Automated Fetal Cardiac Function Parameters in Congenital Heart Disease
Conditions: Congenital Heart Defect, Cardiac Function
Sponsor: Anna Erenbourg

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Protocol Title: 
Automated fetal cardiac function evaluation in pregnancy affected by 
congenital heart disease 
 
 
 
Version Number: 5 
Date of Protocol: 30 September 2022 
HREC Reference: 2022/ETH00943 
 
 
 
LIST OF INVESTIGATORS 
Co-ordinating Principal investigator/Research (CPI): Professor Alec Welsh (Contact Person) 
Royal Hospital for Women and UNSW, School of Clinical Medicine 
Level 0, Royal Hospital for Women, Barker Street (Locked Bag 2000), NSW 2031 
Phone: (+61) 2 9382 6214 
Email: alec.welsh@unsw.edu.au 
 
 
Co-Investigators (NSW, Australia):  
Professor Tracie Barber  
UNSW School of Mechanical & Manufacturing Engineer 
Ainsworth Building, Level 4, Room 401A, Kensington Campus, NSW 2031 
Phone: (+61) 2 9385 4081 
Email: t.barber@unsw.edu.au   
 
Dr Anna Erenbourg 
UNSW School of Clinical Medicine 
Level 0, Royal Hospital for Women, Barker Street (Locked Bag 2000), NSW 2031 
Phone: (+61) 2 9382 6214 
Email: a.erenbourg@unsw.edu.au 
 
Co Investigators (International Participating Centres): 
Dr Vera Cecotti (Lead for participating centre in Mayotte, France) 
Centre Hospitalier de Mayotte  
Phone: (+39) 349 123 86103 
Email: veradott@gmail.com  
 
Dr Stefano Faiola (Lead for participating centre in Milan, Italy) 
Department of Obstetrics and Gynaecology, Buzzi Children’s Hospital 
University Department of Clinical Sciences, University of Milan  
Phone: (+39) 339 594 9455 
Email: stefanofaiola@hotmail.com  
 
 

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Dr Ilaria Fantasia (Lead for participating centre in Aquila, Italy) 
Department of Life, Health and Environmental Sciences, University of L’Aquila 
Obstetrics & Gynaecology Unit, San Salvatore Hospital 
Phone: (+39) 328 092 2893 
Email: ilariafantasia@gmail.com  
 
Professor Tamara Stampaljia (Lead for participating centre in Trieste, Italy) 
Maternal and Child Health Institute - IRCCS Burlo Garofolo  
Department of Medicine, Surgery and Health Science, University of Trieste 
Phone: +393485165666 
Email: tamara.stampalija@burlo.trieste.it  
 
Dr Hagai Avnet (Lead for participating centre in Tel Aviv, Israel) 
Sheba Medical Center   
 
Phone: (+972) 587829812 
Email: hagai.Avnet@sheba.health.gov.il  
 
Professor Beata Radzymińska-Chruściel (Lead for participating centre in Cracow, Poland) 
Fetal Cardiology Unit, Medical Center, Ujastek, Cracow, Department of Pediatric Cardiology, Institute of 
Paediatrics, Faculty of Medicine, Jagiellonian University Medical College, Cracow, Poland 
Phone: (+48) 795 532 367 
Email: bearad63@gmail.com 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 

