating advanced intensive care support (33). In some situations, death or adverse

neurosensory impairment may ensue (34, 195). The approach to cardiovascular care in these

patients is limited by the lack of reliable or readily available physiologic measures of

cardiopulmonary health and suboptimal thresholds for therapeutic intervention, driven in part by

a paucity of information regarding physiological cardiac adaptation after birth and the interaction

with changes in pulmonary circulation.

Animal experiments have demonstrated a progressive fall in PVR over the first 48-72

hours after birth in response to lung recruitment and increased alveolar oxygen concentration

(196). As PVR falls, the direction of flow across the ductus arteriosus (DA) and foramen ovale

(FO) becomes increasingly left-to-right. This is soon followed by the closure of the DA in most,

ductus venosus in many and lastly FO. These changes and the specific relationship with time

after birth have been poorly documented in human neonates. The impact of the transitional

period on myocardial function and cardiac output is also not well understood.

We have recently published normative data for echocardiography derived indices of RV

and LV ventricular function, pulmonary hemodynamics and shunt characteristics on day one and

two of age for a cohort of 50 healthy term neonates (197, 198). We demonstrated that PVR

reduced over the first two days of age, while cardiac function indices remained unaffected.

However, in those studies we did not evaluate infants within the first 12 hours of life, when the

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transitional circulatory changes are expected to be highest. The objective of this study was to

delineate, in a time sensitive manner, the natural history of PVR, cardiac output, myocardial

function and transitional shunts over the first 24 hours following birth.

6.2 Additional methods

Inclusion and exclusion criteria: Women with singleton, low risk, uncomplicated pregnancies

who presented at Mount Sinai Hospital during the study period in labor, between 37 and 42

completed weeks of gestation and had no evidence of intra-uterine growth restriction, placental

dysfunction or fetal distress were approached for consent. Our exclusion criteria were based on

the conditions which could potentially impact physiological postnatal adaptation and included

maternal conditions such as diabetes, hypertension, use of antidepressant medications, abnormal

results of the first trimester screen or integrated perinatal screen, any congenital anomaly

identified in the fetus on an anatomy ultrasound, known genetic anomaly, abnormal Doppler

profile in the umbilical artery on the most recent antenatal ultrasound and clinical suspicion of

chorioamnionitis. Further, among recruited neonates, we excluded infants after birth if their

umbilical arterial or venous cord pH was < 7.0, if they had an Apgar score of ≤ 5 at 5 minutes of

age, required any active resuscitation other than drying and stimulation, were noted to have

dysmorphic features or if required admission to the neonatal intensive care unit. In addition, we

excluded any infant with a congenital heart defect on study echocardiogram other than a PDA

and/or PFO.

Exposure: A total of 15 neonates were prospectively recruited and completed the study

procedure. This was a pre-defined sample size of convenience. Expectant parents of prospective

eligible neonates were approached for consent during labour and the consenting process was

completed before delivery. Each recruited neonate underwent four sequential echocardiograms at

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the following time points: < 0.5 hours, 2 to 3 hours, 7 to 10 hours and 22 to 24 hours of life. For

the first echocardiogram, once consent was obtained, the ultrasound machine was set up inside

the labour room next to the infant warmer. Following delivery and a brief period of skin to skin

care with their mothers, all recruited neonates were brought to the warmer, dried, covered by

warm towels and had neonatal electrocardiograph electrodes applied before undergoing the first

study echocardiogram. This was achieved in all 15 cases at < 30 mins of age. Subsequent scans

were performed at the bedside in the Mother and Baby unit at the pre-specified postnatal age as

above. It was ensured that infants were in a non-agitated quiet state at the time of scans and

simultaneous electrocardiograph recording were also obtained. Each study echocardiogram was

< 20 mins in duration and consisted of recording images in RAW DICOM data format for offline

measurements. All 60 scans performed for this study were digitally stored and analyzed off-line

in a random order. Measures of PVR, ventricular outputs and shunt characteristics were obtained

at each time point. Functional parameters, however, were only obtained at the first and the last

scan. This was done to minimize scanning load on study participants and the specific time points

were chosen based on the rationale that the changes in myocardial loading conditions were

expected to be least at the first and highest at the last scan.

In addition to the measures of PVR mentioned above, for this study we also calculated

PAAT and RVET indexed to the total duration of cardiac cycle, expressed in percentage (PAATi

and RVETi respectively). This was done because PAAT and RVET demonstrated a strong

negative correlation with heart rate, which incidentally changed significantly over the study

period. The following list of echocardiographic indices were employed in this study.

Table 6.1: List of echocardiography derived indices employed in this study

Pulmonary vascular resistance

• Pulmonary artery acceleration time (PAAT) • Pulmonary vascular resistance index (PVRI) = Right ventricular ejection time

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(RVET)/ PAAT • PAAT indexed to total duration of cardiac cycle and expressed as percentage

(PAATi) Pulmonary blood flow

• Right ventricular stoke volume (RVSV) • Right ventricular output (RVO)

Systemic blood flow

• Left ventricular stoke volume (LVSV) • Left ventricular output (LVO)

Shunts • Patent ductus arteriosus (PDA) • Patent foramen ovale (PFO)

Right ventricular (RV) function

Systolic function • Tricuspid annular plane systolic excursion (TAPSE) • Fractional area change measured from RV-focused apical 4 chamber view (FAC-4C) • Fractional area change measured from RV-focused apical 3 chamber view (FAC-3C) • Tissue Doppler derived peak systolic myocardial velocity measured on RV lateral

wall just below the tricuspid annulus (RV s’) • Peak longitudinal strain measured using speckle tracking echocardiography from the

RV lateral wall (GLS-4C) and inferior wall (GLS-3C) Diastolic function

• Isovolumic relaxation time measured using tissue Doppler imaging (IVRT’) • Ratio of tricuspid valve early (TvE) and late (TvA) inflow velocities

Left ventricular (LV) function

Systolic function • Ejection fraction (EF) measured using Simpson’s biplane method • Tissue Doppler derived peak systolic myocardial velocity measured on LV lateral

wall just below the mitral annulus (LV s’) • Average of the peak longitudinal strain measured using speckle tracking

echocardiography from LV-focused apical 4, 3 and 2 chamber views (LV GLS) Diastolic function

• Isovolumic relaxation time measured using tissue Doppler imaging (IVRT’) • Ratio of mitral valve early (MvE) and late (MvA) inflow velocities

Global heart function (calculated for both ventricles)

• Myocardial performance index measured using tissue Doppler imaging [MPI’ = (IVRT’ + isovolumic contraction time)/duration of systole]

• Ratio of total duration of systole and diastole measured using tissue Doppler imaging (systole’: diastole’)

Measures of cardiac function were measured at 1st scan performed at < 0.5 hours of age and again at 4th scan performed between 22-24 hours of age. Other parameters were measured at each scan time.

Subsequent confirmation of reproducibility of results involved performing a qualitative,

time-specific, post-hoc comparison of parameter values with those obtained in a previous

prospective observational study conducted at our institute (197, 198). It was important to confirm

the representativeness of obtained measures in another larger validation cohort as our primary

derivation cohort consisted of a small number of neonates.

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Additional statistical considerations: The association between heart rate and indices of PVR was

analyzed using a linear model [outcome= a + b HR +e, where b (estimate) is the coefficient of

HR and the autoregressive covariance (e) structure was used to account for the repeated

measures]. We confirmed the resolution of association with heart rate for PAAT after being

converted to PAATi, as described above (Table 6.2). However, RVETi continued to show

association with heart rate. For validation, values from the D2 scans were compared with D1

scans using paired Student’s t-test or Fisher’s exact tests, as appropriate.

Table 6.2: Association between indexes of PVR and heart rate

Variable Estimate (sdr) p value

PAAT (msec) -0.22 (0.05) <.0001 RVET (msec) -0.993 (0.10) <.0001 PAATi (%) 0.017 (0.11) .105 RVETi (%) 0.08 (0.022) .001 PVRI, mean (SD) -0.0012 (0.005) .820 PAAT pulmonary artery acceleration time; RVET right ventricular ejection time; PAATi and RVETi PAAT and RVET expressed as percentage of total duration of cardiac cycle respectively; PVRI pulmonary vascular resistance index.

6.3 Results

We studied 15 neonates (9 males), born at a mean (SD) gestation and BW of 40 ± 0.8 weeks and

3.5 ± 0.5 Kg respectively. All but three were delivered vaginally. The median (IQR) Apgar score

at 5 minutes of age was 9 (9, 9) and mean cord pH was 7.2 ± 0.1. There were significant changes

observed in the indices of PVR and blood flow during the first 24 hours of life (Table 6.3). Heart

rate was highest at first scan but came down by the time of 2nd scan; then remained unchanged on

subsequent evaluations.

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Table 6.3: Sequential measurements of echocardiographic indexes of pulmonary vascular resistance and pulmonary and systemic blood flow demonstrating transitional circulatory changes during the first day of life in healthy human neonates

Scan 1 Scan 2 Scan 3 Scan 4 Age (hrs) 0.4 ± 0.1 hrs 2.7 ± 0.2 hrs 8.2 ± 0.6 hrs 22.7 ± 0.7 hrs p PAAT (msec) 33 ± 4 35 ± 4 42 ± 5*¶ 55 ± 7* ¶† <.001 RVET (msec) 166 ± 21 186 ± 21* 208 ± 13* ¶ 216 ± 18* ¶ <.001 PAATi (%) 8.4 ± 1.1 7.4 ± 1.4* 8.1 ± 1.3 10.7 ± 1.4*¶† <.001 PVRI (RVET:PAAT) 5.1 ± .8 5.3 ± .8 5.0 ± .6 4.0 ± .5* ¶† <.001 PA VTI (cms) 8.3 ± 1.4 9.6 ± 2.0* 10.1 ± 1.4* 11.6 ± 1.6 * ¶† <.001 RVSV (mls) 5.3 ± 1.7 6.0 ± 1.7* 6.3 ± 1.4* 7.2 ± 1.3* ¶† <.001

RVSV (mls/Kg) 1.6 ± .6 1.8 ± .6* 1.9 ± .6* 2.1 ± .6* ¶† <.001

RVO (mls/kg/min) 235 ± 87 222 ± 78 218 ± 76 250 ± 78 .043

Ao VTI (cms) 7.5 ± 1.6 9.5 ± 2.2* 10.3 ± 1.9* 10.9 ± 1.7* <.001 LVSV (mls) 3.3 ± .8 3.9 ± 1.1* 4.2 ± .9* 4.2 ± 1.1* <.001 LVSV (mls/Kg) 0.9 ± .2 1.1 ± .2 1.2 ± .2* 1.2 ± .3* <.001 LVO (mls/kg/min) 145 ± 35 138 ± 31 140 ± 34 143 ± 43 NS RVO:LVO 1.7 (1.2, 1.9) 1.7 (1.2, 1.9) 1.5 (1.1, 1.8) 1.7 (1.3, 2.1)† .028 HR (beats/min) 144 (141, 172) 124 (114, 133)* 115 (104, 124)* 113 (107, 129)* <.001

Analysis performed using one way repeat measure ANOVA. Results presented as mean ± SD or median (IQR). * p<.05 vs. baseline, ¶p<.05 vs. 2nd scan, †p<.05 vs. 3rd scan. PAAT pulmonary artery acceleration time; RVET right ventricular ejection time; PAATi and RVETi PAAT and RVET indexed to total duration of cardiac cycle and expressed as percentage respectively; PVRI pulmonary vascular resistance index; RVSV right ventricular stoke volume expressed both in mls as absolute number and mls/kg when index to infant’s body weight; RVO right ventricular output; LVSV left ventricular stoke volume expressed both in mls as absolute number and mls/kg when index to infants body weight; LVO left ventricular output; HR heart rate. NS = p>.10.

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Indices of PVR: Despite the reduction in heart rate, PAAT remained similar on initial two scans;

subsequently it increased for the rest of the study period. PVRI, however, only demonstrated a

reduction on the last scan. This was in relation to changes in RVET, which continued to increase

over the first 3 scans before stabilizing. PAATi, on the other hand, demonstrated a reduction on

the 2nd scan between 2 to 4 hours of life, before showing a progressive increase for rest of the

study period (Figure 6.1). RVETi also decreased on 2nd scan before returning to baseline values.

Gross sequential changes were also noticeable in the pulmonary artery pulse wave Doppler

contour, with mid or late systolic notching being a frequent finding till 4 hours of life Figure 6.2.

Figure 6.1: Changes in echocardiographic measures of pulmonary vascular resistance (PVR) during the first day of life in healthy human neonates. While PAAT started increasing from 3rd scan onwards (A), PVRI only demonstrated changes on 4th scan (B). This discrepancy was due to a simultaneous increase in RVET during the first 8 hours of age (C). Indexing PAAT to total duration of cardiac cycle (PAATi) made it independent of heart rate and RVET. PAATi showed a reduction at 2nd scan compared to 1st scan, before demonstrating a consistent increase for the rest of the study period. Analysis was performed using one way repeat measure ANOVA. The hollow triangles represent mean values while vertical lines represent 95% confidence intervals observed at each scan. * p<.05 vs. baseline, ¶p<.05 vs. 2nd scan, †p<.05 vs. 3rd scan. PAAT pulmonary artery acceleration time; RVET right ventricular ejection time; PVRI pulmonary vascular resistance index.

