Development and Validation of a Prognostic Model of Postnatal Circulation in Fetuses With a Diagnosis of Pulmonary Atresia-critical Stenosis With Intact Ventricular Septum A Prospective Observational Cohort Study
Conditions: Pulmonary Atresia With Intact Ventricular Septum, Congenital Heart Disease, Fetal Cardiac Disorder
Sponsor: Hospital Universitario 12 de Octubre

 
 
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Research program 
 
Development and validation of a prognostic model of postnatal 
circulation in fetuses with a diagnosis of pulmonary atresia-
critical stenosis with intact ventricular septum 
 
A prospective observational cohort study 
 
 

 
 
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Table of contents 
 
Background ....................................................................................................................... 3 
Paradigm ........................................................................................................................... 4 
Hypothesis ........................................................................................................................ 4 
Primary objective .............................................................................................................. 4 
Secondary objectives ........................................................................................................ 4 
Study design ..................................................................................................................... 5 
Inclusion criteria ............................................................................................................... 6 
Exclusion criteria .............................................................................................................. 6 
Echocardiographic examinations ...................................................................................... 7 
Data collection and on-line database ................................................................................ 7 
Electronic case report forms (eCRF) ............................................................................ 8 
Core laboratory ............................................................................................................. 8 
Data analysis and statistical support ............................................................................... 10 
Ethics .............................................................................................................................. 17 
Publication of results ...................................................................................................... 17 
Significance and clinical relevance ................................................................................ 18 
Funding ........................................................................................................................... 18 
 
 

 
 
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Background 
Pulmonary atresia (PA)/critical stenosis (CS) with intact ventricular septum (PA/CS-IVS) 
is a rare congenital heart disease (CHD),1 that presents heterogeneously with severe 
potential implications both for the fetus and the newborn. It implies a significant 
obstruction of the right ventricle (RV) outflow tract, interfering with right ventricular 
development which subsequently will develop a variable degree of hypoplasia. 2 
Prognosis is conditioned by the possibility of achieving a primary repair with 
biventricular circulation (BV) or a one-and-a-half ventricle solution 3 vs. a palliative 
approach bound to a univentricular (UV) circulation in which both survival and quality 
of life are significantly impaired.  
The identification of fetuses that will ultimately be candidates for palliative surgeries/UV 
circulation in postnatal life is key for many reasons: first, it allows for better counseling; 
secondly, it is of great clinical relevance since it allows tailoring fetal follow-up, 
optimizing delivery in a tertiary-care center and, in selected cases, offer the possibility of 
prenatal treatment. Fetal pulmonary valvuloplasty aims to improve RV function and 
reducing the risk of UV circulation, but it is not exempt from risks.4 Therefore, the 
identification of fetuses at the highest risk of UV circulation (hypothetically those who 
would benefit most from this therapy) is of utmost interest, especially between 20 and 28 
weeks, which is when this intervention would be performed. 
Predicting UV circulation prenatally is still a challenge. A systematic review and meta-
analysis showed that certain commonly measured fetal cardiac variables have strong 
associations with postnatal treatment pathway choice.5 Several individual parameters and 
multiparametric scores have been proposed for the prediction of postnatal circulation, 
most of them based on small series of cases.6,7 A recent external validation of available 
models found that multiparametric models targeting UV and non-BV circulation have a 
relatively good performance with AUCs ranging between 0,80 and 0,89 with low false 
positive rates, but they underestimate the risk of a UV outcome. However, said validation 
could only be performed in a small group of fetuses (between 15 and 58, depending on 
the model). Furthermore, it was highlighted that there was high heterogeneity in the 
parameters evaluated and reported, precluding studies from being pooled together.8  
To address the lack of uniform criteria and optimal prognostic models when evaluating 
these fetuses, we are undertaking a prospective observational cohort study to predict the 

 
 
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type of postnatal circulation at diagnosis when the natural history of PA-CS/IVS is not 
altered. 
Paradigm 
PA-CS/IVS may progress to right heart hypoplasia with UV circulation after birth. 
Different prognostic models of postnatal circulation have been developed in small cohorts. 
Although multiparametric models have shown a relatively good performance, they 
underestimate the risk of UV circulation. 
Hypothesis 
A prospectively developed model can accurately predict transplantation-free survival 
with a biventricular or a one-and-a-half repair at 2 years postnatal age in fetuses with 
suspected PA-CS/IVS between 16 and 28 weeks of gestation. 
Primary objective 
To develop and validate a prognostic model for the prediction of transplantation-free 
survival with a biventricular or a one-and-a-half repair at 2 years postnatal age in non-
intervened fetuses with PA-CS/IVS between 16 and 28 weeks of gestation. 
Secondary objectives 
a) To develop and validate a prognostic model for the prediction of transplantation-free 
survival with a biventricular or a one-and-a-half repair at 1 year of postnatal age in 
non-intervened fetuses with PA-CS/IVS between 16 and 28 weeks of gestation. 
b) To develop a second model including variables obtained during prenatal follow-up to 
predict transplantation-free survival with a biventricular or a one-and-a-half repair at 
2 years postnatal age in fetuses with PA-CS/IVS. 
c) To develop a final model to predict the risk of right ventricle dependent coronary 
circulation 
d) To evaluate prenatal and postnatal outcomes in non-intervened fetuses with a 
confirmed postnatal diagnosis of PA-CS/IVS. The following outcomes will be taken 
into account: 
- 
Intrauterine death 
- 
Neonatal/Infant death in the first 2 years 

 
 
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- 
Number and type of required postnatal surgical and catheter interventional 
procedures 
- 
O2 saturation at 30 days  
- 
Need for cardiac transplantation in the first two years 
- 
Presence of RV dependent coronary circulation confirmed by cath or autopsy 
Study design 
This is an international prospective observational cohort study including non-intervened 
fetuses with a diagnosis of PA-CS/IVS between 16+0 and 28+6 weeks of gestation.  
A baseline fetal ultrasound examination will be recorded for all included cases. After such 
initial diagnosis, the number of mother/fetus/infant examinations will not be different 
from the number of examinations that would otherwise be recommended for someone 
choosing not to be part of this study, but only one additional follow-up scan performed 
preferrably at 32-34 weeks and at least 8 weeks apart from the baseline diagnostic scan 
will be recorded in the study. Finally, in liveborn cases, the first complete postnatal 
echocardiographic examination will be included as well.  
The participation in the study will not affect the treatment mothers and fetuses receive 
during pregnancy, nor how the infant is examined and treated after birth. That is, if during 
follow-up a patient is offered prenatal intervention according to local criteria, the case 
will be excluded from analysis, but the decision to offer such treatment should not be 
conditioned by the participation in the study. 
Although the individual characteristics of each patient should be the final determinants 
of the surgical approach, a consensus in general management according to the following 
recommendations should be agreed upon (Annex 1) and all of the following postnatal 
treatment options need to be available: 
1. Neonatal intensive care unit 
2. Neonatal and pediatric cardiac surgical unit 
3. Neonatal and pediatric catheterization laboratory  
The primary aim of the study is to develop a prognostic model for the type of postnatal 
circulation (transplantation-free survival with a biventricular or a one-and-a-half repair at 
2 years postnatal age vs univentricular palliation or transplanted). In order to reduce the 
risk of selection bias, missing data, and inter-variability between participating centers, 
data will be collected in a prospective and organized way, and there will be a minimum 
consensus in management. 