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Study Summary 
Study title: Automated Fetal Cardiac Function Parameters in Congenital Heart Diseases 
Synopsis: Congenital heart disease (CHD) is the most commonly diagnosed fetal malformation, affecting 
between 0.8 and 1.2% of livebirths worldwide (1). As with any other fetal cardiac injury, CHD can result in 
initial cardiac remodelling and dysfunction and later cardiac failure and hydrops (2).  
A small number of studies have looked at fetal cardiac function parameters in fetuses affected by CHD (3-
19). These studies suggest the complementary role of fetal cardiac function evaluation to the classical 
morphological assessment. Fetal cardiac parameters could potentially increase the predictive value of the 
cardiovascular profile score (CPS) to detect hydrops and cardiac failure (20,21). However, a few 
methodological limitations impede drawing any definitive conclusions on this matter (3-6, 8-14, 16,19).  
In the last decades fetal functional cardiac parameters such as myocardial performance index (MPI) and 
tricuspid/mitral annular plane systolic excursion (TAPSE/MAPSE) have been applied in research settings (3-
19). The lack of standardisation and the need for improvement of their repeatability and ease of 
measurement are crucial factors limiting their clinical application (22,23). Automation of these parameters 
could be the next step to overcome such limitations (22-24).   
Objectives: This study will provide evidence as to whether or not, in pregnancy affected by CHD, a 
significant difference in terms of automated fetal cardiac function parameters could be detected when 
compared to healthy pregnancies. 
• 
Primary objective: to compare automated Pulse Wave Doppler (PWD) MPI and Spatio-Temporal 
Image Correlation (STIC) tricuspid/mitral/septal annular plane excursion (TAPSE, MAPSE and 
SAPSE) in fetuses affected by CHD versus healthy fetuses.  
• 
Secondary objectives: to estimate whether these automated functional cardiac parameters could 
improve the predictive value of the cardiovascular profile score in case of hydrops. 
Study design: An international multicentre prospective observational study. An Australian lead study 
carried out in seven teaching hospitals across Europe and in Sydney. 
Planned sample size: Total n = 495 (CHD Group: n = 165; Control Group: n = 330 healthy pregnancies) 
Selection criteria: Singleton pregnancies diagnosed with CHD between 19+6 and 36+6 gestational weeks; 
and healthy singleton pregnancies at 19+6 and 27+6 gestational weeks. 
Study procedure: Recruited pregnant women (CHD and healthy pregnancies) will be offered one or two 
fetal cardiac function evaluations, depending on gestational age at recruitment, to evaluate automated 
fetal functional parameters in fetuses affected by CHD. In a second phase a nested case-control study will 
be carried out evaluating hydrops fetuses matched to no hydrops.  
Each fetal cardiac exploration will include the evaluation of automated pulsed wave doppler (PWD) 
myocardial performance index (MPI), spatio-temporal image correlation (STIC) tricuspid/mitral/septal 
annular plane excursion (TAPSE, MAPSE and SAPSE), alongside cardiac morphometric assessment and 
doppler evaluation of flow across the atrioventricular, aortic and pulmonary valves.    
Statistical considerations: Comparisons between cases and controls in baseline characteristics will be 
performed using two-sample t-tests, Wilcoxon rank-sum tests or Pearson Chi-squared tests, as appropriate.  
Analyses of changes in fetal function parameters over time, and the comparison of these changes between 
cases and controls will employ generalised linear mixed models, as appropriate for the parameter. 
Logistic regression will be used to estimate the association between fetal cardiac parameters and the 
incidence of hydrops. Receiver-operating-characteristics-curve analysis will be carried out to assess 
functional cardiac parameters compared to the routinely used cardiovascular profile score to predict 
cardiac failure in fetuses with congenital heart disease. 
Duration of the Study: 5 years 
 

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Contents 
Background .......................................................................................................................................... 5 
Disease Background....................................................................................................................................... 5 
Rational for Performing the Study ................................................................................................................. 5 
Study Objectives................................................................................................................................... 6 
Primary Objective .......................................................................................................................................... 6 
Secondary Objective ...................................................................................................................................... 6 
Expected outcomes of the study ................................................................................................................... 7 
Study design ......................................................................................................................................... 7 
Design ............................................................................................................................................................ 7 
Study Groups ................................................................................................................................................. 7 
Number of Participants ................................................................................................................................. 7 
Number of Centres ........................................................................................................................................ 7 
Duration ......................................................................................................................................................... 8 
Participant Selection ............................................................................................................................ 8 
Recruitment and Screening ........................................................................................................................... 8 
Inclusion Criteria ............................................................................................................................................ 8 
Exclusion Criteria ........................................................................................................................................... 8 
Study Outline........................................................................................................................................ 8 
Investigation Plan .......................................................................................................................................... 8 
Study Procedure Risks ................................................................................................................................. 10 
Sample Size Calculation ............................................................................................................................... 11 
Data Analysis Plan ........................................................................................................................................ 11 
Safety.................................................................................................................................................. 12 
Adverse Event Reporting ............................................................................................................................. 12 
Serious adverse event reporting ................................................................................................................. 12 
Data Safety and Monitoring Board .............................................................................................................. 12 
Dissemination of results and publication policy .......................................................................................... 13 
Storage and Archiving of Study Documents ...................................................................................... 13 
References.......................................................................................................................................... 15 
 
