1Thomas L, et al. BMJ Open 2021;11:e044924. doi:10.1136/bmjopen-2020-044924 Open access Multicentre prospective observational study exploring the predictive value of functional echocardiographic indices for early identification of preterm neonates at risk of developing chronic pulmonary hypertension secondary to chronic neonatal lung disease Laura Thomas,1,2 Michelle Baczynski,3 Poorva Deshpande,1,4 Ashraf Kharrat,1,4 Sébastien Joye,5 Faith Zhu,1 Daniel Ibarra- Rios,6 Prakesh S Shah,1,4 Luc Mertens,7 Robert P Jankov,8 Xiang Y Ye,9 Elaine Neary,10 Joseph Ting,11 Michael Castaldo,11 Philip Levy,12,13 Aisling Smith,14 Afif F El- Khuffash,14 Regan E Giesinger,15 Patrick J McNamara,15 Dany E Weisz,2,4 Amish Jain 1,4 To cite: Thomas L, Baczynski M, Deshpande P, et al. Multicentre prospective observational study exploring the predictive value of functional echocardiographic indices for early identification of preterm neonates at risk of developing chronic pulmonary hypertension secondary to chronic neonatal lung disease. BMJ Open 2021;11:e044924. doi:10.1136/ bmjopen-2020-044924 ► Prepublication history for this paper is available online. To view these files, please visit the journal online (http:// dx. doi. org/ 10. 1136/ bmjopen- 2020- 044924). Received 16 September 2020 Revised 08 March 2021 Accepted 17 March 2021 For numbered affiliations see end of article. Correspondence to Amish Jain; amish. jain@ sinaihealth. ca Protocol © Author(s) (or their employer(s)) 2021. Re- use permitted under CC BY- NC. No commercial re- use. See rights and permissions. Published by BMJ. ABSTRACT Introduction Although chronic pulmonary hypertension (cPH) secondary to chronic neonatal lung disease is associated with increased mortality and respiratory and neurodevelopmental morbidities, late diagnosis (typically ≥36 weeks postmenstrual age, PMA) and the use of qualitative echocardiographic diagnostic criterion (flat interventricular septum in systole) remain significant limitations in clinical care. Our objective in this study is to evaluate the utility of relevant quantitative echocardiographic indices to identify cPH in preterm neonates, early in postnatal course and to develop a diagnostic test based on the best combination of markers. Methods and analysis In this ongoing international prospective multicentre observational diagnostic accuracy study, we aim to recruit 350 neonates born <27 weeks PMA and/or birth weight <1000 g and perform echocardiograms in the third week of age and at 32 weeks PMA (early diagnostic assessments, EDA) in addition to the standard diagnostic assessment (SDA) for cPH at 36 weeks PMA. Predefined echocardiographic markers under investigation will be measured at each EDA and examined to create a scoring system to identify neonates who subsequently meet the primary outcome of cPH/death at SDA. Diagnostic test characteristics will be defined for each EDA. Pulmonary artery acceleration time and tricuspid annular plane systolic excursion are the primary markers of interest. Ethics and dissemination Ethics approval has been received by the Mount Sinai Hospital Research Ethics Board (REB) (#16-0111- E), Sunnybrook Health Sciences Centre REB (#228-2016), NHS Health Research Authority (IRAS 266498), University of Iowa Human Subjects Office/ Institutional Review Board (201903736), Rotunda Hospital Research and Ethics Committee (REC-2019-008), and UBC Children’s and Women’s REB (H19-02738), and is under review at Boston Children’s Hospital Institutional Review Board. Study results will be disseminated to participating families in lay format, presented to the scientific community at paediatric and critical care conferences and published in relevant peer- reviewed journals. Trail registration number NCT04402645. INTRODUCTION Background Chronic pulmonary hypertension (cPH) is a debilitating and often life limiting condition Strengths and limitations of this study ► In a large prospective cohort of extremely premature neonates, quantitative echocardiographic mark- ers are being acquired longitudinally using a stan- dardised methodology. ► Simple and reliable point- of- care tests to charac- terise pulmonary vascular resistance (pulmonary artery acceleration time) and right ventricular func- tion (tricuspid annular plane systolic excursion) are being systematically evaluated and shared with the clinical and scientific community to facilitate early diagnosis of chronic pulmonary hypertension. ► A robust sample size calculation to guide the recruit- ment strategy and ‘blinded’ analysis of echocardio- grams is an integral part of the study design. ► The final diagnosis of chronic pulmonary hyperten- sion, against which new early diagnostic tests are being evaluated, is based on qualitative diagnostic criteria, as per current standard of care. o n A p ril 5 , 2 0 2 1 b y g u e st. P ro te cte d b y co p yrig h t. h ttp ://b m jo p e n .b m j.co m / B M J O p e n : first p u b lish e d a s 1 0 .1 1 3 6 /b m jo p e n -2 0 2 0 -0 4 4 9 2 4 o n 3 1 M a rch 2 0 2 1 . D o w n lo a d e d fro m http://bmjopen.bmj.com/ http://orcid.org/0000-0002-1413-2966 