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Figure 6.2: A sequentially obtained pulse wave Doppler trace of the main pulmonary artery from one of the study infants, showing typical changes observed during the first 24 hours of age in healthy neonates following successful postnatal transition. A simultaneous increment in right ventricular ejection time (RVET) resulted in an unchanged pulmonary vascular resistance index (PVRI = RVET/PAAT) on the first three scans. Mid- and/or late-systolic notching was a frequent finding on first two scans.

Indices of cardiac outputs: Overall, changes in indices of cardiac outputs were relatively small

over the study period. Measures of RV stroke volume (PA VTI, RVSV) demonstrated an

increase between 1st and 2nd and again between 3rd and 4th scan, while LV stroke volume

parameters (Ao VTI, LVSV) increased only between 1st and 2nd scan. The right and left

ventricular output per minute and their ratio, however, remained unchanged during the first 24

hours of life.

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Natural history of Transitional Shunts (Table 6.4): Transductal shunt was bidirectional in all

patients on the first scan with a maximum duration of right to left shunting being 40% of the total

shunt duration. The proportion of infants with bidirectional shunts, as well as duration of right to

left shunting in bidirectional shunts reduced over the study period. Cessation in transductal flow

was seen in 67% patients by the time of the last scan, with most closing between 7 and 24 hours

of age. The predominant demonstrable flow across the PFO was left to right in most patients at

each time point, the proportion of which increased with time over the study period.

Table 6.4: Natural history and flow characteristics of patent ductus arteriosus (PDA) and patent foramen ovale (PFO) during first day of life in healthy human neonates

Scan 1 Scan 2 Scan 3 Scan 4 Age (hrs) 0.4 ± 0.1 hrs 2.7 ± 0.2 hrs 8.2 ± 0.6 hrs 22.7 ± 0.7 hrs PDA Closed 0 0 3 (20%) 10 (67%) Small restrictive with left to right shunt

0 3 (20%) 7 (47%) 5 (33%)

Bidirectional shunt 15 (100%) 5 (33%) 1 (7%) 0 Growing shunt# 0 7 (47%) 4 (27%) 0 Percentage duration of right to left shunt in bidirectional shunts* [mean(SD; range)]

32 (5; 22-40)% 24 (5; 17-30) % 23% (only one bidirectional

shunt)

-

PFO No flow visualized 5 (33%) 4 (26%) 2 (13%) 2 (13%) Right to left shunt 1 (6%) 0 0 0 Bidirectional shunt 1 (6%) 1 (6%) 1 (6%) 0 Left to right shunt 8 (53%) 10 (67%) 12 (80%) 13 (87%) #Growing shunt was defined as shunt pattern which is almost entirely left to right but had a small right to left component at end-diastole. *= (duration of right to left shunt/total shunt duration) x100. Results presented as number (percentage). No patient had unrestrictive left to right shunt across patent ductus arteriosus. SD standard deviation.

Indices of Myocardial Performance (Table 6.5): In comparison to the 1st scan, significant

increase was observed by the 4th scan time in indices of RV systolic (TAPSE, FAC-4C, GLS-3C)

and global (systole’: diastole’) function; with least overlap seen in values of TAPSE and GLS-3C

between two time points (Figure 6.3). The GLS-3C was lower in comparison to GLS-4C at first

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scan, but increased to comparable values by end of first day of life (Figure 6.4). There were no

change in indices of LV systolic performance, however, a small but significant rise observed in

diastolic performance (MvE: MvA and systole’: diastole’ ratios).

Table 6.5: Changes in cardiac function in healthy neonates during first the day of life illustrating myocardial adaptation associated with physiological postnatal transition

Scan 1 Scan 4 Mean difference (95% CI)

p

Age 0.4 ± 0.1 hrs 22.7 ± 0.7 hrs - Right Ventricle Systolic function

TAPSE (cm) .72 ± .10 .86 ± .09 - .14 (-.18, -.10) <.001 FAC-4C (%) 18.2 ± 6.8 29.1 ± 14.3 -10.9 (-20.3, -1.5) .027 FAC-3C (%) 33.7 ± 7.9 37.6 ± 5.2 -3.9 (-8.6, .8) .097 RV-s’ (cm/s) 6.7 ± 1.3 6.2 ± .8 .5 (.3, -.03) NS GLS-4C (%) 20.6 ± 3.8 22.7 ± 3.8 -2.0 (-4.4, .4) .089 GLS-3C (%) 17.0 ± 2.5 22.0 ± 2.7 -5.0 (-7.3, -2.6) .001

Diastolic function

IVRT’ (ms) 61 ± 17 51 ± 13 10 (-1, 20) .069 TvE: TvA 1.02 ± 0.54 0.77 ± 0.16 0.26 (-0.12, 0.66) NS

Global function

MPI’ .57 ± .21 .46 ± .16 .12 (-.01, .25) .074 Systole’: diastole’ 1.33 ± .19 1.0 ± .20 .33 (.16, .49) .001

Left Ventricle Systolic function

Ejection fraction (%) 65 ± 7 65 ± 8 -.3 (-4.7, 4.2) NS LV-s’ (cm/s) 5.3 ±1.0 5.2 ± .9 .1 (-.5, 0.7) NS LV-GLS (%) 21.7 ± 2.4 22.1 ± 2.1 -.4 (-2.2, 1.4) NS

Diastolic function

IVRT’ (ms) 48 ± 13 52 ± 7 -4 (-10, 3) NS MvE: MvA .97 ± .25 1.23 ± .30 -.26 (-.48, -.03) .028

Global function

MPI’ .60 ± .16 .61 ± .17 -.01 (-.12, .10) NS Systole’: diastole’ 1.27 ± .22 .97 ± .28 .30 (.13, .46) .002

Analysis performed using paired T-test. Results presented as mean ± SD or median (IQR). (‘) indicates that parameter was measured using tissue Doppler imaging. TAPSE tricuspid annular plane systolic excursion; FAC fractional area change measured from right ventricular (RV) focused apical-4-chamber view (FAC-4C) and RV apical-3-chamber view (FAC-3C); s’ peak systolic myocardial velocity measured from the RV lateral wall just below the tricuspid valve annulus for the right ventricle and from the left ventricular (LV) lateral wall just below the mitral valve annulus for the left ventricle; GLS peak longitudinal strain measured using speckle tracking echocardiography; for the right ventricle GLS was measured from the lateral wall (GLS-4C) and inferior wall (GLS-3C), while for the left ventricle GLS was measured from LV-focused apical 4, 3 and 2 chamber views and then averaged to obtain LV-GLS-global; IVRT’ isovolumic relaxation time; TvE and TvA tricuspid valve early and late inflow velocities respectively; MPI’ myocardial performance index [= (IVRT’ + isovolumic contraction time)/ejection time]; MvE and MvA mitral valve early and late inflow velocities respectively; 95% CI 95% confidence intervals. NS = p>.10.

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Figure 6.3: Box plot graphs of key echocardiographic measures of right ventricular (RV) systolic function at <0.5 hours and 22-24 hours of life in healthy human neonates following successful postnatal transition. Among the tested variables, TAPSE and GLS-3C demonstrated maximum change between the two time points. Analysis was performed using paired t-test. TAPSE tricuspid annular plane systolic excursion; FAC-4C fractional area change measured from RV focused apical-4-chamber view; GLS peak longitudinal strain measured using speckle tracking echocardiography from the RV lateral wall (GLS-4C) and RV inferior wall (GLS-3C).

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Figure 6.4: Comparison of peak global longitudinal strain measured using speckle tracking echocardiography from right ventricular lateral wall (GLS-4C) and inferior wall (GLS-3C) at < 0.5 hours and 22-24 hours of age in healthy neonates following uncomplicated postnatal transition. Note that while GLS-4C remained similar at two time points, GLS-3C was significantly lower shortly after birth but increase to values similar to GLS-4C by the end of first day of life.

Validation of measured parameters: The validation cohort consisted of 50 healthy newborns who

underwent sequential echocardiograms at 15 ± 2 and 36 ± 2 hours of age for a previous study.

The mean gestational age and birth weight was 39.3 ± 1.2 weeks and 3.5 ± 0.5 Kg respectively.

Longitudinal changes were observed between two time points in the same parameters as for the

derivation cohort; LVSV and LVO did not change (Table 6.6). Qualitatively, the values obtained

from this historic cohort closely fitted with the trajectory of changes observed in the derivation

cohort in an age-specific manner.

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Table 6.6: Measurements of echocardiographic indexes of pulmonary hemodymanics, pulmonary and systemic blood flow and characteristics of patent ductus arteriosus (PDA) and patent foramen ovale (PFO) obtained from a cohort of 50 healthy term neonates who underwent serial echocardiograms on day 1 and 2 of age for a previous prospective observational study.

Age 15 ± 2 hrs 35 ± 2 hrs p

PAAT (ms) 50 ± 17 59 ±15 <.001

RVET (ms) 216 ± 24 216 ± 23 NS

PAATi (%) 9.6 ± 2.8 12.0 ± 2.7 <.001

PVRI (RVET:PAAT) 4.8 ± 1.3 3.8 ± .8 <.001

RVSV (mls/kg) 2.1 ± .6 2.5 ± .8 <.001

RVO (ml/kg/min) 246 ± 66 307 ± 90 <.001

LVSV (mls/kg) 1.09 ± .20 1.10 ± .23 NS

LVO (mls/kg/min) 128 ± 28 137 ± 28 NS

HR (beats/min) 119 ± 15 124 ± 17 .042

PDA closed, n(%) 28 (56%) 48 (96%) <.001

PFO closed, n(%) 9 (18%) 13 (26%) NS

All PDAs were small, restrictive and shunting left to right. All PFOs were shunting left to right. PAAT pulmonary artery acceleration time; RVET right ventricular ejection time; PAATi and RVETi PAAT and RVET expressed as percentage of total duration of cardiac cycle respectively; PVRI pulmonary vascular resistance index; RVSV right ventricular stoke volume expressed both in mls as absolute number and mls/kg when index to infants body weight; RVO right ventricular output; LVSV left ventricular stoke volume expressed both in mls as absolute number and mls/kg when index to infants body weight; LVO left ventricular output; HR heart rate. NS = p>.10. Note that the data matched closely with the results generated by the study involving sequential echocardiography assessments during first day of age in a time-specific manner supporting the reproducibility of those observations.

Inter-rater reliability of hemodynamic measures:

The inter- and intra-observer measurement variability for functional variables have been

published by our group (197, 198); measurement variability for hemodynamic indices was

calculated for this study (Table 6.7).

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Table 6.7: Intra- and inter-observer variability of indices of pulmonary vascular resistance and blood flow

Variable Intra-observer Variability Inter-observer variability ICC

(95% CI) Mean Bias (95%CI) ICC

(95% CI) Mean Bias (95%CI)

PAAT .90 (.76, .96)

.58 (-11.54, 12.70)

.83 (.55, .94)

4.26 (-10.17, 18.69)

RVET .90 (.76, .96)

-1.05 (-23.49, 31.39)

.78 (.51, .91)

7.05 (-27.06, 41.17)

PA diameter

.93 (.83, .97)

.00 (-.17, .17)

.48 (.06, .74)

-.06 (-.28, .17)

PA VTI .99 (.98, .99)

-.03 (-.67, .60)

.98 (.94, .99)

-.11 (-1.13, .91)

Aortic diameter

.93 (.81, .97)

.01 (-.03, .06)

.87 (.67, .95)

-.01 (-.07, .05)

Aortic VTI

.97 (.93, .99)

.02 (-.71, .75)

.98 (.92, .99)

.15 (-.64, .93)

ICC: Intraclass correlation coefficient; CI: confidence interval; Bias calculated using Bland Altman analysis. p value for all ICC < .001.

6.4 Discussion

The postnatal transition represents a time-period during which neonates are exposed to

sudden and complex changes in cardiopulmonary physiology, resulting in increased cardio-

respiratory vulnerability and risk of complications. Echocardiography is now a routinely

employed investigation for diagnostic and monitoring purposes in neonates suffering from

dysregulation of transitional physiology (199); however, the interpretation of various

echocardiographic indices is challenged by a relative paucity of reliable data in a healthy

population during this time period. The clinical care of these sick neonates is further hampered

by a lack of understanding of normal physiological adaptation in the immediate period after

birth. In this prospective, observational, physiologic study of healthy newborns, we sequentially

measured echocardiographic parameters of PVR, myocardial function, cardiac output and fetal

shunts and describe how these change over the first 24 hours of life. We confirmed that the first

day of life is characterized by significant circulatory changes, consistent with the anticipated

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overall fall in PVR and progressive closure of the DA. In addition, our study has provided

several novel insights in various aspects of transitional physiology in humans as discussed

below.