 
 

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Background 
Disease Background 
Congenital heart disease is the most frequently diagnosed fetal malformation afflicting approximately 0.8% 
to 1.2% of live births worldwide (1). Generally congenital heart disease is defined as a structural 
abnormality of the heart and (or) great vessels that is present at birth (25). 
Recent studies have shown that in the initial stages of an insult, the fetal heart usually manages to adapt, 
undergoing subclinical changes in order to maintain optimal and efficient function (2).  
These changes can include genome expression, molecular, cellular and interstitial variations, defined as 
cardiac remodelling and manifested clinically as changes in size, shape and function of the heart after 
cardiac injury (26).  
Medical imaging investigations have defined normal and abnormal patterns of cardiac remodelling leading 
to a better understanding of how congenital cardiac malformations alter the hemodynamic transition to 
the extrauterine environment (27).  
In most clinical situations, fetal cardiac dysfunction is subclinical. However, if the insult is severe enough or 
persists, impaired cardiac pumping and myocardial relaxation can occur, leading to clinical cardiac failure, 
which in utero essentially manifests as fetal hydrops (Figure 1) (2). 
 
 
Figure 1: Cardiac injury development 
Rational for Performing the Study 
Only a few studies have looked at the variation of cardiac functional parameters in fetuses affected by 
congenital heart diseases (3-19). These studies demonstrated that fetal cardiac function evaluation could 
be a valuable complement to morphological examination, since significant differences were identified 
between healthy and affected fetuses (3-6, 8-14, 16,19).  
The limited number of studies and their methodological limitations (small sample size, retrospective design, 
heterogeneity of congenital heart diseases included etc.) impede the ability to draw definitive conclusions 
on this matter (3-19). This suggests that more evidence is needed to start employing routinely functional 
parameters to evaluate fetuses affected by congenital heart diseases.  
During the last decades a heterogeneous group of cardiac function parameters and different ultrasound 
techniques have been used to evaluate fetal cardiac function. Previous studies on fetal cardiac function in 
congenital heart diseases evaluated mainly the value of morphometric measurements, the myocardial 
performance index (MPI) and/or tricuspid/mitral annular plane systolic excursion (TAPSE/MAPSE) carried 
out manually (3-19).  
Myocardial performance index is a parameter capable of assessing overall cardiac function because it 
encompasses the systolic and diastolic components of the cardiac cycle and can be used in order to assess 
the two ventricles separately (28).  
Atrioventricular (AV) plane displacement is often expressed as mitral annular plane systolic excursion 
(MAPSE), septal annular plane systolic excursion (SAPSE) and tricuspid annular plane systolic excursion 
(TAPSE) and it is a major contributor to cardiac pumping and a measure of longitudinal cardiac function. 
Congenital Heart 
Disease  
= 
Fetal Cardiac Injury 
Phase 1: 
 
Subclinical Fetal 
Cardiac Dysfunction 
Phase 2: 
 
Clinical Cardiac Failure  
(Hydrops) 