 
 
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Cases will be recruited in referral centers regardless of their offering of fetal pulmonary 
valvuloplasty. However, only non-prenatally-intervened cases will be used for analysis. 
Fetal and postnatal echocardiographic examinations will be reviewed by a core laboratory 
to confirm eligibility for inclusion and identify potential measurement errors. 
Data from at least one fetal echo (the first diagnostic evaluation at the referral center) and 
one postnatal (the first one performed after birth) will be collected, each containing a 
comprehensive set of two-dimensional and Doppler measurements. Additionally, one 
additional follow-up scan performed at least 8 weeks apart from the baseline diagnostic 
scan (ideally between 28 and 32 weeks) will be included in the study as well. Analysis of 
the prenatal change of dimensions of the right heart structures and selected hemodynamic 
parameters will enable comparisons between centers regardless of off-protocol center-
specific postnatal treatment policies. 
Inclusion criteria 
All of the following echocardiographic criteria need to be satisfied between 16+0 and 
28+6 weeks 
- 
Absence of flow at the pulmonary valve (PA) or presence of thickened and domed. 
pulmonary valve cusps with a pinhole jet of flow.9 
- 
Doppler evidence of ductal-dependent pulmonary circulation. 
- 
Intact ventricular septum. 
Exclusion criteria 
- 
Poor imaging windows and incomplete/poor quality scan 
- 
Termination of pregnancy 
- 
Cases initially included that undergo prenatal pulmonary valvuloplasty later on in 
pregnancy.  
- 
Unconfirmed PA-CS/IVS at birth. 
- 
Functional PA-CS/IVS (Ebstein malformation, monochorionic twins) 
- 
Any associated cardiac defect except persistent left superior vena cava and 
aberrant right subclavian artery. 
- 
Any significant (i.e that might influence outcome) extracardiac anomaly and/or 
known genetic syndromes. Also, if such a condition is present at inclusion but 
diagnosed only after birth, the case will be retrospectively excluded. 

 
 
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Echocardiographic examinations 
There will be at least two echocardiographic examinations in the study: the baseline 
prenatal and the first neonatal ultrasound examinations. The protocol for both prenatal 
and postnatal examinations is described in Annex 2, and the requested parameters can be 
found in Annex 3. 
The recorded ultrasound examinations are as follows: 
1. Baseline fetal ultrasound examination between 16+0 and 28+6 weeks 
2. Follow-up fetal ultrasound examination at 32-34 weeks of pregnancy and at least 
8-week apart from the initial diagnostic scan.  
3. The first complete echocardiographic examination after birth (before the first 
postnatal intervention). 
Data will be used for  
1. Inclusion criteria (baseline fetal echo) 
2. Development of primary aim prognostic model (baseline fetal echo). 
3. Development of secondary aim prognostic model: longitudinal assessment of 
cardiac growth and hemodynamics  
Z-scores 
For the study purpse, z-scores will be according to the charts indicated below. These do 
not need to be the same as the ones used in clinical practice in different centers although 
they will be asked which one has been used. 
Z-scores for fetal cardiac measurements will be calculated by gestational age 10 . 
Gestational age will be calculated according to crown-rump length11 in the first trimester 
and biparietal diameter in the second trimester (if the prior is not available)12 
Z-scores for postnatal cardiac measurements will be derived by body surface area (Boston, 
https://zscore.chboston.org) 
Data collection and on-line database 
Collection of the clinical data and echocardiographic measurements will be through an 
Electronic Case Report Form developed in an online database managed by RedCap13, 
hosted by the Instituto de Investigación 12 de Octubre (imas12). 
RedCap is compatible with most modern web browsers, including updated versions of 
Chrome, Safari and Firefox.  

 
 
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For detailed descriptions of RedCap security, workflow, and eCRF functionality, please 
see the RedCap website (https://www.project-redcap.org/) 
Electronic case report forms (eCRF) 
There are 8 tabs in the eCRF, each dedicated to a different aspect of the evaluated case. 
A description of every recorded variable in the eCRF is included in Annex 3. 
1. eCRF Inclusion criteria 
All inclusion and no exclusion criteria need to be satisfied for a case to be included. 
2. eCRF Baseline echo 
Include baseline echo if inclusion/exclusion criteria satisfied as well as reported 
counseling. 
3. eCRF Additional follow-up fetal echos 16+0 until 28+6 
Addicional exam separated at least a 8-weeks from the original one. 
4. eCRF last Follow-up fetal echo 16+0 until 42+0 weeks 
The last follow-up echo (if it exists) available before delivery. 
5. eCRF Pregnancy outcomes 
Termination of pregnancy, intrauterine death, or stillbirth. 
Gestational age and mode of delivery. Newborn’s weight, height, and Apgar score 
6. eCRF Neonatal echo 
First complete study after birth and before the first postnatal intervention, surgery 
or catheterization.  
7. eCRF Neonatal discharge 
Performed procedures before discharge 
Type of circulation and clinical diagnoses at discharge 
8. eCRF Follow-up at 2 years 
All cardiac surgical and catheter interventional procedures from initial discharge 
to 2 years follow-up. Outcomes will include the type of circulation, clinical 
diagnoses, neurological sequelae, and need for cardiac transplantation.  
 
Core laboratory 
At least one case per participating center including all required prenatal and neonatal 
videos will be uploaded to a core laboratory FTPS server managed by University of 
Gothenburg (IT Department, Khamees.Elkhateeb@gu.se). This will serve to establish 
that all centers are performing measures according to the same protocol.  