Indices of Pulmonary Vascular Resistance (PVR): Our results indicate, that in humans, despite a

reduction in the ratio of pulmonary to systemic pressures, as suggested by changes in ductal

shunt pattern, the overall fall in RV afterload may occur at a slower rate until 7-10 hours of life,

after which the rate of fall accelerates. In fact, the net effect of circulatory changes may even be

resulting in a rise in RV afterload over the first 4 hours of life, as suggested by the observed

decrease in PAAT on the second scan after adjustment for heart rate (PAATi). This can be

explained by changes in ductal shunt pattern; specifically, we noted a change from a

bidirectional pattern in all infants at 1st scan, thereby exposing the right ventricle to the

cumulative effect of PVR and SVR, to a predominantly left to right shunt in the majority of

infants on 2nd scan. Previous validation studies for PAAT as a marker for PVR were conducted

in adults and older children (171, 172). Our findings suggest that in newborns, instead of PVR

alone, PAAT may be an estimate of overall RV afterload, which in the presence of an open DA

will also be affected by SVR. We speculate that during early hours of transitional circulation, the

fall in PVR may be negated by the removal of exposure of the right ventricle to systemic

circulation secondary to changes in DA, which may result in a net transient increase in RV

afterload. This, however, will have to be confirmed in future studies as we did not collect

measures of SVR.

Another concern with the use of PAAT in newborns is its strong negative correlation with

heart rate (200), which can impact its clinical utility, particularly for heart rates greater than 100

beats/min (201). Indexing PAAT to cardiac cycle duration resolved this heart rate dependency

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and appears to make it more sensitive to detect changes in neonatal pulmonary circulation.

PAATi, however, is a novel index and need further validation, ideally by comparison with

invasive definitive investigations; though, in neonates correlation with expected physiology or

clinical outcomes may be more feasible. PVRI, although independent of heart rate, was

insensitive to early pulmonary hemodynamic changes; this was due to concurrent increase in

RVET observed on the first 3 scans in this study. RVETi also decreased significantly on 2nd scan,

however, its interpretation is hampered by a residual association with heart rate despite being

indexed to cardiac cycle duration.

Indices of Myocardial Function: One of the novel and interesting finding of our study was the

presence of relatively lower values for RV systolic (TAPSE, FAC-4C, GLS-3C) and global

(systole’: diastole’) functional parameters during the first 30 minutes of life. All indices

increased significantly by 24 hours of age, reaching values comparable to published data

measured at 12 - 18 and 30 - 40 hours of life (197). While these results may indicate load-

dependency of echocardiographic measures, they may also suggest that it is physiological to

have transiently low RV function during the initial hours after birth. The fact that results

obtained from multiple parameters corroborated and their increase coincided with the

physiological reduction in PVR suggest load dependency of the neonatal right ventricle. This

may relate to the known properties of the immature heart, which significantly differs from adults

by having a lower compliance and higher proportion of non-contractile tissue (202). We

hypothesize that, similar to neonatal left ventricle which has been shown to have an increased

susceptibility to afterload (94), neonatal right ventricle may also be inherently vulnerable to

dysfunction on exposure to persistently high afterload conditions. Our observation of lower

GLS-3C on the first scan which increased to values similar to GLS-4C by 24 hours of age

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suggest a regional effect of increased afterload on the right ventricle. Whether GLS-3C is more

sensitive to afterload and hence detect changes earlier than GLS-4C in neonates will have to be

examined in future studies. Contrary to RV function, LV functional indices remained relatively

unchanged over the first 24 hours of life, demonstrating values comparable to published data

(198).

Indices of cardiac output: In this study, we found that although stroke volumes for both

ventricles increased between scans one and two, because of higher heart rate immediately after

birth, cardiac outputs per minute remained unchanged. Although sequential evaluations at similar

time points during transition has not been reported before, studies performed at different time

points on the first day of life have documented RVO and LVO in healthy term neonates (203-

208). Few recent studies conducted using modern imaging equipment and similar measurement

technique have reported values comparable to our study (205-207). Noori et al reported RVO

[range 300-340 ml/min/kg] > LVO [range 165-212 ml/min/kg] when measured sequentially at 2,

5 and 10 min of life in 20 term neonates (205). Popat et al reported mean values of RVO 212

ml/min/kg and LVO 190 ml/min/kg at 4 hours of age from 21 term babies (206). A comparative

evaluation of LVO between neonates born by normal vaginal delivery vs caesarian section

performed by Coskun et al showed no difference between the subgroups, with overall mean LVO

of 350 mls/min and 265 mls/min at 1 and 24 hours of age respectively; values indexed to infant’s

weight were not reported (207). Interestingly, similar to our study, previous reports have

consistently found RVO to be greater than LVO on the first day of life. In a fetal

echocardiography study, Mielke et al also reported a median RVO: LVO ratio of 1.4 from 222

normal human fetuses, which was independent of gestational age (25). We found similar RVO:

LVO ratio on first postnatal day, underlining the persistence of RV dominance during this time.

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Variance between RVO and LVO during the transitional period may be explained by relative

volumes of shunt across the PDA and PFO, neither of which is can be directly quantified by

echocardiography. We hypothesize that the higher RVO: LVO relates to the increased left to

right transatrial shunting, as suggested by earlier closure of PDA but persistence of PFO in the

majority of study infants.

Natural History of Transitional Shunts: In this study, we confirmed the findings of previous

investigators that in healthy term infants, by 24 hours of age, the ductus arteriosus is closed in

the majority of cases and demonstrate restrictive left to right flow pattern shunt in the remaining

before undergoing closure by 40 hours of age (209-212). We, like other recent observations, also

demonstrate a bidirectional ductal shunt in all neonates shortly after birth (205, 213). In addition,

we quantified the percentage bidirectional flow and found it to be ≤ 40% of total shunt duration

in all cases, suggesting pulmonary pressures to be below systemic level by 30 minutes of life

(214). Our results suggest that presence of bidirectional ductal shunt is an unusual occurrence in

healthy neonates after 10-15 hours of age, while PFO shunt may be left to right in the majority

irrespective of age.

Importance of this study: While animal experiments have greatly enhanced our knowledge

regarding the overall transitional physiology (24), data from these may not always be applicable

to humans in the clinical settings due to inter-species variability. For instance, sentinel work by

Rudolph in a canine experimental model showed that birth is followed by a progressive fall in

PVR; however, in the same experiment trajectory of change was reported to be slower in

newborn goats (196). This was in contrast to the experiments in lambs, where PVR changes were

reported to be more dramatic at birth (215). Studies have reported invasive pulmonary artery

pressure measured from healthy human neonates during the initial 72 hours of age (216).

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Although these studies were of questionable methodology, their results suggested a slower fall in

pulmonary pressures in human neonates, particularly during the initial 10-12 hours of age; this is

in line with our non-invasive assessments. With the availability of high fidelity imaging

equipment, as demonstrated in this study, it may now be feasible to acquire physiological data

directly from humans, which in addition to confirming the expected physiology may also provide

novel, species specific insights, as well as provide clinically applicable control data. For instance

recent studies in human fetuses have shown that in comparison to fetal lambs, human fetuses

shunt less percentage of blood via ductus venosus and have a higher ratio of pulmonary blood

flow to cardiac output (25, 217), thus highlighting the feasibility and importance of acquiring

human data.

In conclusions, Comprehensive neonatal echocardiography may be used to elicit time-specific

cardio-pulmonary adaptive changes associated with postnatal transition in human neonates and

may provide quantitative and pragmatic measures of hemodynamic alterations and cardiac

function. The immediate postnatal period in humans is characterized by significant fall in PVR,

however, the trajectory of fall in net RV afterload may be slower during the initial 10 hours of

life. Cardiac outputs remains stable throughout this period, at least in part, due to compensation

of lower stroke volumes during the initial hours after birth by high heart rate. The first 24 hours

of life is also associated with significant increase in RV performance, which temporarily relate to

fall in PVR and ductal closure patterns; we speculate that these adaptive changes may indicate an

inherent vulnerability of the neonatal right ventricle to high afterload conditions. These data lay

the foundation for future studies of disease states associated with an abnormal transition and may

facilitate the identification of thresholds for therapeutic interventions.

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Chapter 7: Cardiac Function and Ventricular Interactions in Persistent

Pulmonary Hypertension of the Newborn*

*Draft of manuscript to be submitted to Circulation Imaging

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7.1 Introduction

Persistent pulmonary hypertension of the newborn is a disorder of postnatal transition,

accounting for a significant proportion of admissions to tertiary NICUs (33, 34). Infants with

PPHN often require advanced and sometimes prolonged cardio-respiratory support and are at

significant risk of mortality and morbidities, which despite widespread use of iNO has remained

unchanged over the past 2 decades (33, 34, 38). Cardiac dysfunction could be a potential

contributor. In older patients with PAH, adaptation of RV function in response to acute or

chronic pressure loading is a major determinant of clinical outcomes (218). Development of RV

dysfunction will result in decreased RVO and unfavourable interventricular interactions with the

left ventricle. A pressure-loaded, dilated right ventricle causes leftward shift of the

interventricular septum; this can result in reduced LV filling, further compromising LVO (37, 99,

185, 219). Whether similar mechanisms are at play in PPHN are unknown.

The neonatal right ventricle may be better poised to compensate for increased RV afterload

related to the physiologic neonatal RV hypertrophy, making unfavourable LV-RV interaction

less likely. On the other hand, however, immaturity of the neonatal myocardium, which is

characterized by high collagen content and lower calcium handling capacity, may make RV

function more load dependent (220). Another factor in PPHN is the presence of right to left

shunting of blood via intra- and extra-cardiac shunts. The persistence of ductus ateriosus may

offload the right ventricle in patients with PPHN but at the expense of pulmonary blood flow,

LV preload and systemic oxygen saturation. A right to left shunt across foramen ovale may

contribute to LV preload and LVO but will result in lower RVO and pulmonary blood flow.

We have recently described normative data for various indices of RV and LV size and

function in a representative cohort of healthy term neonates for the first two days of age by using

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a comprehensive echocardiographic imaging protocol (197, 198). This protocol is now the

clinical standard in our institution. In the present study, our aim was to identify

echocardiographic parameters of biventricular size, function, and hemodynamics in neonates

presenting with PPHN during the first 3 days of life using the imaging protocol as previously

described (197, 198). We also examined the relationship between key echocardiographic

findings and adverse clinical outcomes. We hypothesized that PPHN would be associated with

RV systolic and diastolic dysfunction.

7.2 Additional methods

This was a retrospective cohort study primarily aimed at describing the echocardiography

correlates and investigate inter-ventricular interaction in neonates with PPHN.

Inclusion and exclusion criteria: This study included infants born at a gestational age ≥ 35

weeks, with a birth weight ≥ 2500 grams and admitted with a diagnosis of PPHN to the NICU of

the Hospital for Sick Children, Toronto, over a 3 year period (2012 to 2015). Eligible infants

were identified by screening the listed diagnoses in the unit’s admission logbook and the

electronic echocardiography database during the study period. The identified infants had to

satisfy the following criteria for inclusion in the study: need for respiratory support with a

fraction of inspired oxygen of ≥ 0.40; confirmed echocardiography diagnosis of PPHN [defined

as presence of bidirectional or right to left shunting across the patent ductus arteriosus (PDA)

and/or patent foramen ovale (PFO), and/or a tricuspid regurgitant (TR) jet with an estimated

right ventricular systolic pressure (RVsP) greater than 1/2 systemic systolic pressure beyond 12

hours of age]; availability of a functional echocardiogram performed using a comprehensive

imaging protocol as previously described above and stored as raw data to allow detailed offline

analysis; absence of congenital heart defect with the exception of a PDA, PFO or small

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ventricular septal defect; absence of major developmental lung disorders such as congenital

diaphragmatic hernia, large abdominal wall defects, renal defects with oligohydramnios; absence

of known chromosomal anomaly. Clinical data collection: We collected the following clinical

details: gestation, birthweight, cord pH, cardiopulmonary resuscitation (CPR) at birth, 5 minute

Apgar score, use of surfactant, inhaled nitric oxide (iNO) treatment and duration, duration of

invasive ventilation and oxygen therapy, inotrope use, length of hospital stay and need for

extracorporeal membrane oxygenation (ECMO) or death.

Control group: From the echocardiographic data collected on day 1 and 2 of age in 50 well term

neonates for studies described in chapter 4 and chapter 5 (197, 198), the control comparative

group of 50 neonates was constituted by pooling data from 25 day 1 and 25 day 2 scans at

random while including only one scan per infant.

Exposure: Presence of PPHN on a comprehensive echocardiogram during the first 3 days of age.

Additional statistical considerations: Receiver operating curve analysis was performed to

investigate the utility of key echocardiographic indices (p value < 0.1 on univariate analysis) to

predict the composite outcome of death before discharge or need ECMO. Sensitivity, specificity,

positive and negative predictive values were computed where appropriate.

Sample size calculation: The mean ± SD of TAPSE in the control group was used to determine

the sample size for this study. To detect a 10% deference in mean TAPSE among PPHN

neonates, with an alpha of 0.05 and power of 90%, we needed 42 neonates in each group. Hence,

our aim was to include all eligible neonates during the study period based on above mentioned

eligibility criteria with the minimum of 42 PPHN neonates.

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7.3 Results

Study population

Forty nine infants with PPHN were included in the study. Of these 38 were diagnosed

based on PDA shunt (6 right to left, 32 bidirectional), 5 based on bidirectional PFO shunt and the

remaining 6 using RVSP calculated from tricuspid regurgitant jet; which was overall measurable

in 37 (76%) cases. Cases were compared to 50 healthy controls, none of whom had tricuspid

regurgitation or right to left or bidirectional shunting across the PDA if present (20%). Table 7.1

outlines demographic features and age at scan, which were similar between groups, and clinical

features of interest for the study cohort. Cases demonstrated high RVSP and commonly required

advanced cardio-respiratory therapies including high frequency ventilation, iNO and inotropes.