http://crossmark.crossref.org/dialog/?doi=10.1136/bmjopen-2020-044924&domain=pdf&date_stamp=2021-03-31 NCT04402645 http://bmjopen.bmj.com/ 2 Thomas L, et al. BMJ Open 2021;11:e044924. doi:10.1136/bmjopen-2020-044924 Open access in which sustained vasoconstriction, vascular hypoplasia and arterial wall remodelling are associated with high and often progressive increases in pulmonary vascular resis- tance (PVR) and pulmonary arterial pressure, resulting in chronic exposure of the right ventricle to high afterload and ultimately leading to right ventricular (RV) failure.1–4 The data from studies in adults and children indicate that early diagnosis and management focused on preservation of RV performance is a major determinant of treatment success and longevity.5–10 Recognising its importance, the US National Heart, Lung and Blood Institute has recently identified ‘determinants of RV function and failure’ as a key priority for research.5 9 10 In neonates, cPH is inex- tricably linked with developmental disorders of the lung, most commonly chronic neonatal lung disease (CNLD), a frequent complication following extreme preterm birth. It is characterised by inflammation and impaired alveolar development and pulmonary angiogenesis.11–13 CNLD accounts for ~12% of all cases of cPH diagnosed during childhood and for the vast majority during infancy.4 14 15 In Canada, CNLD affects 50%–60% of all extremely low birth weight neonates (ELBW, birth weight <1000 g) resulting in more than 800 new cases each year, of which 20%–30% will be diagnosed with cPH during their stay in the neonatal intensive care unit (NICU).16 Over the last decade, several retrospective and a few prospective studies consistently demonstrated an asso- ciation of CNLD/cPH with higher predischarge and postdischarge mortality, longer duration of mechanical ventilation oxygen therapy, hospitalisation and higher rates of adverse neurodevelopmental outcomes compared with ELBW survivors with CNLD alone.3 17–24 The inci- dence, severity and resultant mortality of cPH in CNLD is generally proportional to the severity of parenchymal lung disease.25 A recent meta- analysis reported the pooled incidence of cPH to range from 4% (95% CI: 1% to 7%) in mild CNLD (ie, oxygen dependency ≥28 days but not at 36 weeks postmenstrual age, PMA), to 33% (95% CI: 21% to 44%) in severe CNLD (ie, oxygen dependency ≥28 days and need for ≥30% oxygen or positive pressure ventilation at 36 weeks PMA).26 Patients with CNLD/ cPH had higher odds of mortality versus only CNLD (OR: 5.29 (95% CI: 2.07 to 13.56)). In stark contrast to the expanding literature on clinical outcomes associated with cPH, there is little data to guide day- to- day manage- ment, including best practices regarding diagnostic and therapeutic strategies. To date, there is no treatment trial targeting cPH in preterm neonates. While expert bodies have published consensus- based guidelines to provide some direction to clinicians, it remains unknown whether a particular approach can ameliorate the extra burden of disease imposed by cPH on CNLD.5 The optimal approach to the surveillance of cPH remains unclear and is a major barrier to timely and effective diagnosis and treatment in this population.27 With this background, this project was envisioned to fill some of the key knowledge gaps in the diagnostic practices relating to cPH in this population. Importance of early identification of cases Although CNLD and its severity correlate with the risk of cPH, the diagnosis of CNLD is typically not made until 36 weeks PMA; currently, this is also the characteristic age when cPH evaluation is undertaken clinically using echocardiography. Late diagnosis, however, is a signifi- cant barrier for therapeutic trials to demonstrate clinical benefits. Similarly, early prophylactic treatment of all extreme premature infants is an undesirable approach, as it exposes patients without disease to pharmacotherapies. This will adversely affect the risk- benefit profile for ther- apies, increase treatment costs and necessitate a larger sample size for clinical trials. Several potential therapies are available, and, despite lack of trial data, are already being used in clinical practice, although often as rescue therapy for severe disease.1 5 28 29 Day- to- day clinical care is significantly hampered by the absence of a diagnostic test that allows case identification early in disease course. In early stages, the disease may be more functional, deter- mined by sustained pulmonary vasoconstriction and rela- tively less by anatomical remodelling, and may be more amenable to treatments. Thus, the overall aim of this study is to systematically develop quantitative echocardio- graphic diagnostic criteria which will allow for the iden- tification of neonates with significant pulmonary vascular disease early in postnatal life (figure 1). A significant reduction in lag between disease onset and diagnosis will offer new avenues for testing therapeutic interventions; a necessary prerequisite for trials to have the maximum chance of success. Need for quantitative echocardiographic diagnostic test for early cPH diagnosis in premature neonates Challenges