 
 
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Required video of prenatal scan: echocardiographic assessment of the fetal heart from 
which the following measurements are drawn 
• Right ventricle length (mm) 
• Left ventricle length (mm) 
• Right ventricle inlet (mm) 
• Left ventricle inlet (mm) 
• Tricuspid valve (mm) 
• Mitral valve (mm) 
• Pulmonary valve (mm) 
• Aortic valve (mm) 
• Pulmonary annulus (mm) 
• Aortic annulus (mm) 
• Right ventricle inflow duration (ms) 
• Cardiac cycle duration (ms) 
• Tricuspid regurgitation velocity (m/s) 
• Tricuspid regurgitation (mild/moderate/severe): a sample of one of the three 
will suffice 
• Pulmonary antegrade flow (yes/no): a sample of one of the two will suffice 
• Atrial flow by color Doppler (normal; bidirectional; right-to-left; none-intact): 
a sample of one of the three will suffice 
 
Required video of postnatal scan: echocardiographic assessment of the neonatal heart 
from which the following measurements are drawn 
• Tricuspid annulus (4 chamber view) (mm) 
• Mitral annulus (4 chamber view) (mm) 
• Tricuspid annulus (PEEL posterior) (mm) 
• Right ventricle morphology ( 4 chamber view): unipartite/bipartite/tripartite 
qualitative evaluation (a single one will suffice) 
• Right ventricle diastolic diameter (parasternal long axis view) (mm) 
• Pulmonary annulus diameter (parasternal short axis view) (mm)  
• Pulmonary artery diameter (parasternal short axis view) (mm) 
• Right pulmonary artery (parasternal short axis view) (mm) 
• Left pulmonary artery (parasternal short axis view) (mm) 
• Interatrial shunt (subcostal view): qualitative (none/small/moderate/large) a 
single one will suffice 
• Tricuspid inlet jet (scale 40-60cm/s) (4 chamber view): quantitative 
• Tricuspid inlet jet / mitral inlet jet ratio (4 chamber view) 
• Tricuspid regurgitation jet (vena contracta, maximum scale) (4 chamber view): 
quantitative 
• Interatrial 
shunt 
(subcostal 
view): 
qualitative 
(right-left/left-
right/bidirectional/no shunt) a single one will suffice 
• Antegrade pulmonary flow: yes/no (cualitative) a single one will suffice 
• Ventriculo coronary connections: yes/no a single one will suffice 
• TAPSE (4 chamber view, mm) 
 

 
 
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The core lab will have an audit function. During the study period, a random recording or 
one in which data from the eCRF needs confirming can be asked to be uploaded to the 
core lab for further evaluation. A data transmission form (DTF) should be included with 
each submitted exam containing data on RedCap-generated patient code, date of upload 
to server, make and model of the ultrasound machine, and name of echocardiographer. 
Echocardiographic variables will be reported to RedCap by the submitting center and 
measurements will be repeated on uploaded echoes by the core lab without knowledge of 
submitted data. Submitted measurements differing by more than 20% from corresponding 
core lab measurements will be re-measured by a second reviewer, the core lab PI. If there 
is continued disagreement, the core lab will contact the submitting center for a review of 
the original measurement, and if this was in error the submitted value will be replaced by 
the core lab value provided that the image quality is acceptable. If that is not the case, the 
value will be excluded from the analysis. A similar approach will be used for variables 
based on qualitative or semiquantitative measurements such as the magnitude of 
regurgitant jets on Color Doppler and ventricular function. 
Data analysis and statistical support  
Study details 
Objectives:  
- 
Primary: To develop and validate a prognostic model for the prediction of 
transplantation-free survival with a biventricular or a one-and-a-half repair at 2 
years postnatal age in non-intervened fetuses with suspected PA-CS/IVS between 
16 and 28 weeks of gestation. 
- 
Secondary:  
a. To develop a second model including variables obtained during prenatal 
follow-up to predict transplantation-free survival with a biventricular or a 
one-and-a-half repair at 2 years postnatal age in fetuses with suspected 
PA-CS/IVS 
b. To evaluate prenatal and postnatal outcomes in non-intervened fetuses 
with a confirmed postnatal diagnosis of PA-CS/IVS. The following 
outcomes will be taken into account: 
o Intrauterine death 
o Neonatal/Infant death 
o Number of required postnatal procedures 

 
 
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o Need for oxygen support 
o Need for cardiac transplantation 
o Presence of RVDCC 
Design: This is a prospective cohort study to develop a model to predict between 16 and 
28 weeks of gestation, transplantation-free survival with a biventricular or a one-and-a-
half repair at 2 years postnatal age. 
The number of examinations of mother/fetus/infant in this study is not different from the 
number of examinations that will be recommended for someone choosing not to be part 
of this study. Participation in the study should not affect the treatment mothers and fetuses 
receive during pregnancy, nor how the infant is examined and treated after birth. The aim 
of the study is to, in a prospective and organized way, collect and evaluate multi-center 
data in order to reduce the risk of selection bias, missing data, and inter-variability 
between participating centers. Women will be recruited from referral centers with at least 
5 prenatally diagnosed cases per year. Fetal and postnatal echocardiographic 
examinations will be reviewed and remeasured by a core laboratory to confirm eligibility 
for inclusion and identify potential measurement errors. Data from at least the diagnostic 
prenatal echo and the first neonatal one will be collected, including, if available, all exams 
between 16+0 and 42+0 weeks (with at least a 6-week interval). Analysis of change in 
dimensions of the right heart structures and selected hemodynamic variables from the 
point of study inclusion until just before the first postnatal catheter or surgical procedure 
provides an opportunity to make an unbiased evaluation with respect to off-protocol 
center-specific postnatal treatment policies. 
Inclusion/exclusion criteria:  
Inclusion criteria 
All of the following echocardiographic criteria need to be satisfied between 16+0 and 
28+6 weeks 
- 
Absence of flow at the pulmonary valve (PA) or presence of thickened and domed 
pulmonary valve cusps with a pinole jet of flow14 
- 
Doppler evidence of ductal-dependent pulmonary circulation 
- 
Intact ventricular septum 
Exclusion criteria 
- 
Termination of pregnancy 
- 
Cases initially included that undergo prenatal pulmonary valvuloplasty later on in 
pregnancy.  

 
 
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- 
Unconfirmed PA-CS/IVS at birth 
- 
Functional PA-CS/IVS (Ebstein malformation, monochorionic twins) 
- 
Any associated cardiac defect except persistent left superior vena cava and 
aberrant right subclavian artery 
- 
Any significant (i.e, that might influence the outcome) extracardiac anomaly 
and/or known chromosomal aberration. Also, if such a condition is present at 
inclusion but diagnosed only after birth the case will be retrospectively excluded 
Randomization: Not applicable 
Sample size: Based on the results of two systematic reviews, there were 8 potential 
variables identified for model development. Considering these 8 variables and assuming 
a prevalence of the evaluated outcome (biventricular or one and a half solution at two 
years of age) of 75% in our setting, the minimum sample size required for new model 
development based on user inputs would be 289, with 217 events, a shrinkage of 0.9 and 
24.1 Events per Predictor Parameter 15 . However, after carrying out a feasibility 
evaluation, considering the low prevalence of the disease and that the study should be 
carried out only in referral centers, it would not be possible to reach this figure in a 
reasonable time since this recruitment would take an excessively long time, increasing 
costs and would ultimately end up being exposed to changes in the state of the art of 
disease management and that the model would become obsolete once its development is 
completed. Therefore, it is proposed to include 200 cases in the study, of which 160 will 
be used for the development of the model and 40 for validation. This would imply a 
reduction of 16 events per parameter and widen the confidence interval of the resulting 
prediction. However, these 16 events far exceed the classical rule of thumb of 10 per 
parameter and even the 15 proposed by Harrell. Furthermore, this sample size is 
considered sufficient for the development of the proposed artificial intelligence models, 
where the low number of inputs (8) allows their development in a reasonable manner with 
120 patients. Considering that the model developed on the largest population to date has 
used 38 patients and the foregoing, a sample size of 200 patients represents a great 
advance over what is available.  
Framework: Development of a multivariable prediction model based on TRIPOD 
statement16 and PROGRESS group recommendations17.  
 