Table 7.1: Baseline demographics of neonates with PPHN and controls, and clinical outcomes of neonates with

PPHN

PPHN (n= 49 ) Controls (n= 50) p value

Gestation (Weeks) 39.3 ± 1.7 39.7 ± 1.20 .18

Birth Weight (Kg) 3.37 ± 0.56 3.50 ± 0.44 .22

Male gender 25 (51%) 33 (66%) .16

Age at scan (days) 1.7 ± 0.8 1.5 ± 0.5 .22

Heart rate (Beats/min) 131 ± 17 122 ± 17 .02

RVSP (mmHg) 43 [35 – 55] - -

5 Minute Apgar Score 6 [4 – 9] 9 [9 – 9] <.01

CPR at Delivery 12 (25%) 0 (0%) -

Surfactant use 9 (18%) 0 (0%) -

Cord pH 7.11 [6.97 – 7.22] 7.24 [7.17 – 7.27] <.01

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Primary diagnosis

Parenchymal lung disease

(meconium aspiration/ pneumonia)

Idiopathic

Hypoxic ischemic encephalopathy

30 (61%)

13 (27%)

6 (12%)

Total body hypothermia for hypoxic ischemic encephalopathy

19 (39%)

Invasive ventilation 47 (96%)

High frequency ventilation 24 (49%)

iNO Treatment 35 (71%)

Positive response to iNO 27/35 (77%)

Inotrope Use 43 (88%)

ECMO or Death 6 (12%)

iNO Duration (Days)* Ω 2 [1 – 10]

Days of Invasive Ventilation Ω 6 [4 – 10]

Day of Oxygen Use Ω 9 [4 – 16]

Length of Hospital Stay Ω 14 [9 – 21]

Values are presented as mean ± Standard Deviation, median [interquartile range], and count (%). *presented as median [Range]. Ωonly for neonates who survived without ECMO. None of the controls had a significant tricuspid regurgitant jet to measure RVSP. PPHN persistent pulmonary hypertension of the newborn; PAAT pulmonary artery acceleration time; RVET right ventricular ejection time; PVRI Pulmonary vascular resistance index; RVSP Right ventricular peak systolic pressure; CPR cardiopulmonary resuscitation; iNO inhaled nitric oxide; ECMO extracorporeal membrane oxygenation.

Right and Left Ventricular Function and inter-ventricular interactions

In comparison to controls, PPHN neonates demonstrated differences in several indices of

RV and LV function; with more changes seen in RV indices (Table 2 and 3). To evaluate the

confounding effect of a global perinatal ischemic insult, inter-group comparison was also

performed after exclusion of neonates diagnosed with hypoxic ischemic encephalopathy; this did

not alter the results significantly. The right ventricle in PPHN showed a small but significant

increase in linear dimensions (basal diameter in long axis view and distal diameter of the outflow

tract), but RV areas measured in the apical 4-chamber view were not different. RV areas

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measured from apical 3-chamber view, pulmonary artery diameter as well as LV volume were

lower in PPHN. Both the right and the left ventricle in PPHN neonates demonstrated inferior

systolic, diastolic and global performance in comparison to healthy controls.

Table 7.2: Echocardiographic indices of right ventricular (RV) function in infants with PPHN vs. Controls

Variable A. PPHN (all patients, n= 49 )

B. PPHN (without HIE,

n=30)

C. Controls (n= 50)

p value

A vs. C

p value B vs. C

RV Dimensions Basal Diameter – 4C (cm)

1.75 ± 0.29 1.69 ± 0.28 1.75 ± 0.21 .99 .99

Basal Diameter – Long axis (cm)

2.43 ± 0.28 2.43 ± 0.32 2.14 ± 0.25 <.01 <.01

Outflow Tract – Distal (cm)

1.29 ± 0.22 1.27 ± 0.19 1.03 ± 0.10 <.01 <.01

Pulmonary Artery Diameter (cm)

0.84 ± 0.12 0.82 ± 0.12 0.94 ± 0.09 <.01 <.01

End-Diastolic Area – 4C (cm2)

4.28 ± 1.12 4.09 ± 1.11 4.27 ± 0.69 0.99 0.42

End-Systolic Area – 4C (cm2)

3.40 ± 1.06 3.23 ± 1.11 3.20 ± 0.60 0.25 0.66

End-Diastolic Area – 3C (cm2)

5.46 ± 1.56 5.20 ± 1.27 6.57 ± 1.07 <.01 <.01

End-Systolic Area – 3C (cm2)

3.52 ± 1.08 3.25 ± 0.94 4.07 ± 0.67 .01 <.01

RV-Specific Functional Measurements FAC – 4C (%) 20.80 ± 10.74 19.94 ± 11.40 25.26 ± 6.99 .02 .03 FAC – 3C (%) 35.53 ± 9.30 37.5 ± 9.95 37.90 ±6.48 .21 .87 FAC– Global (%) [=(FAC-4C + FAC-3C)/2]

29.64 ± 7.66 29.81 ± 8.52 31.67 ± 4.62 .18 .35

TAPSE (mm) 6.81 ± 1.92 7.05 ± 1.71 9.25 ± 1.30 <.01 <.01 Tissue Doppler Imaging (TDI) Measurements s’ (cm/s) 5.81 ± 1.68 6.12 ± 1.52 6.62 ± 1.14 .01 .15 e’ (cm/s) 5.71 ± 1.80 6.17 ± 2.02 7.70 ± 1.68 <.01 <.01 a’ (cm/s) 6.44 ± 1.98 6.50 ± 2.17 8.92 ± 2.51 <.01 <.01 Systolic Time (ms) 185 ± 34 182 ± 24 212 ± 25 <.01 <.01

Diastolic Time (ms) 175 ± 42 175 ± 45 203 ± 47 <.01 .01 IVRT (ms) 65 ± 22 58 ± 19 46 ± 12 <.01 <.01 e’: a’ 0.94 ± 0.30 1.00 ± 0.32 0.95 ± 0.24 .83 .52 TvE: e’ 8.51 ± 2.93 7.84 ± 2.47 6.07 ± 1.42 <.01 <.01 Systole’: Diastole’ 1.10 ± .27 1.09 ± 0.25 1.10 ± 0.30 .97 .86 Myocardial Performance Index

0.65 ± 0.37 0.59 ± 0.21 0.41 ± 0.14 <.01 <.01

Pulsed Wave Doppler and Output TvE (cm/s) 46 ± 11 44 ± 10 45 ± 9 .70 .83 TvA (cm/s) 53 ± 12 53 ± 11 55 ± 12 .29 .49 TvE: TvA 0.90 ± 0.27 0.86 ± 0.28 0.85 ± 0.16 .35 .88 PAAT (ms) 36 ± 10 34 ± 8 54 ± 17 <.01 <.01

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RVET (ms) 175 ± 25 171 ± 17 214 ± 24 <.01 <.01 PVRI (RVET: PAAT) 5.17 ± 1.42 5.20 ± 1.20 4.30 ± 1.00 <.01 <.01 Pulmonary artery VTI cm/s

8.42 ± 2.66 8.11 ± 2.03 11.40 ± 3.00 <.01 <.01

Stroke Volume (ml) 1.46 ± 0.69 1.41 ± 0.59 2.39 ± 0.76 <.01 <.01 Right Ventricular Output (ml/kg/min)

189 ± 87 187 ± 78 291 ± 97 <.01 <.01

Deformation Parameters (STE) GLS – 4C (%) -16.9 ± 5.4 -18.3 ± 5.9 -21.2 ± 5.6 <.01 .04 GLS – 3C (%) -17.7 ± 6.0 -18.8 ± 5.7 -21.8 ± 5.0 <.01 .06 Overall GLS (%) [=(GLS-4C + GLS-3C)/2]

-16.9 ± 5.4 -18.6 ± 5.4 -21.6 ± 4.6 <.01 .04

Data Presented as Mean ± standard Deviation or median (interquartile range). Mean difference (95% confidence interval) only shown for parameters which differed significantly between groups. 4C apical 4-chamber view; 3C apical 3-chamber view; FAC fractional area change; TAPSE tricuspid annular plane systolic excursion; s’/e’/a’/ systolic/early diastolic/late diastolic velocities of basal segment of RV free wall; IVRT Isovolumic relaxation Time; TvE/TvA early/late tricuspid inflow velocities; VTI Velocity Time Index; STE speckle tracking echocardiography; GLS peak global longitudinal strain.

Table 7.3: Echocardiographic indices of left ventricular (LV) function in infants with PPHN vs. Controls Variable A. PPHN

(all patients, n= 49 )

B. PPHN (without HIE,

n=30)

C. Controls (n= 50)

p value

A vs. C

p value

B vs. C LV Dimensions End-Diastolic Diameter (cm)

1.71 ± 0.27 1.73 ± 0.28 1.73 ± 0.24 .73 .97

LV Length – 4C (cm) 3.09 ± 0.34 3.08 ± 0.33 3.09 ± 0.27 .99 .82 End Diastolic Volume – 4C (ml)

4.93 ±1.79 5.15 ±1.79 6.45 ± 1.23 <.01 <.01

Sphericity index (= EDV-4C/Length)

1.57 ± 0.48 1.65 ± 0.47 2.09 ± 0.42 <.01 <.01

LV-Specific Functional Measurements Simpson’s Ejection Fraction – 4C (%)

49 ± 12 51 ± 11 55 ± 9 .01 .10

Simpson’s Ejection Fraction – 2C (%)

49 ± 10 49 ± 12 55 ± 9 <.01 <.04

Simpson’s Ejection Fraction – Biplane (%)

49 ± 7 50 ± 7 55 ± 6 <.01 .01

Shortening Fraction (%) 41 ± 9 41 ± 9 40 ± 8 .49 .46 Tissue Doppler Imaging (TDI) Measurements Lateral Wall s` (cm/s) 5.29 ± 1.62 5.50 ± 1.64 4.79 ± 0.77 .09 .05 e’ (cm/s) 5.23 ± 1.74 5.30 ± 1.65 6.61 ± 1.50 <.01 <.01 a’ (cm/s) 5.67 ± 2.20 5.81 ± 2.39 5.41 ± 1.22 .52 .43 Systolic Time (ms) 162 ± 29 157 ± 22 187 ± 20 <.01 <.01 Diastolic Time (ms) 171 ± 44 174 ± 48 193 ± 35 .01 .09 IVRT’ (ms) 73 ± 23 70 ± 22 52 ± 11 <.01 <.01 e’: a’ 1.02 ± 0.42 1.01 ± 0.35 1.27 ± 0.38 <.01 <.01 MvE: e’ 11.00 ± 5.33 10.52 ± 4.37 9.12 ± 2.98 .06 .83 Systole’: Diastole’ ratio 1.01 ± 0.35 0.96 ± 0.29 1.00 ± 0.21 .82 .65

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Myocardial Performance Index

0.86 ± 0.38 0.82 ± 0.29 0.61 ± 0.16 <.01 <.01

Septal Wall s` (cm/s) 3.85 ± 1.22 4.09 ± 1.22 3.65 ± 0.55 .35 .09 e’ (cm/s) 4.01 ± 1.34 4.10 ± 1.45 4.81 ± 1.08 <.01 .04 a’ (cm/s) 4.47 ± 1.59 4.52 ± 1.72 4.35 ± 0.94 .67 .65 e’: a’ 0.95 ± 0.32 0.97 ± 0.34 1.14 ± 0.39 .01 .05 Pulsed Wave Doppler and Output MvE (cm/s) 51 ± 15 51 ± 13 57 ± 9 .03 .06 MvA (cm/s) 50 ± 17 51 ± 17 52 ± 9 .62 .73 MvE: MvA 1.08 ± 0.39 1.10 ± 0.45 1.11 ± 0.27 .60 .83 Aortic VTI cm/s 10.5 ± 3.25 10.8 ± 2.65 10.07 ± 1.59 .41 .15 Stroke Volume (ml) 1.03 ± 0.44 1.14 ± 0.46 1.10 ± 0.19 .35 .59 Left Ventricular Output (ml/kg/min)

135 ± 61 153 ± 62 134 ± 26 .92 .12

Deformation Parameters (STE) GLS – 4C (%) -15.6 ± 3.9 -16.5 ± 3.5 -21.1 ± 2.4 <.01 <.01 GLS – 3C (%) -16.7 ± 4.5 -18.3 ± 4.2 -20.8 ± 2.9 <.01 .01 GLS – 2C (%) -16.7 ± 3.1 -17.5 ± 3.2 -22.3 ± 3.3 <.01 <.01 Overall GLS (%) [(GLS-4C + GLS-3C + GLS-2C)/3]

-16.7 ± 3.3 -17.8 ± 3.1 -21.4 ± 2.0 <.01 <.01

Data Presented as Mean ± Standard Deviation. Mean difference (95% confidence interval) only shown for parameters which differed significantly between groups. 4C apical 4-chamber view; 2C apical 2-chamber view; s’/e’/a’/ systolic/early diastolic/late diastolic velocities of basal segment of LV free wall and interventricular septum respectively; IVRT Isovolumic relaxation Time; MvE/MvA early/late mitral inflow velocities; VTI: Velocity Time Index; STE speckle tracking echocardiography; GLS peak global longitudinal strain.