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Longitudinal cardiac function executed by longitudinal myocardial fibers is considered the first affected 
when hypoxia occurs (23). 
The translational potential of fetal cardiac functional parameters is constrained by the lack of standardised 
methodology for demarcation of the time periods used in their calculation and the need for improvement 
of its repeatability and ease of measurement (22,23). Machine learning application to fetal cardiology and 
automation of the measurement may be considered as the next step towards such improvement leading to 
increase the applicability of fetal cardiac function parameters in standard clinical practice (22-24).  
The term ‘automation’ refers to the act of taking measurements from Doppler waveforms. In conventional 
imaging, this is undertaken manually by the sonographer or sonologist, introducing a number of potential 
points for human error where cursor positions are chosen. In contrast, automation refers to the technique 
of using engineering techniques to select the correct point(s) for measurement of Doppler indices. This has 
been previously shown by our group to b technically possible and to result in perfectly repeatable 
interpretation of waveforms (ICC 1.0).  
Furthermore, fetal cardiac parameters could potentially be of great value at identifying fetuses at risk of 
developing hydrops and cardiac failure (20,21). A cardiovascular profile score has been recognised as a 
valuable support to select fetuses at risk of developing hydrops (29-31).    
As far as we are aware, the predictive value of automated cardiac parameters and their additional 
specificity and sensitivity at estimating the risk of hydrops and cardiac failure has not been previously 
explored. This could be of great value in improving the cardiovascular score profile predicting value of fetal 
cardiac failure.     
Study Objectives 
The aim of the study is to determine the value of automated functional cardiac parameters in the 
evaluation of fetal congenital heart diseases.  
In previous studies published by our group automated PWD-Mod MPI and STIC MAPSE/TAPSE were 
validated in healthy pregnancies (32,33). Automated PWD-MPI and STIC MAPSE and TAPSE have been 
shown to be the most useful indicators and most reliably achievable automated cardiac functional 
parameters in a normal fetal population by previous work by our research group (personal 
communication). 
We now intend to apply these parameters to both healthy pregnancies and those affected by congenital 
heart diseases. We also wish to progress application of our offline engineering techniques to automation of 
measurement of other Doppler indices.  
Primary Objective 
Our primary objective is to evaluate automated PWD-MPI and STIC TAPSE SAPSE and MAPSE comparing 
fetuses affected by congenital heart disease (CHD) to reference values across the fetal healthy population. 
More specifically the aim is to estimate whether there is a significant difference in each parameter 
between fetuses with CHD overall compared to healthy fetuses and then by subgroups of different CHDs. 
Furthermore, the analysis will be looking at the variation of these parameters over time comparing healthy 
fetuses to CHD overall and by subgroups.   
Secondary Objective 
Our secondary objective is to estimate whether automated functional cardiac parameters could improve 
the predictive value of cardiovascular profile score in case of hydrops; facilitating earlier or optimised 
intervention in the form of elective delivery. 

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Expected outcomes of the study 
The expected outcome of the study would be to demonstrate a significant difference in terms of fetal 
cardiac dysfunction between the two groups. It is anticipated that the standardisation provided by 
automation of measurement will make this difference easier to detect.  
If this is shown to be correct, the automated parameters could be used routinely in fetal cardiac evaluation 
of fetuses affected by congenital heart disease. This could inform and support further research to evaluate 
the predictive value of these parameters to help identify fetuses at higher risk of developing incipient 
hydrops/heart failure at need of immediate further interventions.     
Study design 
Design 
An international multicentre prospective observational study will be carried out in seven Teaching Hospitals 
in Europe and Australia.  
Study Groups 
A cohort of pregnant women whose fetus is detected and diagnosed with an isolated cardiac anomaly will 
be approached and recruited during an anomaly scan carried out up to 36+6 weeks gestation (CHD Group). 
A corresponding cohort of pregnant women without any congenital heart diseases will be recruited during 
the second trimester anomaly scan and up to 27+6 weeks of gestation (Control Group). Patients will be 
offered to participate in the study after inclusion/exclusion criteria verification. Informed consent will be 
discussed and signed with the researcher in each centre. 
Number of Participants 
CHD Group: n = 165. Control Group: n = 330 healthy pregnancies. 
Number of Centres 
This will be an Australian SESLHD/UNSW lead study with international centre participants assisting in data 
collection. Each study site will perform the same procedures as internationally published literature (34-57) 
to ensure uniformity. The following is the list of centres and expected number of participants: 
1. Royal Hospital for Women, Randwick, NSW (Lead Centre); CHD Group: 20; Control Group: 40 
2. Centre Hospitalier de Mayotte, Mayotte, France; CHD Group: 25; Control Group: 50 
3. Department of Obstetrics and Gynaecology, Buzzi Children’s Hospital, University Department of 
Clinical Sciences, University of Milan (Milan, Italy); CHD Group: 35; Control Group: 70 
4. Department of Life, Health and Environmental Sciences, University of L’Aquila, Obstetrics & 
Gynaecology Unit, San Salvatore Hospital (Aquila, Italy); CHD Group: 10; Control Group: 20 
5. Maternal and Child Health Institute - IRCCS Burlo Garofolo, Department of Medicine, Surgery and 
Health Science, University of Trieste (Trieste, Italy); CHD Group: 20; Control Group: 40 
6. Sheba Medical Center (Tel Aviv, Israel); CHD Group: 20; Control Group: 40 
7. Fetal Cardiology Unit, Medical Center, Ujastek, Cracow, Department of Pediatric Cardiology, 
Institute of Paediatrics, Faculty of Medicine, Jagiellonian University Medical College (Cracow, 
Poland); CHD Group: 35; Control Group: 70. 
Ethics approval for each international site will be applied for separately in collaboration with the lead 
researcher from each participating centre. 