Study population and participants 

 
 
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Screening data: All pregnant women with a fetus with suspected PA-CS/IVS will be 
screened for eligibility. If they meet all the inclusion criteria and none of the exclusion 
ones, they will be offered to participate. A complete count of screened, eligible, and 
included participants will be detailed in the final report of the study. 
Eligibility: As described in the prior section, cases must meet all the inclusion criteria, 
and none of the exclusion ones. 
Recruitment: A flow chart of participants from baseline through completion of the study 
will be presented in the final report. 
Withdrawal/Follow-up: Rates and reason for withdrawals, as well as lost-to-follow-up 
rates and account for these individuals, will be reported accordingly in the analysis 
(removed from final analysis). Participants found not eligible after inclusion 
(unconfirmed postnatal diagnosis) will also be removed.  
The timing of withdrawal will also be reported for participants that withdraw. 
Study populations 
- 
Full analysis set for All Enrolled: All included fetuses, regardless of their intention to 
treat at birth. 
- 
Full Analysis Set for Live Births Intended for Postnatal Treatment: All enrolled 
fetuses that have satisfied inclusion/exclusion criteria that also are live at birth will be 
included in the Full Analysis Set at birth population. Infants wrongly included in the 
study (not confirmed postnatal diagnosis) will not be part of the study population, but 
will be described as excluded cases. 
Full analysis set (FAS) 
 
 
 
 
 
 
 
 
 
Chronogram 
Contact with reference centers and assignment of local principal investigators (4 months): 
February 2024 – May 2024. 
Included 
fetuses at 
16+0 – 
28+0 
FAS 
enrolled 
FAS birth 
Exclusion criteria during follow-up 
or at birth
Termination of 
pregnancy 
Intrauterine death 

 
 
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Publication of the study protocol (5 months): February – June 2024 
Evaluation of perinatal care policies in the different participating centers, analysis and 
publication of results (6 months): June 2024 – December 2024 
Patient recruitment (2 years): in our center it will begin immediately after approval of the 
Ethics Committee expected in February; February 2024 until December 2025. In the 
different participating centers, the same process will be carried out, varying the 
recruitment start date depending on the acceptance of their Ethics Committee, 
maintaining the inclusion deadline of December 2025. 
Collection and analysis of variables (48 months): Parallel to recruitment and up to 2 years 
after the birth of the last newborn in December 2026 
Development of the first predictive model for one-year results and publication of results 
(6 months): January – June 2026 
Statistical analysis, writing of manuscripts, and publication of final data (6 months): 2027 
and the beginning of the following year – February 2028. 
Presentation and publication of results (3 months): May 2028 
 
Study variables 
A list of all included variables is depicted in Annex 3.  
Baseline patient characteristics: Participants will be described according to the baseline 
variables 
Predictors: After a careful literature review, the following 8 variables will be considered 
for inclusion in a multivariate predictive model. The measurements used for model 
development will be those from the inclusion echo according to the protocol described in 
Annex 2. 
- 
Tricuspid valve z-score 
- 
Presence of ventriculo coronary connections 
- 
Right ventricle/Left ventricle length ratio 
- 
Tricuspid valve/Mitral valve inlet ratio 
- 
Pulmonary annulus / aortic valve ratio 
- 
Tricuspid regurgitation (none, mild, moderate, severe) 
- 
Right ventricle inflow duration/ Cardiac cycle duration (%) 
- 
Pulmonary atresia vs critical stenosis 
Neonatal variables: as described in Annex 3 
Follow-up at 2 years variables: as described in Annex 3 

 
 
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Statistical analysis 
Continuous variables will be presented as mean (standard deviation) or median 
(interquartile range) if non-normally distributed. Normality will be tested with the 
Shapiro-Wilk test. Categorial variables will be presented as count (percentage). 
The main analysis will be conducted on the FAS enrolled population, and additional 
analysis on the FAS for Live Births Intended for Postnatal Treatment 
Results will be stratified by the primary outcome. Univariate comparisons between 
variables will be performed using the appropriate tests (Student´s t-test or Mann–Whitney 
U-test for continuous and chi-square or Fisher’s exact test for categorical variables). 
Analysis of longitudinal change of left heart size and function will be conducted in an 
exploratory manner for the second model. This will be investigated using mixed effects 
regression models for repeated data, with normal distribution for z-scores and binomial 
distribution for binary variables, handling time in the study both as distinct visits and 
continuous (gestational age) as found most appropriate. Linear and non-linear models will 
be investigated. Model selection will be based on the lowest goodness of fit statistics, e.g. 
Akaike’s Information Criterion.  
Confidence intervals and p values: 95% confidence intervals and p values will be reported 
for statistical models to aid in interpretation. Statistically significant p values will be 
considered when <0.05 in two-tailed distributions. 
Adherence and protocol deviations: This is an observational study. However, a change of 
prognosis that implies the performance of fetal intervention will be considered a major 
protocol deviation. The clinical monitors of the study will review the list, and the 
finalization of the major protocol deviations will be done at the clean file meeting. 
The number of patients with major protocol deviations will be summarized 
Statistical Interim Analysis: No interim analysis is planned until patient recruitment and 
follow-up have concluded, as this is not an interventional study. 
Timing of final analysis and outcome assessments: All outcomes will be analyzed 
collectively. All outcomes can occur at any time point during the 2-year surveillance 
period.  
Model development: The criteria for transparent reporting of a multivariable prediction 
model for individual prognosis or diagnosis (TRIPOD) will be followed for model 
development (Annex 4). A prediction model using logistic regression will be performed, 
with the outcome “transplantation-free survival with a biventricular or a one-and-a-half 

 
 