The systolic functional indices which demonstrated least overlap in values between groups

were TAPSE for the right ventricle and GLS for both the right and the left ventricle (Figure 7.1

and 7.2). PPHN neonates also had lower RV stroke volume and output, however, LV output was

not different.

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Figure 7.1: Box plot graph showing comparison of tricuspid annular plane systolic excursion (TAPSE) between neonates with persistent pulmonary hypertension of newborn (PPHN) and healthy controls.

Figure 7.2: Box plot graphs for overall global peak longitudinal strain for the right and the left ventricle in neonates with persistent pulmonary hypertension of newborn (PPHN) and healthy controls.

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In neonates with PPHN, moderate but significant inter-ventricular linear correlations

were observed between functional measures of both ventricles; in the whole cohort, as well as

after exclusion of neonates with a diagnosis of HIE (Table 7.4). The strongest correlations were

found between the RV and LV GLS and the RV and LV MPI (Figure 7.3). Functional indices

did not correlate with PAAT or PVRI, while RVSP showed correlation with overall RV-GLS

and FAC-global correlated with the duration of right to left shunt across PDA.

Table 7.4. Pearson’s Product-Moment correlation coefficients for linear correlations between left ventricular (LV) and right ventricular (RV) functional and hemodynamic parameters in patients with PPHN after excluding neonates undergoing total body hypothermia for hypoxic ischemic encephalopathy (n=30)

Septal s’

LV s’

LV-EF

LV-GLS

LV MPI’

LVO LV-IVRT’

SI PVRI RVsP PDA-RL

RVO

RV s’ .49 .44 -.48 .53

FAC-global

.69 .75

TAPSE .68 .62 .45 -.51 .41 .40 .47

RV-GLS .68 .61 .69 .62 .55

RV-MPI’ .62 -.46 .56 -.46 -.58

RVO .45 -.52 .49 -.50 x

RV-IVRT’

.75 -.46 -.49

PDA-RL .79 .48 x

Correlation coefficient (r) are shown if p<0.05. Significant linear correlations were observed between functional and hemodynamic measures of both ventricles. Persistent PPHN pulmonary hypertension of newborn; TAPSE tricuspid annular plane systolic excursion; s’ peak systolic velocity measured at basal segment of RV free wall, LV free wall and interventricular septum respectively; GLS global peak longitudinal strain; FAC fractional area change; IVRT isovolumic relaxation time; MPI myocardial performance index; RVO right ventricular output; LVO left ventricular output; PVRI pulmonary vascular resistance index; RVsP right ventricular peak systolic pressure; PDA R-L duration of right to left shunt across patent ductus arteriosus measured as percentage of total cardiac cycle time; EF ejection fraction (Simpson’s); SI sphericity index (EDV-4C/LV length). There were no significant correlations with pulmonary artery acceleration time or LV end diastolic volume. Note similar analysis when conducted using data from the whole cohort revealed stronger inter-ventricular correlations than above but weaker correlation between functional measures and those indicative of severity of pulmonary pressures (data not shown above).

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Figure 7.3: Among the inter-ventricular linear correlations observed between various functional indices, the strongest correlation was found between right ventricular (RV) and left ventricular (LV) overall global peak longitudinal strain (GLS) and RV and LV myocardial performance index measured using tissue Doppler imaging (MPI’). Correlation coefficient was calculate using Pearson’s product moment correlation.

Correlation with adverse outcome

Comparison of echocardiographic parameters during the first three days of life revealed

lower TAPSE and RV and LV systolic myocardial velocities for neonates who subsequently died

or needed ECMO compared to those who survived without ECMO, though these differences did

not reach statistical significance (Table 7.5). Of these parameters, lower TAPSE was most

predictive of adverse outcome. A cut off of TAPSE of 6 mm had a sensitivity of 83%, specificity

of 74%, negative predictive value (95% CI) of 97 (86, 99) % and positive predictive value of 42

(25 - 61) % (Figure 7.4).

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Table 7.5: Comparison of echocardiographic indices between PPHN neonates who subsequently died or needed ECMO vs. those who survived until discharge without ECMO

Variable Death or ECMO (n=6) Survived until discharge (n=43) P Value

FAC-G (%) 22 (13, 29) 19 (12, 31) .94

TAPSE (mm) 5.2 (3.9, 5.7) 7.5 (6.0, 8.4) .05

RV s’ (cm/s) 4.4 (4.0, 5.3) 6.2 (5.0, 6.8) .08

TvE: e’ 10.8 (9.3, 11.2) 8.0 (6.6, 9.9) .28

RV IVRT’ (ms) 55 (51, 73) 64 (51, 80) 1.0

RV MPI’ 0.76 (0.60, 0.87) 0.57 (0.39, 0.70) .11

RV GLS (%) 16 (14, 18) 17 (15, 22) .67

RVO (mL/kg/min) 113 (94, 198) 187 (139, 245) .15

RVSP (mmHg) 33 (30, 45) 47 (37, 55) .17

PAAT (ms) 39 (30, 43) 33 (28, 40) .47

PVRI 4.3 (3.7, 5.6) 5.2 (4.2, 6.4) .33

Sphericity Index 1.7 (1.1, 1.9) 1.5 (1.2, 1.8) .84

LV EDV-4C (mL) 5.5 (2.9, 7.0) 4.7 (3.5, 6.0) .82

Ejection fraction (Simpson’s)

49 (43, 54) 49 (42, 58) .79

LV s’ (cm/s) 4.4 (3.5, 4.5) 5.5 (4.3, 6.3) .06

MvE: e’ 11.7 (10.9, 12.8) 9.5 (7.8, 13.0) .21

LV IVRT’ (ms) 77 (72, 91) 65 (54, 80) .15

LV MPI’ 0.76 (0.70, 0.94) 0.74 (0.62, 0.89) .58

LV GLS (%) 15 (14, 17) 17 (15, 19) .35

LVO (mL/kg/min) 110 (83, 116) 124 (98, 177) .15

All comparisons were made using Mann Whitney U test and results are presented as median (interquartile range). (‘) indicate that measurements were obtained using tissue Doppler imaging. FAC-G global fractional area change; TAPSE tricuspid annular plane systolic excursion; RV right ventricular; LV left ventricular; s’/e’ systolic/early diastolic velocities of basal segment of RV free wall and LV free wall respectively; TvE early tricuspid inflow velocity; MvE early mitral inflow velocity; IVRT’ isovolumic relaxation time; MPI’ myocardial performance index; GLS overall global peak longitudinal strain; RVO right ventricular output; RVSP Right ventricular peak systolic pressure; PAAT pulmonary artery acceleration time; PVRI Pulmonary vascular resistance index; EDV-4C end diastolic volume in apical 4 chamber view; LVO left ventricular output.

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Figure 7.4: A box plot graph comparing TAPSE for neonates with persistent pulmonary hypertension of newborn (PPHN) who survived until discharge without ECMO (n=43) vs. those who either died or needed ECMO (n=6). A cut off of TAPSE of 6 mm (dotted line) had a negative predictive value (95% CI) of 97 (86, 99)% and positive predictive value of 42 (25 - 61)%.

Influence of Sex

LV functional parameters were lower in females (n=24) in comparison to males (n=25)

[GLS-4C (14.4 ± 3.5 vs. 16.8 ± 4.0%; p = .04); LV-IVRT’ (77 ± 24 vs. 64 ± 18 ms; p = .03); LV

systole’: diastole’ (1.11 ± 0.40 vs. 0.89 ± 0.22; p=.04)]. No other echocardiographic or clinical

differences were observed between males and females; with both groups having a 12% mortality

(n=3 in each group).

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PDA, total body hypothermia, CPR at birth and iNO treatment

RV and LV functional parameters were lower in patients with a PDA shunting either

bidirectional or right to left (n=38) in comparison to neonates without a PDA (n=10) [median

(IQR) FAC-3C [33 (27, 38) vs 43 (37, 49)%, p<.01]; RV GLS [15 (13, 17) vs. 22 (19, 23)%,

p<.01]; RV systole’: diastole’ [1.02 (0.84, 1.21) vs. 1.25 (1.15, 1.40), p=.03]; LV s’ [4.7 (4.1,

5.9) vs. 6.1 (5.8, 7.0) cm/s, p<.01]; LV systole’: diastole’ [0.92 (0.72, 1.11) vs. 1.24 (1.03, 1.33),

p=.01]; MvE: MvA [1.07 (0.90, 1.23) vs. 0.91 (0.79, 0.96), p=.03]; LVO [114 (91, 163) vs. 170

(127, 210), p=.01]]. Within neonates with PDA, a right to left shunt was associated with lower

values of functional indices than bidirectional shunt (data not shown).

PPHN neonates who were receiving treatment by total body hypothermia (n=19)

demonstrated lower RV systolic [pLS-4C (14.7 ± 3.6% vs. 18.2 ± 5.9%; p=.01)], RV diastolic

[IVRT (78 ± 21 ms vs. 58 ± 19 ms; p<.01)] and LV systolic [GLS (14.9 ± 3.1 vs. 17.8 ± 3.1);

p=.02; LVO (106 ± 48 vs. 153 ± 62 ml/min/kg; p<.01] performance. Additional exposure to CPR

at birth (n=8) vs. hypothermia without needing CPR (n=11) was associated with further

worsening in diastolic performance of both the right ventricle [TvE: e’ (7.1 ± 4.4 vs. 11.3 ± 1.6;

p=.02)] and the left ventricle [IVRT’ (75 ± 19ms vs. 84 ± 33ms; p=.02)]. No differences were

observed in PAAT, PVRI and RVSP in relation to any of the tested variable. Treatment with iNO

or responsiveness to iNO did not correlate with any echocardiographic parameter except a lower

PVRI in PPHN neonates who received iNO (data not shown).

7.4 Discussion

Our study looked at functional parameters for LV and RV function in infants with PPHN.

We confirmed that during the first three days of life, PPHN is associated with multiple adverse

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alterations in biventricular systolic and diastolic function. TAPSE and longitudinal strain are the

indices of RV systolic function most affected in PPHN, FAC-4C was marginally lower. This is

keeping with the previous report by Zakaria et al, in which 30 neonates with pulmonary

hypertension [median (IQR) age at scan 6 (1-11) days] demonstrated lower TAPSE, but not FAC

(43). Though controls were of more advanced age in this study [age at scan 10 (5, 52) days].

Similar to our study, RV s’ was also found to be low in PPHN, while strain was not measured.

The preservation of FAC in PPHN may reflect the neonatal right ventricle’s effort to preserve its

ejection fraction in the presence of a high afterload, however, it may also indicate a limitation of

our sample size or a high measurement variability associated with FAC calculations, which

requires manually tracing the endocardial borders in a heavily trabeculated neonatal right

ventricle; also previously reported by us (197). Although FAC may be maintained by right

ventricle’s radial or circumferential function as opposed to the longitudinal function detected by

other parameters, this will have to be investigated in future studies. Nevertheless, our findings of

lower RVO in PPHN in spite of preserved FAC and a lack of association between FAC values

and adverse clinical outcomes suggest against utility of FAC in this patient population.

One of the significant finding of this study was the presence of biventricular cardiac

dysfunction in PPHN, with changes seen in systolic and diastolic function of both ventricles. In

addition, use of a comprehensive approach allowed us to investigate and describe the presence of

inter-ventricular correlations between several functional parameters. These findings may be

explained by one or a combination of factors including common underlying etiology responsible

for high pulmonary pressures and cardiac dysfunction, impact of RV dysfunction on the left

ventricle via adverse ventriculo-ventricular interactions and secondary effect of cardiorespiratory

instability and associated treatment strategies on cardiac function such as, hypoxia, acidosis, use

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of high frequency ventilation and inotropes. Although it was not feasible to delineate individual

contribution of each of these factors, our study provide some important clues. Hypoxic ischemic

encephalopathy and its treatment with hypothermia was an important confounding etiology as

these infants had suffered a significant perinatal global ischemic insult and had a rectal

temperature ranging from 33.5 to 34.5 degrees at the time of the scan. Our results, however,

remained unchanged when analysis was repeated after excluding these neonates, suggesting

cardiac dysfunction in PPHN to be relatively independent of the underlying etiology. Several

mechanisms are known to govern adverse inter-ventricular interactions in pulmonary

hypertension including, shared myocardial fibres between ventricles, dysfunction of the shared

septal wall, lower pulmonary blood flow resulting in low LV filling, transmission of intra-cavity

diastolic pressure via interventricular septum and mechanical effects due to large RV size,

leftward bowing of the interventricular septum and pericardial constraint (14, 73, 75, 84, 221,

222). We did not find significant RV dilatation in neonates with PPHN. This suggests that in

PPHN, early in disease process, adverse inter-ventricular interaction, if at all at play, is not

secondary to mechanical factors or pericardial constraint. Whether adverse inter-ventricular

interactions exists in PPHN and is mediated by other mechanisms, as suggested by our finding of

lower LV volume (indicating reduced LV filling) and septal diastolic dysfunction or whether

cardiac dysfunction is secondary to overall clinical instability frequently seen in this disorder and

not directly related to the high pulmonary pressures, will have to be examined in future studies;

these should ideally be investigated in experimental animal models. Our finding of a relative lack

of association between functional parameters and markers of severity of pulmonary vascular

disease (PAAT, PVRI, RVSP) as well as treatment with iNO points towards latter to be the case.