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Duration 
We estimate the duration to be a total of five years. Considering the sample size calculation and the 
incidence of cases in the different participating centres, we estimate the recruitment phase of the project 
will be 2-3 years. With data collection, analysis, reporting and follow up to an additional two years. 
Participant Selection 
Recruitment and Screening 
Pregnant women will be recruited from each participating centre during their second or third trimester 
morphology scan.  
They will be approached by a member of the research team (Dr Erenbourg) and not by their treating 
doctor. 
The Control Group will be mostly recruited at the time of the second trimester morphology scan; the CHD 
Group during a second/third trimester anomaly scan with collaboration and referral from the hospital fetal 
cardiologist.  
Healthy patients will be approached in the waiting room after their second trimester ultrasound scan, 
offered to participate and given a copy of a patient information sheet and consent form. CHD patients will 
be approached and offered to participate after consultation with perinatal cardiologists. A week after the 
first contact, we will phone patients to verify their intention to take part and to organize the fetal cardiac 
function follow up. Flyers will be placed at the hospitals and further pregnant women interested in 
participating in the study can contact the Research Investigators for further information. 
After checking for exclusion and inclusion criteria, patients will be invited to participate accordingly. If 
women wish to take part, the consent form will be read, discussed and signed with the local researcher.  
Inclusion Criteria 
Inclusion criteria for the CHD Group are as follows: singleton pregnancies; gestational age between 19+6 
and 36+6 weeks gestation, determined by the last menstrual period and confirmed by first trimester 
ultrasound; isolated congenital cardiac anomaly diagnosed. 
Inclusion criteria for the Control Group are as follows: singleton pregnancies; gestational age between 
19+6 and 27+6 weeks gestation, determined by the last menstrual period and confirmed by first trimester 
ultrasound; no congenital cardiac anomaly diagnosed. 
Exclusion Criteria 
Exclusion criteria for both groups are as follows: fetuses whose mothers have comorbidities that have 
been proven to potentially affect cardiac function including intrahepatic cholestastis (34), pre-gestational 
and gestational diabetes (58-61) and preeclampsia (62-64); growth restricted fetuses defined as estimated 
fetal weight or abdominal circumference <3rd percentile for GA (63, 65-69); fetuses with other structural 
extracardiac anomalies at ultrasound examination; fetuses affected by any diagnosed genetic 
abnormalities. 
In a second phase a nested case-control study will be carried out evaluating hydropic fetuses matched to 
no hydrops.   
Study Outline 
Investigation Plan 
Participating patients will be offered a fetal cardiac function echocardiography between 27+6 and 29+6 
weeks gestation and another between 34+6 and 36+6 gestational weeks (Figure 2). If a patient from the 