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repair at two years postnatal age”. The eight preselected variables will be evaluated in a 
univariate analysis comparing both groups, and those with p<0.10 will be entered into a 
logistic regression model. 
Following primary model development, models will be internally validated to decrease 
optimism through bootstrapping with 1000 iterations.  
Model performance: Model performance will initially be investigated by assessing 
overall performance, classification, and discrimination. Overall performance will be 
evaluated through Nagelkerke R, in which the amount of variability in outcomes is 
explained by the prediction model, and the Brier score, a performance measure for the 
distance between observed and predicted outcome. Classification will be assessed by 
sensitivity, specificity, positive predictive value, and negative predictive value. 
Discrimination, which describes the ability of the prognostic model to distinguish 
between patients with and without the outcome, will be estimated with the area under the 
receiver operating characteristic curve (AUROC) and graphed the sensitivity against 1-
specificity for consecutive cutoffs for the predicted probability of an outcome.  
Model validation: The predictive ability of the models will be subsequently tested based 
on discrimination, calibration, and measures of overall performance. The measures of 
overall performance and discrimination will be the same as those described in the 
development phase. Calibration refers to the agreement between observed and predicted 
probability. Models will be assessed graphically and estimated with the intercept and 
slope calibration. In case of perfect fit of the model to the data, calibration intercept is 
equal to 0 and slope equal to 1. Calibration will be quantified by the calibration slope 
(with 95% confidence intervals) and graphically by plotting the predicted risk against the 
observed outcome using a loess smoother.   
Missing Data: All data will be tested for missing values prior to analyses. Missing data 
will be categorized as missing completely at random, missing at random, or missing not 
at random. If missing data is below 5%, a complete case analysis will be performed. If 
missing data is above 5%, multiple imputation will be performed. Imputation iterations 
will be at a minimum of 20 iterations and will proportionally increase with the percentage 
of greatest missing data. Missing outcomes will be marked prior to imputation, with 
outcomes imputed. However, imputed outcomes will be excluded from analyses. It 
should be noted if data is categorized as missing not at random, multiple imputation will 
not be performed on these data, and will be potentially excluded from analyses.  
Additional Analyses: 

 
 
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Machine Learning  
Following primary model development, internal validation, and assessing model 
performance, four machine learning models (Random Forest, Support Vector Machine 
Regression, Gradient Boosting Machine, and Artificial Neural Networks) will be 
developed to predict the primary outcome. All machine learning models will incorporate 
all the same predictors used to develop the logistic regression model. An iterative matrix 
process will be performed to assess optimal tuning hyperparameters for all machine 
learning models, with root mean square error utilized to determine optimal tuning. All 
models will then be internally validated to reduce overfitting with 10-fold cross-
validation. Machine learning models will also be used to determine the importance and 
weight of predictors to help inform clinical practice and decision making.  
Harms:  
N/A. This is an observational cohort; not a randomized clinical trial 
Statistical Software and support 
The computer software programs Stata and Python will be used for all analyses. Statistical 
support will be provided by the Statistics Service of the imas12 Research Institute. 
Ethics 
The study was approved by the Institutional Review Board in Hospital 12 de Octubre, 
Madrid (pending). Each participating institution/country (as required) will submit an 
application for ethical approval. The consent form used for the ethical application in 
Spanish is available in an English version. 
Publication of results 
The results will be reported in different papers. Co-authorship will be based on the criteria 
published by ICMJE (18). 
Steering group 
An international steering group will be assembled for the development of the project. At 
least one representative from every participating country will be invited to join the 
steering committee. 

 
 
Research protocol v2.0 26.Dec.2023 
 
Significance and clinical relevance  
The predictive accuracy of current models has been recently evaluated, multiparametric 
ones being good for the prediction of biventricular outcomes but underestimate the risk 
of a univentricular outcome. Furthermore, most models have been developed on small 
cohorts across long periods of time, making management and prognosis heterogeneous 
and models at high risk of overfitting. Finally, the role of pulmonary valvuloplasty has 
been considered as well and even though it seems it allows better ventricular growth, 
candidate selection is key as it is a high-risk procedure. 
Therefore, the first goal towards proper counseling and management of these pregnancies 
should be based on developing and validating a model aiming to solve the described 
pitfalls. From then on, patient selection would be more appropriate and a randomized 
clinical trial could be designed to evaluate the benefit of prenatal fetal intervention.   
Funding 
Instituto de Salud Carlos III (Spanish Ministry of Economy, Industry, and 
Competitiveness): grant number PI23/00776 
 

 
 
Research protocol v2.0 26.Dec.2023 
 
Annex 1. Proposed postnatal management  
1. The newborn will be admitted to the intensive care unit and started on PGE1 treatment (0.01-0.03ug/kg/min). 
2. Within 48h after delivery, a complete anatomical and functional cardiological evaluation will be performed according to the protocol 
described in Annex 2 (postnatal echo) 
3. Postnatal management will be guided according to the following flow-chart 
 
Right ventricle
Right ventricle coronary 
dependent circulation
Univentricular 
physiology
(do not open 
pulmonary valve)
Mild or moderate hypoplasia
(consider biventricular approach)
Open the 
pulmonary valve
Situation after arterial 
duct closure/PGE 
withdrawal 
Sat O2> 75%
Follow-up
Probable biventricular 
physiology
Sat O2 < 75%
Ductal stent/ S-P Shunt
One and a half physiology: interatrial communication closure, closure of source of additional pulmonary 
flow (if it has not happened  spontaneously), open RVOT and superior cavopulmonary anastomosis
Severy hypoplastic
(atresia/severe tricuspid hypoplasia)
Univentricular 
physiology
Ductal stent (do not 
open pulmonary 
valve)
S-P Shunt (do not 
open pulmonary 
valve) 

 
 
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*** Criteria for progression to of one and a half ventricle physiology during follow-up 
(the decision will be made in a variable amount of time and will depend mainly on two 
factors): 
- 
Severe hypoxemia after ductal/S-P shunt closure 
- 
Insufficient pulmonary valve and significant systemic venous congestion 
o Restrictive RV physiology 
▪ The following echocardiographic parameters will be assessed 
• Systemic venous congestion (hepatic veins and inferior 
vena cava dilation, reversed flow), congestive hepatopathy. 
• Significant right atrial dilation (mild >10cm2/m2; moderate 
10-15cm2/m2; severe >15cm2/m2). 
• Interatrial communication with bidirectional shunt and 
evident right-left shunt. 
• Restrictive filling pattern (pulsed-Doppler, repeated e 
<<<a wave without variability with respiratory cycle) 
o Non-treatable severe tricuspid anomalies (insufficiency and/or stenosis)  
*** The decision-making process will include, in the majority of cases, an interatrial 
communication closure test (evaluation of hemodynamic tolerance, right atrial pressure) 
*** If restrictive RV physiology characteristics persist and the closure of the interatrial 
communication is not well tolerated, a superior cavopulmonary and subsequent total 
cavopulmonary connection might be considered even if initially a biventricular 
physiology was opted for. 
 