in the clinical evaluation of at- risk premature neonates include associated parenchymal lung disease, which often masks the symptoms of evolving cPH and RV dysfunction,30 31 non- applicability or delayed appearance of specific clinical signs (eg, raised jugular venous pres- sure and systemic venous congestion) and non- feasibility of a gold standard test (right heart catheterisation) due to small patient size and high risk for complications.10 32–34 Cardiac MRI, a clinical reference investigation for the assessment of RV function in older patients, is cumber- some and not feasible during early disease in the majority of patients due to clinical instability and need for out- of- facility transport. When feasible, these investigations can only be performed sporadically, late in the disease course and are not suitable for longitudinal assessments. Two- dimensional echocardiography is a safe, quick, well- tolerated, non- invasive bedside investigation which is ideally suited for longitudinal assessments. Although it is considered the clinical investigation of choice for the assessment of heart function and pulmonary haemo- dynamics in neonates, it has not been utilised to its full potential in neonatal cPH. Previous studies have advanced our understanding of cPH in neonates; however, their key limitation is the use of qualitative subjective diagnostic criteria—‘flat o n A p ril 5 , 2 0 2 1 b y g u e st. P ro te cte d b y co p yrig h t. h ttp ://b m jo p e n .b m j.co m / B M J O p e n : first p u b lish e d a s 1 0 .1 1 3 6 /b m jo p e n -2 0 2 0 -0 4 4 9 2 4 o n 3 1 M a rch 2 0 2 1 . D o w n lo a d e d fro m http://bmjopen.bmj.com/ 3Thomas L, et al. BMJ Open 2021;11:e044924. doi:10.1136/bmjopen-2020-044924 Open access interventricular septum’ in the majority of cases. This index represents a qualitative assessment for the loss of normal concave curvature of the septal wall between the right and the left ventricle; this is thought to reflect RV systolic pressure >1/2 systemic systolic pressure.35 36 Pres- ently, based on above studies, this criterion is used as the standard method to diagnose cPH in NICUs. Though easily assessable in the majority of patients and specific for severe disease, it is unsuitable for early diagnosis or close monitoring of disease progression.18 For these consider- ations, these criteria are not usually relied on by clinicians in older patients.37 Only one- third of neonates eventu- ally diagnosed with cPH could be identified using these criteria in the fourth week of life.18 Many quantitative echocardiographic markers relevant to the study of cPH are now widely available for use in neonates, in partic- ular those representing RV function and PVR.38 Several of these have been validated and shown to be of prognostic significance with clearly identified cut- off values for clin- ical use in adults with cPH.39 This protocol seeks to address at least three of the domains to improve diagnostic preci- sion and clinical monitoring of cPH in preterm infants recently identified by the Paediatric Pulmonary Hyper- tension Network.27 These areas are: (1) the standardisa- tion of echocardiographic characterisation of cPH, (2) the use of echocardiography to identify a risk profile for early prediction of risk, diagnosis and follow- up and (3) enhanced assessment of RV performance and afterload using novel quantitative echocardiographic techniques. This will be the first study systematically examining the predictive value of novel echocardiographic markers to develop a diagnostic test for early diagnosis of cPH in the infants with gestational age (GA) <27 weeks or BW <1000 g. RESEARCH HYPOTHESIS AND STUDY OBJECTIVES We hypothesise that quantitative echocardiographic indices of RV function and PVR will allow early diagnosis of cPH prior to 36 weeks PMA in extreme premature infants; thus, reducing the time to diagnosis compared with contemporary clinical practice. More specifically, we hypothesise that pulmonary artery acceleration time (PAAT, a simple marker of PVR validated in adults and children against invasive measurements) and tricuspid annular plane systolic excursion (TAPSE, a routinely used marker of RV function) either alone or in combi- nation will be the most useful parameters for early case identification. The primary objective of this study is to evaluate the ability of relevant quantitative functional echocardio- graphic indices, in particular PAAT and TAPSE, to iden- tify preterm neonates with cPH early in postnatal life, develop a diagnostic test based on the best combination of parameters and define its diagnostic characteristics (sensitivity, specificity, positive and negative likelihood ratios). Our secondary objectives are (1) To test the rela- tionship between the developed diagnostic test and clin- ical outcomes known to be affected by cPH (mortality and respiratory morbidities). This is important for clin- ical corroboration of our new criteria and will provide the necessary preliminary data to enable sample size calculations for subsequent therapeutic trials, targeting improvement in clinical outcomes. (2) To longitudinally Figure 1 In