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congenital heart disease cohort is recruited after 29+6 weeks gestation, a fetal cardiac function 
echocardiography evaluation between 34+6 and 36+6 weeks gestation will be offered.  
At the stage patients will come in to carry out fetal cardiac function echocardiography they will already 
know whether the baby is affected by congenital heart disease since malformation screening is carried out 
between 18 and 20 gestational weeks and will have already been undertaken. In the remote case of 
detecting an undiagnosed fetal malformation during one of the research scans, patients will be reassured, 
and appointments arranged at MFM clinic for further consultation along with notification of to their own 
treating doctor. Dr Erenbourg is a fetal medicine practitioner and Professor Welsh is Professor of Maternal-
Fetal Medicine so in a number of cases immediate counselling will be available.  
Every participant will be assigned a study number following recruiting order of participation. De-
identification will be undertaken at image acquisition by the RHW doctors prior to image review by the 
engineers. Researchers will access an online patients’ form system, collect the assigned patients' number 
and add the necessary outcomes information. Ultrasound images will be saved locally in the ultrasound 
machine by the same assigned patient’s study number and uploaded to a specific onedrive folder created in 
Sydney. 
The research ultrasound scans will be performed by a trained sonographer using either a clinical ultrasound 
system or a research ultrasound system (Verasonics Vantage 256 Ultrasound System: VVS) that is TGA 
approved for research imaging. The machine used clinically is made by one of the common commercial 
manufacturers (in this case General Electric E10) and is an identical model to that used in the Department 
of Matenal-Fetal Medicine. The machine used for research use that we intend to use is manufactured by a 
company called Verasonics and the machine is Vantage 256. It uses the same fundamental electronic 
circuitry and transducer design as conventional commercial machines, and in fact uses the same 
transducers as the commercial machines. However, the way that the ultrasound is delivered differs. Instead 
of transmitting beams as used in conventional ultrasound machines, Verasonics scans the area of interest 
by unfocused waves. The latest allows high quality images with a limited number of compounded plane-
waves, and reduced acquisition time (70,71). There is no difference in the common indices used to monitor 
machine output (the Thermal and Mechanical Indices) from commercial machines. The nature of the 
Verasonics machine means that we have secured TGA approval to use this as a research tool and not one to 
be used for clinical diagnosis. This is the source of the definition of ‘machine approved for research use’. 
Should any concerns arise from a diagnostic perspective during an ultrasound scan, then any images to be 
used for management purposes will be acquired using the clinical (GE E10) machine.  Any information 
generated from the VVS will only be used for research and never for diagnosis or clinical tests. If any 
abnormalities are found while scanning with the VVS, a confirmation scan will be done using our clinical 
ultrasound machine. 
Women will be placed in a semi-recumbent position, as standard for pregnancy ultrasounds. After routine 
biometry, the research fetal cardiac function ultrasound will be carried out. 
Each fetal cardiac function examination will include the following parameters: 
1. 
Fetal biometric parameters (biparietal diameter, head circumference, abdominal circumference, 
femur diaphysis length) 
2. 
Standard fetal Doppler parameters (umbilical artery, medial cerebral artery, ductus venosus) 
3. 
Fetal cardiac heart rate 
4. 
Presence of pericardial effusion or hydrops 
5. 
Cardiac morphometry - all measurements carried out at the end of diastole, with the exception 
of atrial dimensions measured in systole (at their maximum extension) 
- 
Heart/thorax area measurement 
- 
4 heart chambers measurements (apical/basal 4-chambers view in 2D) 

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- 
Atrial and ventricular areas (apical/basal 4-chambers view in 2D) 
- 
Ventricular and atrial sphericity calculation 
- 
Inter-ventricular septum and myocardial walls thickness measurement (transversal 4-
chambers view in 2D or M-mode) 
6. 
Cardiac contractility 
- 
STIC M-Mode stroke volume, ejection fraction and shortening fraction 
- 
Automated STIC MAPSE, TAPSE, SAPSE 
- 
Automated PWD Left and Right Mod-MPI  
7. 
AV valves function evaluation 
- 
Cine-loop evaluation of correct opening and closing 
- 
Anterograde Colour Doppler without regurgitation 
- 
Pulsed Doppler evaluation of flow velocity (monophasic or biphasic) 
- 
Left and right E/A ratio calculation 
- 
If any regurgitation: peak velocity and duration quantified   
8. 
Aorta outflow evaluation 
- 
Aorta artery measurement (at the level of valvular ring in systole) 
- 
Aortic flow evaluation (Colour Doppler evaluation of systolic peak velocity)         
9. 
Pulmonary outflow evaluation 
- 
Pulmonary artery measurement (at the level of valvular ring in systole) 
- 
Pulmonary flow evaluation (Colour Doppler evaluation of systolic peak velocity) 
10. 
V-sign evaluation 
- 
Confirmation of anterograde flow in the entire length of the arteries 
- 
Pulsatility index of aortic isthmus and ductus arteriosus 
All fetal morphometric and functional cardiac parameters will be normalised to Z-score by gestational age 
where possible. Fetal cardiac volumes and 2D images with inadequate quality due to fetal movements, 
presence of acoustic shadows of fetal ribs or spine, and maternal breathing will be excluded.  
If hydrops develops, cardiovascular profile score will be added to the routine cardiac function exploration 
as shown in Table 1.  
The study population will be followed up until delivery and discharge of both mother and neonate.  
Patients’ information will be collected anonymously. Each patient’s history will be evaluated and 
information about previous pregnancies (maternal or fetal diseases during pregnancy) and outcomes (type 
of delivery, maternal and neonatal conditions at birth, long-term outcome of the pregnancy) will be 
collected.  Furthermore, we plan to collect information about the current pregnancy (maternal and fetal 
observations during pregnancy) and outcomes (type of delivery, maternal and neonatal conditions at birth 
and up to hospital discharge of both).     
Study Procedure Risks 
There is no increased risk related to participating in this study. The study uses conventional ultrasound 
machinery as used in routine fetal evaluation, with no alteration in power output (as defined by Thermal 
Index or Mechanical Index). The VVS research ultrasound system is also comparable to conventional 
ultrasounds in terms of risks. 
We anticipate approximately 20-30 minutes scan duration for acquisition of the necessary research data 
which is in keeping with standard ultrasound examinations, and the ALARA (As Low As Reasonably 
Achievable) principle conventionally applied in fetal imaging (73). 
 