Our interesting observation of a lack of RV dilatation in PPHN could either be explained by the

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fact that the observed RV dysfunction in early disease was secondary to factors other than

exposure to high afterload or may relate to the relative immaturity of the neonatal myocardium,

which is known to characterized by a higher proportion of non-compliant tissues such as

collagen (220). Nevertheless, our study demonstrates that PPHN is a global disorder and

highlights the importance of evaluating function for both ventricles irrespective of the underlying

etiology.

Of the studies that have examined the hemodynamics in neonates with PPHN, some have

concluded that low LVO is a common occurrence (37, 99, 219). This is contrary to our finding

and may reflect the fact that previous studies either used older controls or compared their data

with previously published ‘normal’ neonatal reference ranges, which did not account for specific

postnatal age. Our control data was prospectively collected on day 1 and 2 of life. This may also

explain why the LVO values observed in our control cohort were lower than what was

considered as cut off to define abnormal by previous investigators (< 150 mls/min/kg). The

standard validated LVO measurement technique was used in our study, as explicitly described

elsewhere (175, 198). PPHN neonates had lower RVO, which, at least in part, may reflect lower

left to right contribution of the PFO shunt because of the increased right sided pressure in

comparison to controls; all of whom had a left to right shunting PFO. Our results suggests that in

PPHN, PFO shunt along with heart rate may be important factors in maintaining normal range

LVO, which is an indicator of blood flow to pre-ductal organs including brain.

Recent studies have examined the relationship between RV functional indices and outcome

defined as death or need for ECMO in neonates with PPHN (44, 185). Agarwal and colleagues

examined 117 PPHN infants and found RV systole to diastole ratio measured using color flow

Doppler as the best predictor. Malowitz et al reported TAPSE (sensitivity and specificity of 56%

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and 85% respectively for values < 4 mm) and RV GLS (sensitivity and specificity of 52% and

77% respectively for values ≥ -9%) to be most predictive. We also found TAPSE, albeit a

different cut off (6 mm with sensitivity and specificity of 83% and 74% respectively), but not

strain to be associated with adverse outcome. This difference could reflect differences in study

population as evidenced by a high incidence of adverse outcome in these studies (62% and 30%

respectively) and their inclusion of neonates with congenital diaphragmatic hernia. We chose to

exclude these infants along with other developmental lung disorders as these are inherently

different conditions, which are probably better studied as a separate cohort (45). The 12%

occurrence of death or need for ECMO observed in our study is keeping with the previous

epidemiological reports of neonates with PPHN, suggesting representativeness of our disease

cohort (33, 223). Another difference between our study and the study by Malowitz et al is the

methodology used for measurements. In their study, all measurements were performed offline on

compressed DICOM images (30 frames per second) - TAPSE by measuring the difference in the

distance between tricuspid lateral annulus and transducer interface in diastole and systole and

strain by using Tomtec Velocity Vector Imaging software. In comparison, we measured TAPSE

from M-mode images acquired online at the time of scanning (> 1000 frames per second) and

strain offline on raw data images (100 - 120 frame per second) using a different vendor software

(184). A limitation is that our estimated rate of adverse outcome was based on a small number of

infants and will therefore require confirmation in a larger study.

In this study, we identified previously unknown, risk factors associated with functional

echocardiographic abnormalities within the PPHN cohort. These include female sex, hypoxic

ischemic encephalopathy and therapeutic total body hypothermia, need for CPR at birth, and

presence of a ductal shunt as well as right to left shunt across PDA (clinically can be detected by

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presence of pre-ductal saturations higher than post-ductal). Given the small sample size in our

study, these results can only be considered as preliminary. Nevertheless, these findings are in

line with that of Malowitz et al, who found a predominant right to left PDA shunt to be

associated with the need for ECMO or death in PPHN.

The current data suggest from a clinical outcome perspective, TAPSE, a simple bedside

measure of RV function, may be a useful parameter for identifying high risk infants with PPHN.

However, this observation is based on small number of retrospective studies and should be

confirmed prospectively in a larger cohort. A major advantage of using multiple indices for

assessment of ventricular function is that it allows for inter-parameter corroboration, as observed

in the primary and secondary analyses of our study; which may lower the risk of being misled by

a random chance finding. This is particularly important in neonates due to non-feasibility of

invasive investigations and a higher chance of multiplying measurement errors due to small

patient size. While we recognize that measuring all indices described in this study would not be

pragmatic in routine day-to-day intensive care practice, we suggest that echocardiographic

evaluation in PPHN should be as comprehensive as feasible and include both RV and LV

function. Special attention should be paid to TAPSE as it may help identifying infants at the

highest risk of adverse outcome.

We also found that even though indices of the severity of pulmonary hemodynamics were

grossly different in PPHN cohort compared to controls, these or iNO treatment did not correlate

with clinical outcomes. This suggest that PPHN may be similar to pulmonary hypertensive

disorders in older patients, where symptomology and clinical outcomes are now known to be

governed less by the severity of disease than by effect of RV function; which in itself is variable

between individuals and is at least partially independent of the measured pulmonary pressures

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(23, 218). It is plausible that an appropriate evaluation of biventricular function in these high risk

neonates early in the disease course may help delineate underlying pathophysiology and

characterize cardiac dysfunction, and enable titration of therapeutic interventions to individual

patient needs (224). This may well improve patient outcomes, which have otherwise remained

unchanged in spite of the widespread use of pulmonary vasodilator agents. Given the lack of

invasive hemodynamic monitoring in neonates, the non-specific nature of symptoms and

difficulties in eliciting RV specific clinical signs, it is critical for clinicians to have a high index

of suspicion and low threshold for evaluation of RV function in PPHN.

In conclusion, I demonstrate that PPHN is a disorder frequently characterized by global

cardiac dysfunction. In comparison to age appropriate controls, PPHN neonates demonstrate

worsening in several echocardiographic indices of RV and LV systolic and diastolic function

during the first three days of age; which were independent of HIE being the underlying etiology.

TAPSE during the first three days of age identifies neonates at most risk of subsequent adverse

clinical outcome. A pressure loaded neonatal right ventricle during early postnatal life is

characterized by a relative lack of dilatation but demonstrates evidence of significant inter-

ventricular functional correlations.

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Chapter 8: Project summary and clinical significance

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8.1 Project summary and future questions

Right ventricular dysfunction is an established and important predictor of outcomes in

several cardio-respiratory disorders in older patients including congestive heart failure, valvular

heart disease, the post-operative period after cardiac surgery, myocardial infarction and

congenital heart diseases (8, 84). RV dilatation is a predictor of mortality in acute respiratory

distress syndrome (225). Absence of a right ventricle in Fontan’s circulation results in chronic

progressive multi-organ failure secondary to chronic venous congestion (6, 7). It is noteworthy,

however, that patients with Eisenmenger’s syndrome and pulmonary stenosis remain

asymptomatic for decades as the right ventricle adapts to high afterload by undergoing

hypertrophy (86, 87). In no other condition however the relevance of RV function more

appreciated than in PAH. In chronic PAH, irrespective of the underlying etiology and method

used for its assessment (cardiac catherization, MRI, echocardiography), studies have consistently

found indices of RV function to be more predictive of symptoms and clinical outcomes than

indices representing severity of pulmonary vascular disease (15). The ability of the right

ventricle to preserve function varies between patients and disease states and is at least in part is

independent of the degree of pulmonary arterial pressure elevation (13). Significant functional

interactions also exist between the left and right ventricle (221).

Very little is known about heart function in newborn infants in either health or disease

states. The immediate period following birth is unique and is characterized by an increase in

pulmonary blood flow, a rapid (followed by gradual) decrease in PVR and closure of fetal shunts

(196, 215). The specific timing and rate of these events in human neonates are not well known.

The adaptation of an immature heart, in particular the right ventricle, during this phase of

physiological ‘pulmonary hypertension’ has not been studied. This information is clinically

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important as it enables clinicians to understand the pathophysiological alterations associated with

disease states presenting during this high-risk period. Pulmonary hypertensive disorders are not

infrequent in the setting of the NICU (39). Dysregulation of postnatal transition, primarily failure

of the PVR to decrease to normal levels, results in the well-known syndrome of PPHN, which is

characterized by severe hypoxic respiratory failure secondary to persistence of high RV afterload

after birth (226). Despite the widespread use of iNO, the high rates of mortality and morbidities

associated with this disorder have largely remained unchanged over the last two decades (38). In

addition, underlying RV dysfunction is thought to be an important contributor. Over the last

decade, cPHT has been described in the setting of CNLD (227), a complication affecting more

than half ELBW infants (47). A number of prospective and retrospective studies have now

established that cPHT affects 1 in every 3 cases of CNLD and these patients have a higher rate of

mortality, as well as short term respiratory morbidities (55, 56, 179, 228, 229). Most deaths in

this context occur secondary to RV failure, diagnosis is usually made late in the disease course

and is limited by the use of crude qualitative criteria. It remains unclear how - or if - an immature

right ventricle adapts to a chronic gradual increase in afterload. Further, the significance of RV

function and/or dilation, as well as the use of echocardiographic measures for early identification

of high-risk infants for targeted treatment trials remains unexplored.

One of the primary reasons for these knowledge gaps is the difficulty in clinical assessment

of cardiac health in neonates. The symptoms associated with RV failure are nonspecific and may

overlap with those arising secondary to parenchymal lung disease which is a common occurrence

in these patients. Clinical signs of RV failure, frequently utilized in older patients, are difficult to

elicit in neonates. Definitive investigations like cardiac catheterization and CMRI are generally

not feasible and can only be obtained sporadically, late in the disease course. Echocardiography

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remains the clinical 'gold standard' investigation to evaluate heart function for these patients, but

until recently evaluation was limited to qualitative assessments. Recent technological

advancements have enabled the development of echocardiography indices for appraisal of RV

size and function in older patients and now in neonates (197, 198). Normative data for TAPSE

and TDI derived myocardial systolic velocity are well established for both term and preterm

neonates (62, 63). There are, however, limited data regarding feasibility, relative reliability and

normal data for many other indices. Our understanding regarding the neonatal right ventricle and

how it responds to acute and/or chronic exposure to increased afterload, as well as its interaction

with the left ventricle remain unknown.

The overall aim for the studies included in this thesis was, to develop tools to help provide

comprehensive, quantitative, reliable, reproducible and pragmatic evaluation of biventricular

cardiac function and pulmonary hemodynamics in newborn infants. We then investigated the

utility of these tools in advancing our knowledge of neonatal cardiopulmonary physiology in

health and disease; specifically, during the immediate postnatal period to describe normal

cardiovascular adaptation in healthy babies and second, in the setting of PPHN, to describe its

value in a disease state. During my clinical fellowship, prior to starting my graduate studies in

the Department of Physiology, I developed a comprehensive echocardiography imaging protocol

specifically for use in neonates, as described in Chapter 2 and Chapter 3. In addition to

incorporating existing functional indices, I developed novel indices from views which may allow

quantification of RV size and function independent of septal motion, an important confounding

variable during the neonatal period. These previously undescribed indices included the linear

dimension of the ‘depth' of the right ventricle (B-PLAX), the area of the posterior RV cavity

(EDA-3C), the functional contribution of the RV infundibulum (FAC-3C) and longitudinal

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function of its inferior wall (GLS-3C). Subsequently, in the prospective studies described in

Chapter 4 and Chapter 5, I confirmed the feasibility of employing this imaging protocol to

assess RV and LV functional indices during the immediate neonatal period (197, 198). These

studies allowed me to generate relative measurement reliability data for these parameters and

produced a comprehensive set of normal values for the first two days of life in healthy term

neonates. The results obtained from these studies corroborated with previously established data

for TAPSE and TDI, as well as LV longitudinal strain values reported in smaller studies (150,

151); thus supporting the validity of my derivation cohort. In addition, I established the cogency

of these measurements for use in the immediate postnatal period by demonstrating a lack of

appreciable change between the first and second day of life in relation to the anticipated

physiological change in cardiac loading conditions and closure of fetal shunts. The first scan on

day 1 of life, however, in those studies, was performed between 12 to 18 hours of age. Although

this matched the average age at which neonates suffering from disorders of postnatal transition

are commonly evaluated, it was beyond the period when peak changes in loading conditions are

anticipated.