 

 
 
Research protocol v2.0 26.Dec.2023 
 
Annex 2. Echocardiography protocol 
a) Prenatal echocardiography (pending to add images/figures) 
General principles 
Image and video quality should be maximized by adjusting system settings and image 
magnification.  
Doppler flow recording should be optimized using the appropriate transducer, <30º angle 
of insonation, adequate pulse repetition frequency (PRF), gate with and baseline 
adjustment for optimal visualization. Doppler velocities should be measured in cm/s and 
pressure gradients in mmHg. 
2-dimensional measurements should be recorded in millimeters (mm) at the maximal 
dimension of the structure. 
- 
Valve dimensions from hinge point to hinge point 
- 
Vascular structures from inner edge to inner edge 
Image acquisition should be done under the ALARA principle that affect the acoustic 
output and by considering the transducer dwell time and total scanning time.18 
Required images and videos 
Still images and clips should be transferred to the FTPS server using DICOM-format 
Each submitted echo exam should include clips (with and without color), still images of 
measured structures and spectral Doppler registrations. Note, the still image should be 
the still image where measurements were made. A minimum of 3-5 consecutive cardiac 
cycles is required. Obtain images avoiding excessive transducer movement. More data 
may be obtained at the discretion of the Echocardiographer. The following views should 
be included: 
 
4 chamber view 
Diameter of the mitral valve (end-diastole) (mm) 
Diameter of the tricuspid valve (end-diastole) (mm) 
Left ventricle: diameter and inlet length in end-diastole (mm) 
Right ventricle: diameter and inlet length in end-diastole. Subjective 
appreciation of reaching apex and arrangement (uni, bi or tripartite). Subjective 
assessment of systolic function (normal, mild, moderate or severe depression). 
Subjective quantification of endocardial fibroelastosis (none, mild – scattered 
echogenic spots, moderate – non-contiguous echogenic patches or severe – 
contiguous echogenic lining) 
 Presence of ventriculo-coronary connections (number of vcc) 
LV outflow tract view 
Aortic valve and ascending aorta diameter in systole 

 
 
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RV outflow tract view 
Pulmonary annulus and pulmonary trunk diameter in systole 
Three vessel tracheal 
view 
Qualitative evaluation 
 
 
Development of Z-scores for fetal cardiac dimension from echocardiography, 
Schneider, Daubeney et al 2005  
 
Spectral Doppler registrations of 
- 
All four valves: presence of flow, regurgitation or insufficiency assessed both 
quantitatively and qualitatively (none, mild, moderate, severe) 
- 
Tricuspid inflow:  
o biphasic, e and a wave 
o Fused, fusion of e and a wave but not monophasic 
o Monophasic, only a wave 
o No inflow 
o Inflow duration (ms) / Cardiac cycle duration (ms) 
Cardiac function assessment will be done qualitatively including signs of cardiomegaly, 
pericardial effusion or other signs of hydrops fetalis. 
 
 
 

 
 
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b) Postnatal echocardiography (pending to add images/figures) 
The following items will be evaluated in the anatomical and functional postnatal study 
a. Abdominal situs: locate stomach and liver. 
b. Cardiac situs: locate abdominal aorta, inferior cava vein, and concordance 
between abdominal organs and heart. 
c. Evaluate systemic venous drainage: suprahepatic veins, inferior vena cava, 
azygos/hemiazygos veins, superior vena cava, presence/absence left superior 
vena cava, presence/absence innominate vein.  
d. Evaluation of pulmonary venous drainage. 
e. Atrial evaluation: 
i. Right atrium: location, size. 
ii. Left atrium: location, size. 
iii. Interatrial septum: presence or not of defects (patent foramen ovale, 
interatrial communication) and shunt (left-right or right-left).  
f. Atrio-ventricular valves:  
i. Tricuspid valve: implantation of the leaflets, assess the presence of three 
leaflets (anterior, posterior, and septal), their morphology, and function 
(opening, closing). Evaluate the tricuspid annulus (size, implantation) 
and the subvalvular apparatus (first and second-order chords, free 
wall/septal anchorage). Evaluate the filling pattern and the mean gradient 
through the tricuspid (taking into account the possible underestimation in 
relation to the presence or absence of ASD with right-left shunt). 
Assessment of the presence of valvular regurgitation and its degree 
(regurgitation jet study). 
ii. Mitral valve: implantation of the leaflets, study the presence of two 
leaflets (anterior and posterior), their morphology and function (opening, 
closing). Mitral annulus (size, implantation) and subvalvular apparatus 
(cords and papillary muscles). Assess the filling pattern, and the mean 
gradient through the mitral valve (taking into account the possible 
underestimation in relation to the presence or absence of ASD with left-
right shunt). Assessment of the presence of valvular regurgitation and its 
degree (regurgitation jet study). 
iii. Tricuspid/Mitral ratio. 

 
 
Research protocol v2.0 26.Dec.2023 
 
g. Right ventricle: evaluate position, morphology, development (inlet, trabecular, 
and infundibulum; hypoplastic, bipartite, tripartite), and function (systolic - 
TAPSE, shortening fraction; diastolic - filling pattern, right atrium size, assess 
systemic venous congestion). 
h. Left ventricle: Study position, morphology, development, and function (systolic 
- ejection fraction; diastolic - filling pattern, left atrium size).  
i. Interventricular septum: evaluate integrity. 
j. Outflow tracts: assessment of outflow tracts, alignment with the ventricle, 
presence of subvalvular anatomical obstruction (muscular labrum). 
k. Pulmonary valve: annulus (size), description of the leaflets and morphology, 
presence of commissure fusion, movement. 
l. Aortic valve: ring (size), description of the leaflets and morphology, presence of 
commissure fusion, movement. 
m. Main pulmonary artery and branches: size, branches confluence.  
n. Ascending, arch, and descending aorta. Supraaortic branches: size, right/left 
arch, location of the aortic branches.  
o. Arterial duct: morphology, permeability, size, and shunt direction  
*** To evaluate the size of the different structures, according to the newborn size, 
Boston z-scores will be used. (https://zscore.chboston.org;).  
The following references will be used to categorize structures: 
- 
Normal: z-score between -2 to +2 
- 
Mild hypoplasia: z-score between -3 and -2 
- 
Moderate hypoplasia: z-score between -4 and -3 
- 
Severe hypoplasia: z-score < -5 
 
 
 

 
 
Research protocol v2.0 26.Dec.2023 
 
 
VIEW 
Structure 
Dimension / Morphology 
Functional evaluation 
z-score 
Images 
Subcostal 
Right ventricle (RV)   
 
Unipartite (severely hypoplastic) * 
Bipartite (moderately hypoplastic) ** 
Tripartite (normal or mildly hypoplastic) 
*** 
Impaired diastolic function (reversed flow and dilation of suprahepatic 
veins and inferior vena cava) 
 
SC* 
SC** 
SC*** 
 
Interatrial septum 
Interatrial communication: 
- 
< 3mm: small 
- 
3-6mm: moderate 
- 
> 6mm: large 
Flow 
- 
Right-left 
- 
Bidirectional 
- 
Left-right 
 
 
Apical 
Tricuspid valve 
Annulus 
 
*Boston 
 
 
 
Subvalvular apparatus 
Presence or absence of commissural fusion 
Presence or absence of short chords 
 
 
 
 
Inlet jet (measured by color with Nyquist 
scale 40-60cm/s and level of the opening 
of the leaflets.) 
Ratio of tricuspid inlet jet to mitral inlet jet: 
- 
1 normal 
- 
0.7-1 mildly abnormal 
- 
0.4-0.7 moderately abnormal 
- 
< 0.4 severely abnormal 
 