 
 

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Table 1: The cardiovascular profile score (72) 
Category 
2 points 
1 point 
0 point 
Hydrops 
None 
Ascites or pericardial 
or pleural effusion 
Skin oedema 
Heart size (HA/CA) 
> 0.2 and ≤ 0.35 
0.35 - 0.5 
< 0.2 or > 0.5 
Cardiac function 
Normal MV and TV, 
biphasic diastolic 
filling, LV or RV SFs > 
0.28 
Holosystolic TR or LV 
or RV SFs < 0.28 
Holosystolic MR or TR 
dP/dt < 400, 
monophasic diastolic 
filling 
Arterial Umbilical 
Doppler 
Normal  
AEDV 
REDV 
Venous Doppler  
Normal  
DV atrial reversal 
UV pulsations 
AEDV, absent end-diastolic velocity; DV, ductus venosus; HA/CA, heart to chest area ratio; LV, left ventricle; MR, mitral valve; REDV, 
reversed end-diastolic velocity; RV, right ventricle; SF, ventricular shortening fraction; TR dP/dt, change in pressure over time of TR 
jet; TR, tricuspid valve regurgitation; TV, tricuspid valve; UV, umbilical vein  
 
Sample Size Calculation 
The primary outcome of the study is the difference in the mean of automated fetal functional cardiac 
parameters between CHD cases and controls. This will be analysed using a two-sample t-test. 
To estimate our sample size, we used the most commonly applied fetal functional parameter, specifically 
the left ventricle MPI (LV-MPI) as a proxy of all the automated fetal cardiac parameters.  
Due to the rarity of congenital heart disease, we performed the sample size calculation based on recruiting 
two controls for each case. Pooled across cases with isolated pulmonary valve stenosis (n = 16) and controls 
(n = 48), Guirado et al (2018) observed a standard deviation of 0.098 in LV-MPI measurements (4). Using 
this observed pooled SD, we require a total sample of 381 pregnancies (127 CHD + 254 controls) with 
completed measurements to achieve at least 80% power to detect a difference of 0.03 in mean LV-MPI (4), 
with a two-sided type I error rate of 5%.  
We acknowledge that some pilot data may be required to evaluate the limited number of pathological 
cases and therefore some approximations are necessary e.g. for standard deviation within the population. 
For this reason, we have aimed to recruit a larger number of participants (approximately 30%), 165 CHD 
and 330 Controls, allowing also for some patient exclusions due to patient drop out, difficulties in scanning 
due to fetal movements etc, and incomplete data sets. 
The aim would be to recruit sufficient cases to be able to estimate if there is significant difference in terms 
of fetal cardiac function parameters between affected and not affected fetuses to inform further research. 
Data Analysis Plan 
Raw (radio-frequency) ultrasound data generated using the VVS will allow the researchers to analyse the 
signal/image processing that takes place prior to display, enabling refinement of this imaging technique. 
Image analysis will be carried out first manually through optical evaluation and then through the use of 
mathematical algorithms which will recognise and analyse only high-quality images. This could be a 
limitation because automatically only high-quality images will be included (which is not representative of 
real clinical work) but also guarantees that parameters are collected only from almost perfect research 
material (showing true differences if they exist).  