Following this, in another prospective study, described in Chapter 6, I sequentially studied

15 healthy term neonates shortly after birth and ascertained the value of my techniques and

methods by successfully describing detailed cardiopulmonary adaptive changes associated with

birth during the first day of life. Prior to this, our understanding of circulatory changes after birth

was mostly based on animal experiments (24). In this study, I not only confirmed the occurrence

of the expected decrease in PVR and closure of the ductus arteriosus in human neonates but, also

for the first time, provided time-specific, quantitative, physiological circulatory data regarding

postnatal transition in humans. I believe this has a high potential of immediate clinical

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translation. In this study, I acquired a number of novel facts regarding human transitional

circulation. I demonstrated that in humans, despite the reduction of pulmonary to systemic

pressure ratio, the decrease in pulmonary arterial pressure, after the first 20 minutes of age, may

be slower during the first 10 to 12 hours of life. Further, in spite of major changes in loading

conditions and increase in stroke output, the right and left ventricular outputs remained relatively

stable throughout the first day of life. This was owing to the changes in heart rate and

directionality of shunting across the DA and FO. The ratio of the right and left ventricular output

found in this study closely matched a recent observation using similar methods in normal human

fetuses. This observation is in keeping with the persistence of fetal RV dominance during the

first day of postnatal life. In addition to the alterations in indices of PVR and blood flow, I also

observed significant changes in echocardiographic indices of heart function, particularly the right

ventricle. This data suggested a transient physiological reduction in RV function at < 30 minutes

of age in comparison to measurements performed during the second half of the first day. This has

not been described previously and may suggest a relative vulnerability of the neonatal right

ventricle to high afterload conditions immediately after birth. Finally, I confirmed the

representativeness of these values by performing a time-specific comparison with similar data

obtained prospectively from a previous cohort of healthy neonates included in the studies

described in Chapter 4 and Chapter 5.

To investigate the utility of this protocol in a population of babies with abnormal

cardiopulmonary transition, in Chapter 7, I evaluated a retrospective cohort of neonates with

PPHN who had comprehensive echocardiographic evaluation during the first three days of life.

Use of the imaging protocol developed for this work, allowed me to gain several novel insights

into the pathophysiology associated with this serious disorder of postnatal transition. I observed

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that unlike older patients, the neonatal right ventricle, early in the disease process, did not show

significant dilatation even in the presence of dysfunction, suggesting a relative absence of

heterotropic adaptive mechanism during early postnatal life. In addition, I found that PPHN is

characterized by global cardiac dysfunction, a finding which persisted in spite of exclusion of

neonates who developed PPHN secondary to a perinatal ischemic insult. This could either

suggest that the underlying mechanisms resulting in dysregulation of postnatal transition or

subsequent clinical instability may affect both ventricles or the presence of inter-ventricular

functional dependence during early postnatal life where the reduction in LV function is a ‘side-

effect’ of RV dysfunction. Additional novel findings of this study included the presence of

significant linear correlations between echocardiographic functional indices of both ventricles

and the identification of risk factors associated with relative adverse cardiac performance within

the PPHN cohort; these have not been previously described and included, female sex, total body

hypothermia in the setting of hypoxic ischemic encephalopathy, the need for CPR at birth and

the presence of a right to left shunt across a PDA and/or PFO. The finding of a lower TAPSE

during the first three days of life being predictive of subsequent adverse outcome of death or

need for ECMO in neonates with PPHN is important. Although this finding was based on a small

patient sample, it corroborates the finding in a recent report by Malowitz et al in which TAPSE,

longitudinal strain and persistent presence of a right to left shunt across PDA was found to be

predictive of adverse clinical outcome in neonates with severe PPHN. The incidence of adverse

outcome was high in this cohort which, unlike my study, included neonates with congenital

diaphragmatic hernia. Small observational studies conducted using TDI derived myocardial

indices in neonates with congenital diaphragmatic hernia have also reported presence of RV

diastolic dysfunction early in the postnatal course to be predictive of subsequent longer duration

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of need for respiratory support. This supports my observation of the presence of RV dysfunction

and its correlation with adverse clinical outcome in PPHN.

Successful completion of this work has given me the impetus, the necessary reliable

clinical tools and collaborative expertize to continue advancing the knowledge in the ‘untapped’

field of cardiopulmonary physiology in newborn infants. During the conduct of my graduate

studies, I have developed a longitudinal research plan with a clear medium to long term focus on

conducting early interventional randomized control clinical trials, based on carefully described

pathophysiologic mechanisms, targeting RV function in neonatal pulmonary hypertensive

disorders (Figure 8.1). Some of the unanswered questions that I wish to address in my ongoing

and future work and are described in more detailed in Chapter 9 include the following:

• Which of the previously described echocardiographic indices most accurately reflect

RV size and function in neonates with pulmonary hypertension (Validation study)?

• How does a neonatal right ventricle respond to a chronic, but gradually increasing,

exposure to high afterload?

• Can functional indices of RV performance, developed during this work, help early

identification of preterm neonates who subsequently get diagnosed with ‘significant’

chronic pulmonary hypertension?

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Figure 8.1: A schematic presentation of the long term research strategy developed during the conduct of this thesis, highlighting research questions for my ongoing and future work.

8.2 Clinical relevance

The clinical significance of individual studies have been highlighted in the discussion

section of respective chapters. Overall, the studies conducted as a part of this thesis have a high

potential of early clinical translation. Currently, there are major knowledge gaps in our

understanding of cardiovascular physiology in neonatal disease states and its role as a potential

contributor to the high rate of adverse outcomes observed in certain conditions such as acute or

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chronic PHT. This may relate in part to a general lack of clinical awareness and in part to

unavailability of reliable measurement tools. As previously discussed, clinical appraisal of heart

function is challenging in neonates, in particular for assessment of RV function.

Echocardiography is commonly used in a qualitative manner, presumably driven by a

technological lag in imaging equipment, but in light of advancements in imaging techniques this

is now changing. High quality imaging equipment suitable for quantitative estimation of

ventricular function in smallest of neonates is now widely available in many NICUs.

Increasingly, many neonatal intensivists are undertaking training in advanced functional

echocardiography and utilizing cardiac imaging in routine clinical practice. For instance, the

majority of Canadian tertiary NICUs now have a program in targeted neonatal echocardiography,

most established in the last 5 to 7 years (230). Similar trends have also been reported from

Australia and New Zealand (231). Neonatologist-performed functional echocardiograms are

commonly followed by changes in clinical management (232, 233). Current utilization of

quantitative echocardiography indices for decision making by neonatologists, however, is based

on normative data which can be described as patchy at best. This may lead to incorrect

assumptions and in appropriate treatment decisions. For instance, LVO < 150 ml/min/kg has

commonly been considered as a cut off threshold to define “critically low LVO” in neonates

(219). This assumption was based on old publications of neonatal ‘normative data’, which did

not account for specific postnatal age. My systematic prospective study, however, showed that

normal neonates when assessed at rest in their natural environment commonly have LVO below

this cut off in the immediate postnatal period. In addition, interpretation of echocardiographic

variables during the immediate postnatal period is marred by a limited understanding of

physiological cardiovascular adaptation and mechanisms of disease. Current approaches to

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cardiac assessment, definitions of thresholds of concern, standard therapeutic approaches are

often based on dogma and clinical presumptions. There is an urgent need to evolve to a more

thoughtful approach where appropriate diagnostic tools are developed and tested, normality is

understood, pathological deviations from normality are recognized, at risk situations and patients

are timely identified, such that therapies are based on sound physiological principles and their

efficacy can be reliably monitored. High quality, comprehensive and systematic investigations

are critical to advance this field. The studies conducted during the course of my graduate studies

addresses some of these key issues. In the studies conducted, I was able to provide standard

norms for normal heart dimensions that may allow enhanced insight into states of cardiac under-

filling or chamber dilation. Similarly, I describe standard norms of heart function and describe

their interplay with the physiologic modulators of normal postnatal adaptation. These data

provide evidence that suggest that neonatal right ventricle may be vulnerable to dysfunction on

persistent exposure to high afterload. These data may allow for accurate diagnosis of cardiac

dysfunction during this high-risk period. Through my data in the PPHN cohort, I provide a

concrete example of how a pathological state of abnormal transition influences cardiac

performance, which may relate to subsequent adverse clinical outcome. I believe, the knowledge

generated through this body of work will lead to heightened awareness among clinicians

regarding the importance of cardiac function and its assessment in sick neonates, provide tools

and evidence which will inform day-to-day cardiac imaging and its interpretation in the NICUs

and stimulate and guide future efforts to understand cardio-pulmonary pathophysiology and its

clinical relevance in neonatal disease states. All of this may lead to earlier identification and

treatment of at-risk neonates as well as may help plan early targeted interventional clinical trials.

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Chapter 9: Limitations and future directions

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9.1 Limitations

There are several limitations of the studies conducted during the course of this work, as

enlisted below:

Chapters 4 and 5:

• Although this study demonstrated the feasibility of reliably quantifying RV and LV size and

function in neonates, which remained largely unaffected by physiological changes in loading

conditions during postnatal transition, I am unable to comment on the impact of unmeasured

but pathological loading conditions on functional parameters.

• I cannot exclude that factors like sex, mode of delivery and the presence of a PDA may have

had a small effect on cardiac performance. I did perform univariate analysis to test these

factors and found no associations, but this study was not designed to detect such relations.

Nevertheless, all PDAs observed in this study were small and hemodynamically

insignificant.

• My finding of linear relationship between BW and RV and LV dimensions can only be

considered applicable to the neonatal period and for appropriately grown neonates (i.e. BW

between 10th to 90th percentile). Future studies should evaluate a wider range of birth

weights (e.g. small or large for gestational age neonates, premature neonates) and age (e.g.

during first year of life), to further explore the extent of this relationship.

• Similarly, though I did not find any relation between gestational age and functional

parameters, this is likely due to the narrow range of gestational age of our study cohort (37

to 42 weeks); hence, the data generated in this study can only be considered relevant to term

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gestation. Subsequent studies should attempt employing a similar comprehensive imaging

protocol for preterm neonates.

• I did not explore relationship of echocardiographic parameters with body surface area. This

was done to ensure relative accuracy and clinical suitability of the study results. BW is the

most commonly used anthropometric parameter in neonates, while length measurements are

more prone to error, especially in critically ill neonates. Further, the commonly used

formulas for calculating BSA need further validation in neonates (234).

• This study did not investigate the effect of using equipment from different vendors on strain

measurements. The longitudinal strain values reported in this study and may not be

applicable to other vendors (188).

• Although I found similar results on day 1 and 2 of life, due to the timing of scans, I may

have missed the period of peak changes in transitional circulation, which are likely to occur

within the first few hours of age. Nevertheless, the timing of the first scan was based on the

anecdotal experience in sick neonates, where echocardiograms are usually preceded by

medical assessment and stabilization.

• From a normative data point of view, these studies had a relatively small sample size;

however, this still is the largest prospective systematic study in this population with an a

priori aim of establishing normal values.

Chapter 6:

• This observational data is acquired from a small number of neonates and is assumed to be

representative for all populations. There may be sex, racial or other confounders (e.g. mode

of delivery) that we were unable to consider. Nevertheless, although the data acquired in this

study was from a small number of neonates, the fact that it was acquired sequentially and

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closely fitted with a larger historical cohort in a time sensitive manner indicate the validity

of my findings.

• All measurements reported in this study were derived using echocardiography, which is

subject to operator dependent errors and variability in measurement. Invasive catheterization

or magnetic resonance imaging may provide better estimates of systemic and pulmonary

hemodynamics, myocardial function and the influence of loading conditions, but these are

not feasible in the majority of neonates, particularly in healthy states. Although, there are

limitations, echocardiography represents a feasible and in the hands of expert operators, may

provide reliable measures of changes in hemodynamics and cardiac function.

• The data presented in this study can only be considered applicable to the measurement

techniques used, which have been explicitly described in Chapters 3 (197, 198).

Chapter 7:

• Similar to previous studies in this field, my observations are also limited by being

retrospective in nature, which meant a relatively variable age at scan and potential

confounding effect of pre-scan clinical course and therapeutic interventions including

resuscitation at birth, mode of invasive ventilation, inotropes, therapeutic hypothermia and

iNO. This, however, is also reflective of real life clinical setting where stabilization, medical

interventions and transfer to tertiary centres commonly precede imaging.

• Because of the relatively small sample size, I could not examine the independent effect of

high pulmonary pressure versus potential confounding variables mentioned above on cardiac

dysfunction. For the same reason, the results of the secondary analysis can only be

considered as preliminary. It is likely that this study was under-powered to identify all

potential risk factors associated with cardiac dysfunction in PPHN. This study, however, is

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the first report of comprehensive cardiac function assessment in this patient population and

provides novel pathophysiological insights as well as identified previously unreported risk

factors.

• Another limitation of this study is the lack of gold standard tests, namely cardiac

catherization and CMRI. It is thus possible that my finding of a lack of RV dilatation in

PPHN may relate to the relative insensitivity of individual echocardiographic measures to

identify changes in RV size. Use of a comprehensive protocol and corroboration of findings

by several parameters, however, makes this as well as being misled by a chance finding less

likely. Nevertheless, future studies should examine the validity of echocardiographic

measures of cardiac size and function in neonates.

• This being a clinical physiological study performed in a vulnerable patient population using

non-invasive tests, is not able to provide confirmation of the suspected underlying

pathophysiology. For instance, I cannot confirm or rule out the presence of inter-ventricular

functional dependence in PPHN or identify the relative contribution of various potential

mechanisms mediating it. This study, however, provides important clues and highlight areas

for future definitive investigations. These in the context of PPHN will have to be explored

using suitable animal models.

• As echocardiography is not a mandatory investigation at the study centre, I cannot rule out a

degree of selection bias in the patient population, which may consist of more severely

affected neonates. Nevertheless, the results of this study support the importance of early

assessment of cardiac function in neonates with PPHN.