Ap* 
 
 
 
Regurgitation jet (in the neonatal period, study it on the maximum 
Nyquist scale): 
- 
vena contracta (VC) (mm) 
- 
penetrance in the right atrium (<1/2 right atrium, reaches the 
atrial roof). 
- 
Regurgitation mechanism (coaptation deficit, functional 
insufficiency - pressure). 
Classification: 
- 
Mild: VC < 3mm and/or reaches <1/2 of the right atrium  
- 
Moderate: VC 3-6mm and/or reaches >1/2 right atrium 
- 
Severe: VC > 6mm and/or reaches the right atrium roof 
 
Ap** 
 
 
Tricuspid annulus/mitral annulus ratio: 
favourable for biventricular when the 
ratio is > 0.79 
 
 
 
 
Right ventricle 
Unipartite (severely hypoplastic).* 
Bipartite (moderately hypoplastic).** 
Tripartite (normal or mildly hypoplastic). 
*** 
Systolic function: 
TAPSE: normal > 6mm in newborns at term 
Sp wave in Tissue Doppler: normal > 6cm/s in newborns at term 
Diastolic function (filling pattern, E wave, A wave):   
*Boston 
Ap*** 
Ap**** 
Ap***** 

 
 
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- 
Restrictive pattern: E<<<A without respiratory variability.  
*** Diastolic dysfunction in the newborn can be normal  
 
Ventriculo coronary 
fistula 
 
 
 
 
Parasternal 
long left axis 
Right ventricle 
Diastolic diameter 
 
*Boston 
PEEL* 
Parasternal 
long left 
anterior axis 
Right ventricle 
outflow tract 
Infundibulum diameter 
 
*Boston 
 
 
Pulmonary 
annulus/valve 
Diameter 
 
*Boston 
 
 
 
 
Presence/absence of antegrade flow (pulmonary atresia/critical 
pulmonary stenosis). 
 
 
Parasternal 
long left 
posterior axis 
Tricuspid valve 
Annulus 
 
*Boston 
 
 
 
Subvalvular apparatus 
Presence or not of commissural fusion 
Presence or not of short chords 
Regurgitation insufficiency (same as apical view)  
 
 
Parasternal 
short left axis 
Main pulmonary 
artery and branches 
Diameter 
 
*Boston 
 
Left sagittal 
Arterial duct 
Diameter, length, morphology 
Shunt, velocity, direction 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
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SC* 
 
 
SC** 
 
 
 

 
 
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SC*** 
 
 
 
Ap*** 
 

 
 
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Ap**** 
 
 
Ap***** 
 
 

 
 
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Ap* 
 
 
Ap ** Jet IT 
 
 
 

 
 
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PEEL* 
 
 
 

 
 
Research protocol v2.0 26.Dec.2023 
 
 
Annex 3. eCRF 
1. eCRF Inclusion criteria 
Gestational Age at Screening: 
Inclusion criteria: 
o Absence of flow at the pulmonary valve (PA) or presence of thickened and 
domed pulmonary valve cusps with a pinole jet of flow: yes/no 
o Doppler evidence of ductal-dependent pulmonary circulation: yes/no 
o Intact ventricular septum: yes/no 
Exclusion criteria 
o Unconfirmed PA-CS/IVS at birth: yes/no 
o Functional PA-CS/IVS (Ebstein malformation, monochorionic twins): 
yes/no 
o Any associated cardiac defect except persistent left superior vena cava and 
aberrant right subclavian artery: yes/no 
o Any significant (i.e that might influence outcome) extracardiac anomaly 
and/or known chromosomal aberration. Also, if such a condition is present 
at inclusion but diagnosed only after birth the case will be retrospectively 
excluded: yes/no 
▪ If yes, specify anomaly 
Signed consent: yes/no 
Revoked informed consent: yes/no 
 
If yes: revoked further follow-up but allows treatment of information 
gathered up to this point; revoked further follow-up and treatment of information 
gathered up to this point  
 
2. eCRF Baseline echo 
Number of fetuses (count)  
GA of baseline echo 
Right ventricle length (mm) 
Left ventricle length (mm) 
Right ventricle inlet (mm) 
Left ventricle inlet (mm) 
Tricuspid valve (mm) 

 
 
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Mitral valve (mm) 
Pulmonary valve (mm) 
Aortic valve (mm) 
Pulmonary annulus (mm) 
Aortic annulus (mm) 
Evidence of endocardial fibroelastosis: none, mild, moderate, severe 
Evidence of ventriculo-coronary connections: yes/no 
Right ventricle inflow duration (ms) 
Cardiac cycle duration (ms) 
Tricuspid regurgitation velocity (m/s) 
Tricuspid regurgitation (mild/moderate/severe) 
Pulmonary antegrade flow (yes/no) 
Atrial flow by color doppler (normal; bidirectional; right-to-left; none-intact) 
Clinician’s counseling (biventricular outcome, univentricular outcome) 
3. eCRF last Follow-up fetal echo until delivery (min 8 weeks apart) 
GA of last echo 
Right ventricle length (mm) 
Left ventricle length (mm) 
Right ventricle inlet (mm) 
Left ventricle inlet (mm) 
Tricuspid valve (mm) 
Mitral valve (mm) 
Pulmonary valve (mm) 
Aortic valve (mm) 
Pulmonary annulus (mm) 
Aortic annulus (mm) 
The right ventricle reaches the cardiac apex: yes/no 
Right ventricle morphology: uni, bi or tripartite 
Evidence of ventriculo-coronary connections: yes/no 
Right ventricle inflow duration (ms) 
Cardiac cycle duration (ms) 
Tricuspid regurgitation velocity (m/s) 
Tricuspid regurgitation (mild/moderate/severe) 
Pulmonary antegrade flow (yes/no) 

 
 
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Atrial flow by color doppler (normal; bidirectional; left-to-right; none-intact) 
Clinician’s counseling (biventricular outcome, univentricular outcome) 
4. eCRF Pregnancy outcomes 
Termination of pregnancy: yes/no 
Fetal death: yes/no If yes: date of death 
Growth restriction19: yes/no 
Other pregnancy comorbidities (none, hypertensive disorders, gestational 
diabetes, other comorbidities) 
Gestational age at delivery (weeks) 
Newborn’s sex: male/female 
Newborn’s weight (g) 
Newborn’s length (cm) 
5. eCRF Neonatal echo (livebirths Only) 
2D: 
- 
Tricuspid annulus (4 chamber view) (mm) 
- 
Mitral annulus (4 chamber view) (mm) 
- 
Tricuspid annulus (PEEL posterior) (mm) 
- 
Right ventricle morphology ( 4 chamber view): qualitative evaluation  
Unipartite 
Bipartite 
Tripartite 
- 
Right ventricle diastolic diameter (parasternal long axis view) 
- 
Pulmonary annulus diameter (parasternal short axis view) (mm)  
- 
Pulmonary artery diameter (parasternal short axis view) (mm) 
- 
Right pulmonary artery (parasternal short axis view) (mm) 
- 
Left pulmonary artery (parasternal short axis view) (mm) 
- 
Interatrial shunt (subcostal view): qualitative 
None 
< 3mm: small 
3-6mm: moderate 
> 6mm: large 
Color Doppler: 
- 
Tricuspid inlet jet (scale 40-60cm/s) (4 chamber view): quantitative 
- 
Tricuspid inlet jet / mitral inlet jet ratio (4 chamber view) 