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Ultrasound images will be analysed and cardiac function parameters interpreted by SESLHD/UNSW 
researchers based at the Royal Hospital for Women, Randwick. Images will be analysed by a team of fetal 
medicine doctors at Royal Hospital for Women and UNSW engineers (led by Professor Tracie Barber) to 
assure that algorithms are correctly applied to calculate fetal cardiac function parameters.  
Comparisons of interest between cases and controls in baseline characteristics will be performed using 
two-sample t-tests, Wilcoxon rank-sum tests or Pearson Chi-squared tests, as appropriate.  
Secondary outcomes comparing cases and controls at a single time point will be analysed in a similar way to 
the primary outcome, subject to checks of assumptions. Analyses of changes in fetal function parameters 
over time (i.e. between the 27+6-29+6 and 34+6-36+6 week scans), and the comparison of these changes 
between cases and controls will employ generalised linear mixed models, as appropriate for the parameter. 
Logistic regression will be used to estimate the association between fetal cardiac parameters and the 
incidence of hydrops. Receiver-operating-characteristics-curve analysis will be carried out to assess 
functional cardiac parameters compared to the routinely used cardiovascular profile score to predict 
cardiac failure in fetuses with congenital heart disease. 
Statistical analysis will be performed using SPSS version 22.0 (SPSS Inc., Chicago, IL, USA). 
For those cases without complete data acquisition (i.e. intending but not undertaking a second scan), 
analysis will take place for only the isolated value and not for any temporal change. Their single gestational 
data set of ultrasound measurements will still be included in analysis but excluded from any analysis of 
sequential change. 
Safety 
Adverse Event Reporting 
Any potential breach of privacy will be guarded against by de-identification of data following study 
completion and storage of utilised data in either password-protected electronic databases and/or locked 
filing cabinet within the Perinatal Imaging Research Group office at RHW. 
Should there be any concerns or unexpected findings during the research scanning, the patient’s usual 
antenatal carer will be contacted and appropriate follow-up care will be organised by them. 
Serious adverse event reporting 
No serious adverse events are anticipated but should they occur they will be reported through the primary 
investigator, Professor Welsh and the obstetrical team based at the Royal Hospital for Women. 
Data Safety and Monitoring Board 
To assure high quality data collection, images will be collected at each participating centre by experienced 
fetal medicine doctors who have practiced fetal medicine for over 10 years. At RHW this will be Dr Anna 
Erenbourg. Each image will be stored securely. Data will be anonymised and monitored by researchers at 
SESLHD/UNSW. Researchers at UNSW will also review revise and complete data collection in case of 
missing data.   
Professor Alec Welsh as principal investigator will supervise and guarantee the quality of the data. Dr Anna 
Erenbourg will work both as international coordinator and as site researcher at RHW with this work being 
undertaken for her PhD thesis through UNSW.  
Each participating centre will have a site researcher, a fetal cardiologist who will discuss and sign consent 
form, collect ultrasound images and relevant patients’ data. The data collected will be uploaded and 
securely stored onto the UNSW platform, Dr Anna Erenbourg will be responsible for images analysis, data 
analysis and publication drafting supervised by Professor Welsh and Professor Barber.  

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Anna Erenbourg will propose the study to patients, discuss and sign the consent form and collect data in 
Sydney. Once ethical approval for each overseas center will be granted, Anna Erenbourg (under the 
supervision of Alec Welsh and Tracie Barber) will download data from all centers, merge the information in 
a unique database and analyze them with the help of a statistician at UNSW. All the participants will then 
collaborate in drafting manuscripts for publication.  
Outcome data will be accessed only by authorised researchers using an encrypted code for data protection. 
Final drafts for publication will be reviewed by all the authors from each research site.     
Dissemination of results and publication policy 
Results of the study will be published in peer-reviewed scientific journals, presentations at conferences or 
other professional forums. In any publication, patient privacy will be protected and presented in a de-
identified manner.  
Storage and Archiving of Study Documents 
Patients’ data and ultrasound images will be collected at each participating centre and safely secured using 
a UNSW Research Data Management Plan and the UNSW ResToolKit System for an online data 
management using UNSW servers. Patient privacy will be protected by deidentifying data once initial data 
abstraction has occurred, and through storage of data in password-protected computers and servers. 
Identified data will only be accessible to Professor Welsh and Dr Erenbourg. This data will be stored for a 
minimum of 5 years after publication, then the data record will be securely destroyed in accordance with 
the UNSW Records Disposal processes.  
 
 
 
 
 

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Study Flow Chart 
 
 
 
 
 
 
Figure 2: Patient Recruitment and Study Flow Chart 
 
 

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