9.2 Rationale for future directions and clinical significance

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As demonstrated in the studies included in this thesis, there are now a number of reliable

surrogate echocardiographic indices of RV size and function which can be applied to the

neonatal population. This is important as echocardiography is the main non-invasive imaging

technique that is routinely available for neonates and is ideally suited for longitudinal

assessments in an acute care setting. Although the concurrent use of several assessment

techniques may be desirable in research, due to time constraints, it is not pragmatic for day-to-

day intensive care clinical practice. The accuracy of individual markers in their representation of

size and function of the neonatal right ventricle is not known, particularly in the presence of high

afterload. Echocardiography measures depend on geometric assumptions, hence their relative

accuracy may vary based on patient population and underlying disorder. It is therefore important

to investigate the validity of these measures by examining their correlation with a gold standard

test. As highlighted before, cardiac catheterization is an invasive procedure, typically not feasible

in neonates due to the high risk of complications; CMRI is a safe and feasible alternative. CMRI-

derived RV volumes and ejection fraction are validated techniques for quantifying RV size and

function respectively. A major advantage of CMRI is that, unlike echocardiography, the

technique of ventricular volumetry does not rely on geometric assumptions as it segments the

chamber volumes throughout the cardiac cycle through a series of contiguous slices aligned

across the short axis of the heart; making this the current non-invasive gold standard for the

assessment of RV size and function. In addition, cine phase contrast CMRI provides a robust

quantification of pulmonary and systemic blood flow (235). CMRI is devoid of exposure to

radiation, it does not require contrast media and has been shown to be feasible in neonates

without the use of sedation (feed and wrap method) (236). CMRI has been demonstrated to be

feasible, safe and has been successfully used to study and validate echocardiographic

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hemodynamic measures related to the left ventricle and PDA in stable preterm neonates late in

NICU course (236). It is, however, expensive and cumbersome, often requiring out-of-facility

patient transfers and is available only in specialized centers. Nevertheless, CMRI can be

performed in stable and relatively mature infants, making it an ideal method to inform and

improve neonatal echocardiography practices. As a follow up to my previous work, I intend to

undertake a prospective study with the aim of identifying key echocardiography indices for

longitudinal monitoring of RV health in a high-risk population of neonates; specifically, I plan to

evaluate preterm neonates with known cPHT. In this study, I aim to recruit 25 preterm neonates

with moderate-severe CNLD and an echocardiographic diagnosis of cPHT. Each patient will

have a right ventricle-focused echocardiogram (method under investigation) and CMRI (clinical

reference method). Corresponding quantitative indices of RV size, function and blood flow will

be measured for comparison. At the end of the recruitment phase, relationships between

corresponding echocardiographic and CMRI measures will be examined to define the accuracy

and relative reproducibility of echocardiographic measurements. This will be the first study to

appraise the accuracy of echocardiography parameters for RV function in preterm neonates. The

data generated from this study will help to establish a validated and generalizable

echocardiographic surveillance protocol applicable to this patient group. This protocol will

impact clinical care and could benefit future clinical trials. To date, I have secured funding for

this prospective project and will begin recruitment following completion of my graduate studies.

Another objective of my immediate future work, is to investigate the feasibility of earlier

identification of ELBW infants at highest risk of developing cPHT secondary to CNLD. cPHT is

increasingly being recognized as an important determinant of mortality and pre-discharge

respiratory morbidity in ELBW infants. Echocardiography is the clinical ‘gold standard’

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investigation; however, to date, many of the diagnostic criteria used are crude and inadequate for

early identification in the majority of cases. With technological advancements and the

development of newer imaging modalities, as highlighted by my current and other collaborative

work (64, 66, 193), it is now feasible to serially study quantitative changes in PVR and RV

function in the smallest of neonates, thus providing opportunities for early recognition of disease

(Figure 9.1). In this study, my plan is to recruit a cohort of ELBW infants and serially study

changes in PVR and RV function from the third week of age until 36 weeks of corrected

gestational age, when a diagnosis of CNLD and cPHT is typically made. At the end of the

recruitment phase, I will determine sensitivity and specificity of echocardiographic markers

obtained in the third week of age by comparing infants with versus without cPHT. Early

identification of infants at increased risk of cPHT has direct clinical relevance as it raises the

possibility of disease modification. several potential therapeutic agents such as, inhaled nitric

oxide, milrinone (phosphodiesterase 3 inhibitor), sildenafil (phosphodiesterase 5 inhibitor),

prostacyclin and bosentan (endothelin receptor antagonist) are already available (39); however,

the therapeutic impact of these drugs could be limited unless used early in the disease process,

before a more ‘fixed’ increase in PVR secondary to pulmonary vascular remodeling occurs.

Previous physiological studies have demonstrated a partial vasodilatory effect as a result of these

therapies when used later in established disease, presumably because of pulmonary vascular

remodeling (237-239). This will be the first study examining the predictive value of novel

echocardiographic markers in neonatal cPHT. Early identification is likely to have a significant

impact on preventative or curative treatment strategies. I have already obtained all necessary

approvals for this project and aim to initiate recruitment alongside the previously mentioned

validation study soon after completion of my graduate work.

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Figure 9.1: One of my immediate future plan is to refine diagnostic criteria for cPHT in preterm neonates with CNLD and identify echocardiographic parameters which best identify at-risk neonates early in disease course.

In addition to earlier recognition of at-risk patients, the use of our comprehensive imaging

protocol in cPHT patients will facilitate longitudinal assessment of the response of the neonatal

right ventricle to chronic increase in afterload and its associated impact on LV performance. Our

study investigating PPHN has demonstrated a relative lack of heterotropic adaptation in the

immediate neonatal period, in keeping with the lack of reference to RV dilatation in previous

studies. Studies of cPHT in preterm neonates, however, have consistently noted the presence of

RV dilatation, indicating a different response in chronic versus acute pressure loading conditions.

Indeed, this has also been our clinical observation (Figure 9.2). One of my goals in this study of

preterm cPHT will be to systematically evaluate neonatal RV physiology in the presence of

chronic pressure loading.

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Figure 9.2: A parasternal short axis view obtained using 2D echocardiography in a preterm neonate with significant chronic pulmonary hypertension demonstrating severe RV dilatation.

9.3 Final remarks

The importance of incorporating RV function in the study of pulmonary hypertension is

being increasingly recognized as a primary clinical and research target for evaluating the

efficacy of existing therapies and in the development of new ones (1). In spite of commercial

availability and clinical use of a plethora of pulmonary vasodilator therapies, clinical outcomes

of patients with PAH has not shown consistent improvements (23). An overarching focus on

promoting pulmonary vasodilation without considering the impact of employed strategies on

RV functional recovery has been cited by field experts as one of the major reasons behind this

observation. Similarly, the use of pulmonary vasodilators, most notably iNO, have consistently

demonstrated an improvement in acute symptoms of PPHN, but this has not translated into

better survival or long-term clinical outcomes (38, 239). Milrinone, a commonly used inodilator,

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has recently been shown in a small observational study to improve RV function in term neonates

with PPHN when symptoms were not responsive to standard treatment with inhaled nitric oxide

(224); this improvement was temporarily associated with the resolution of clinical symptoms.

Though pulmonary hypertension in neonates has been a subject of pre-clinical and clinical

research for several decades, the field of RV function is still in its infancy; at least in part due to

the long-standing problem of a relative inability to quantify RV function in small neonatal

hearts. With the current availability of reliable measurement tools, this may now change. The

aim of the studies described herein was to address some of these knowledge gaps and advance

the field of clinical cardiopulmonary physiology in neonates, in particular understanding the role

of the right ventricle in pulmonary hypertensive disorders. My current and future planned work,

as described in the previous section, will provide ideal grounds for realizing my medium to

long-term goal of conducting mechanistic therapeutic trials targeting the specific subgroup of

patients at highest risk of adverse outcomes. This will serve two key purposes – first, avoidance

of exposure to unnecessary pharmacological agents for neonates not at high risk of subsequent

adverse outcome will ensure a favourable risk-benefit profile for therapeutic interventions, as

well as reduce treatment costs. Second, targeting a high-risk group of patients will potentially

lower the required sample size for subsequent clinical trials.

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Appendix: Additional figures and data

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Chapter 4

Figure 6.2: Measurement of peak longitudinal strain of RV inferior wall (GLS-3C) using speckle tracking echocardiography from one of the study infants. The endocardial border was manually traced from RV 3-chamber view. The tracking was performed automatically by the software but was visually inspected before accepting results. The pLS values for individual segments (represented graphically by color-coded lines) was averaged to generate an overall pLS-3C.

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Figure 4.3: Bland-Altman plots showing intra- and inter-observer measurement variability was lower for biplane-fractional area change (global-FAC) in comparison to single-plane measurements acquired from RV apical 4 chamber view (FAC-4C) or from RV apical 3 chamber view (FAC-3C).

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Figure 4.4: Bland-Altman plots showing intra- and inter-observer measurement variability was relatively lower for global peak longitudinal strain (GLS-global) obtained by averaging GLS acquired from RV apical 4 chamber view (GLS-4C) and from RV apical 3 chamber view (GLS-3C) in comparison to its individual components.

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Figure 4.5: Bland-Altman plots for inter and intra observer measurement variability for myocardial performance index calculated using time periods measured from color flow Doppler (MPI) and tissue Doppler imaging (MPI’). MPI’ was more reproducible than MPI, particularly for the same observer.

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Figure 4.6: Modest linear correlation observed between RV peak systolic myocardial velocity (s’) measured from TDI and STE-derived peak longitudinal strain of RV lateral wall

Chapter 7

The cardiac functional profile in neonates with PPHN also varied significantly in relation

to the type of shunt pattern at the level of PDA and/or PFO (Table 7.6). A pure right to left shunt

at the level of the PDA (n=6) or PFO (n=7) was associated with inferior cardiac performance. On

the other hand, echocardiograms with a left to right shunt at the level of PFO (n=16) were

characterized by better LV systolic and diastolic indices, even when occurring in association

with a bidirectional or right to left ductal shunt (n=12). Fewer infants with a left to right PFO

shunt needed treatment with inotropes (69% vs. 97%; p=.01). No differences were observed in

PAAT, PVRI and RVSP in relation to shunt patterns.

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Table 7.6: Alterations in echocardiographic indices in relation to the type of shunt pattern in neonates with persistent pulmonary hypertension of the newborn during first three days of age

PDA shunt right to left (n=6) vs. PDA bidirectional/absent (n=43)

PFO shunt right to left (n=7) vs. PFO left to right/absent or bidirectional (n=42)

PFO shunt left to right (16) vs. right to left/ bidirectional/absent (33)

PFO shunt left to right + PDA shunt right to left/ bidirectional (n=12) vs. others (37)

↓ TAPSE

(4.9 vs. 7.5 mm)

↑ RV MPI

(0.70 vs. 0.56)

↓ RV basal diameter – 4C

(1.62 vs. 1.83 cm)

↑ IVS e’/a’

(1.04 vs. 0.87)

↓ RV s’

(3.9 vs. 6.4 cm/s)

↓ Sphericity index (EDV-4C/LV length)

(1.18 vs. 1.67)

↓ RV End Diastolic Area – 4 Chamber

(3.7 vs. 4.3 cm2)

↑ MvE/MvA

(1.09 vs. 0.94)

↓ RV GLS

(13.7 vs. 18.0 %)

↓ LV EDV-4C

(3.47 vs. 5.5 mls)

↑ LV EF

(Simpson’s biplane)

(55 vs. 46%)

↑ RV IVRT

(93 vs. 60 ms)

↓ LV EF (Simpson’s biplane)

(43 vs. 50%)

↑ MvE/MvA

(1.06 vs. 0.94)

↑ RV MPI

(0.84 vs. 0.56)

↓ LV GLS

(13.6 vs. 17.4)

↓ Sphericity index (EDV-4C/LV length)

(1.03 vs. 1.7)

↓ LV EDV-4C

(3.25 vs. 5.22)

↓ LV EF (Simpson’s biplane)

(42 vs. 50%)

↓ LV s’

(4.1 vs 5.6 cm/s)

↓ LV e’

(2.9 vs. 5.1 cm/s)

↑ LV IVRT

(98 vs. 65 ms)

↓ LVO (102 vs. 141 ml/min/kg)

↓ LV GLS

(12.0 vs. 18.4%)

Interpretation: Relatively lower RV and LV systolic,

Interpretation: Relatively lower RV global function

Interpretation: Interpretation:

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diastolic and global function. Higher RV diastolic pressure.

and LV systolic function. Higher RV diastolic pressure.

Relatively less RV dilatation and better LV systolic and diastolic performance.

Relatively better septal and LV diastolic performance.

Results are presented as medians and are only listed if p<0.05. All comparisons were made using Mann Whitney U test. There was no infant with a left to right shunting PDA in this cohort. Exclusion of infants with absent PDA (n=10) or absent PFO (n=7) did not change the results (data not shown). Only three neonates had both PDA and PFO shunting right to left. No differences were observed in indices of pulmonary vascular resistance and RV systolic pressure. RV right ventricular; LV left ventricular; IVS interventricular septum; TAPSE tricuspid annular plane systolic excursion; s’/e’/a’/ systolic/early diastolic/late diastolic velocities of basal segment of RV free wall, LV free wall and interventricular septum respectively; GLS global peak longitudinal strain; IVRT isovolumic relaxation time; MPI myocardial performance index; EDV-4C end diastolic volume in apical 4 chamber view; EF ejection fraction; LVO left ventricular output.

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