 
 
Research protocol v2.0 26.Dec.2023 
 
- 
Tricuspid regurgitation jet (vena contracta, maximum scale) (4 chamber view): 
quantitative 
- 
Interatrial shunt (subcostal view): qualitative  
Right-left 
Left - Right 
Bidirectional 
No shunt 
- 
Antegrade pulmonary flow: yes/no (cualitative)  
- 
Ventriculo coronary connections: yes/no  
 
M Mode: 
- 
TAPSE (4 chamber view, mm) 
 
6. eCRF Neonatal discharge 
Alive: yes/no 
 
If no, date of neonatal death 
Date of discharge 
Add all procedures until discharge 
First postnatal procedure (balloon pulmonary valvuloplasty; surgical valvotomy, 
modified Blalock-Taussig shunt or ductal stent; Atrial septal device or surgical 
closure; Shunt ligation or occlusion; Transplant, tricuspid valve surgery, superior 
cavopulmonary, none (died without procedures), none necessary) 
Date of procedure 
Second postnatal procedure 
 
Date of procedure 
Days at NICU before discharge 
Type of circulation: univentricular, one and a half, biventricular, transplant 
 
7. eCRF Follow-up at 2 years 
Date of last follow-up 
Loss to follow-up (yes/no) 
 
If yes: revoked informed consent, loss of follow up 
Alive (yes/no) 
 
Date of death 

 
 
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Oxygen saturation 
Right ventricle-dependent coronary circulation (yes/no)  
Type of circulation evolution 
Transplant: 
 
From planned univentricular 
 
From planned one and a half  
 
From biventricular 
Univentricular 
 
From planned univentricular 
 
From planned one and a half  
 
From biventricular 
One and a half 
 
From planned univentricular 
 
From planned one and a half  
 
From biventricular 
Biventricular 
 
From planned univentricular 
 
From planned one and a half  
 
From biventricular 
Other sequealae 
 
 
 

 
 
Research protocol v2.0 26.Dec.2023 
 
Annex 4. TRIPOD statement 
Section/Topic 
 
Checklist Item 
Page 
Title and abstract 
Title 
Identify the study as developing and/or validating a multivariable prediction model, the target 
population, and the outcome to be predicted. 
1 
Abstract 
Provide a summary of objectives, study design, setting, participants, sample size, predictors, 
outcome, statistical analysis, results, and conclusions. 
NA 
Introduction 
Background and 
objectives 
Explain the medical context (including whether diagnostic or prognostic) and rationale for 
developing or validating the multivariable prediction model, including references to existing 
models. 
3 
Specify the objectives, including whether the study describes the development or validation of the 
model or both. 
3 
Methods 
Source of data 
Describe the study design or source of data (e.g., randomized trial, cohort, or registry data), 
separately for the development and validation data sets, if applicable. 
9 
Specify the key study dates, including start of accrual; end of accrual; and, if applicable, end of 
follow-up.  
NA 
Participants 
Specify key elements of the study setting (e.g., primary care, secondary care, general population) 
including number and location of centres. 
9 
Describe eligibility criteria for participants.  
11 
Give details of treatments received, if relevant.  
NA 
Outcome 
Clearly define the outcome that is predicted by the prediction model, including how and when 
assessed.  
9 
Report any actions to blind assessment of the outcome to be predicted.  
NA 
Predictors 
Clearly define all predictors used in developing or validating the multivariable prediction model, 
including how and when they were measured. 
12 
Report any actions to blind assessment of predictors for the outcome and other predictors.  
NA 
Sample size 
Explain how the study size was arrived at. 
10 
Missing data 
Describe how missing data were handled (e.g., complete-case analysis, single imputation, multiple 
imputation) with details of any imputation method.  
14 
Statistical 
analysis methods 
Describe how predictors were handled in the analyses.  
13 
Specify type of model, all model-building procedures (including any predictor selection), and 
method for internal validation. 
14 
For validation, describe how the predictions were calculated.  
… 
Specify all measures used to assess model performance and, if relevant, to compare multiple 
models.  
14 
Describe any model updating (e.g., recalibration) arising from the validation, if done. 
… 
Risk groups 
Provide details on how risk groups were created, if done.  
NA 
Development vs. 
validation 
For validation, identify any differences from the development data in setting, eligibility criteria, 
outcome, and predictors.  
… 
Results 
Participants 
Describe the flow of participants through the study, including the number of participants with and 
without the outcome and, if applicable, a summary of the follow-up time. A diagram may be 
helpful.  
NA 

 
 
Research protocol v2.0 26.Dec.2023 
 
Describe the characteristics of the participants (basic demographics, clinical features, available 
predictors), including the number of participants with missing data for predictors and outcome.  
NA 
For validation, show a comparison with the development data of the distribution of important 
variables (demographics, predictors and outcome).  
NA 
Model 
development  
Specify the number of participants and outcome events in each analysis.  
NA 
If done, report the unadjusted association between each candidate predictor and outcome. 
NA 
Model 
specification 
Present the full prediction model to allow predictions for individuals (i.e., all regression 
coefficients, and model intercept or baseline survival at a given time point). 
NA 
Explain how to the use the prediction model. 
NA 
Model 
performance 
Report performance measures (with CIs) for the prediction model. 
NA 
Model-updating 
If done, report the results from any model updating (i.e., model specification, model performance). 
NA 
Discussion 
Limitations 
Discuss any limitations of the study (such as nonrepresentative sample, few events per predictor, 
missing data).  
NA 
Interpretation 
For validation, discuss the results with reference to performance in the development data, and any 
other validation data.  
NA 
Give an overall interpretation of the results, considering objectives, limitations, results from 
similar studies, and other relevant evidence.  
NA 
Implications 
Discuss the potential clinical use of the model and implications for future research.  
NA 
Other information 
Supplementary 
information 
Provide information about the availability of supplementary resources, such as study protocol, 
Web calculator, and data sets.  
NA 
Funding 
Give the source of funding and the role of the funders for the present study.  
NA 
 
*Items relevant only to the development of a prediction model are denoted by D, items relating solely to a validation of a prediction model are denoted by V, 
and items relating to both are denoted D;V.  We recommend using the TRIPOD Checklist in conjunction with the TRIPOD Explanation and Elaboration document. 
 
 
 
 

 
 
Research protocol v2.0 26.Dec.2023 
 
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