early stage of chronic pulmonary hypertension (cPH), the disease is expected to be more functional, determined by sustained pulmonary vasoconstriction and relatively less by ‘fixed’ anatomical remodelling, hence, may be more amenable to treatments. The overall aim of this study is to develop new diagnostic criteria sensitive enough to identify extreme premature neonates with significant pulmonary vascular disease, who subsequently will be diagnosed with cPH secondary to chronic neonatal lung disease. CNLD, chronic neonatal lung disease; PVR, pulmonary vascular resistance o n A p ril 5 , 2 0 2 1 b y g u e st. P ro te cte d b y co p yrig h t. h ttp ://b m jo p e n .b m j.co m / B M J O p e n : first p u b lish e d a s 1 0 .1 1 3 6 /b m jo p e n -2 0 2 0 -0 4 4 9 2 4 o n 3 1 M a rch 2 0 2 1 . D o w n lo a d e d fro m http://bmjopen.bmj.com/ 4 Thomas L, et al. BMJ Open 2021;11:e044924. doi:10.1136/bmjopen-2020-044924 Open access study the myocardial maturation pattern in extreme premature neonates and evaluate how it may be affected by occurrence of cPH. This is important as preliminary work indicates that occurrence of CNLD/cPH may nega- tively impact cardiac maturational patterns in preterm infants.40 However, this observation needs further vali- dation and the relative impact of CNLD versus cPH on cardiac development is not known. (3) To identify clin- ical risk factors associated with occurrence of cPH in this cohort. (4) To establish a large representative cohort of high- risk preterm neonates to pursue future long- term follow- up studies to understand their development and respiratory morbidities during childhood. METHODS AND ANALYSIS Design, setting and study population This study is a prospective, multisite, observational cohort study being conducted in five tertiary NICUs across Canada, the USA, UK and Ireland. The study teams from the six participating sites have been trained in the stan- dardised procedure of enrollment, echocardiography and data collection. At its inception, the study was being conducted at two tertiary NICUs in Toronto, Mount Sinai Hospital (start date 1 November 2017) and Sunnybrook Health Sciences Centre (start date 31 January 2018). Subsequently, to help accelerate study completion, the following additional sites were recruited: Liverpool Women’s NHS Foundation Trust (start date 6 November 2019), the Rotunda Hospital (start date 20 November 2019), the University of Iowa Stead Family Children’s Hospital (start date 6 July 2020) and BC Women’s Hospital and Health Centre (start date 7 September 2020). The study protocol is also currently under review at Boston Children’s Hospital Institutional Review Board. These additional sites were selected based on their knowledge in similar patient populations and study settings as the primary study sites and their similar echocardiography equipment, analysis software and presence of a formal in- house neonatal echocardiography programme led by an experienced neonatologist with formal training in neonatal echocardiography, all of whom are site collabo- rators in this study. Infants are considered eligible for enrollment if they are born at GA <27 weeks and/or with a birth weight (BW) <1000 g, and are cleared by the attending clinical team to approach for informed consent between 10 and 14 days of age. Neonates with major congenital and/or genetic anomalies and congenital heart defects (with the excep- tion of patent ductus arteriosus (PDA), patent foramen ovale (PFO), peripheral pulmonary artery stenosis or small (<3 mm diameter) ventricular septal defects) are not considered eligible for recruitment. Recruitment and study procedures Families of eligible neonates are approached for consent between 10 and 20 days postnatal age. All recruited neonates undergo three serial cardiopulmonary eval- uations: early diagnostic assessments (EDAs) at two predefined time points (study interventions, between 14 and 21 days of age (inclusive) and again between 32nd and 33rd weeks PMA) and a standard diagnostic assess- ment (SDA) at ≥36±2 weeks PMA (standard clinical care). Each EDA consists of collecting relevant clinical data and a focused echocardiogram to collect indices representa- tive of PVR and RV functions, while SDA consists of an echocardiogram to allow assigning the final diagnosis of cPH using the current clinical definition (figure 2). Each assessment is carried out by a cardiac sonographer or fellow trained in neonatal haemodynamics, with infants in a non- agitated state and is coordinated with any one of the routine handling times on that day. In the event of an intercurrent illness or procedure, such as sepsis, necro- tising enterocolitis or treatment for retinopathy, assess- ments are deferred until the neonate is deemed to have recovered by the attending neonatologist. Blinded anal- ysis of all EDA echocardiograms to measure parameters- under investigation (table 1) is completed by a centralised trained senior research sonographer, at arm’s length from the study, who remains blinded to all clinical data. The echocardiogram done for SDA will be analysed by sepa- rate personnel to categorise infant as cPH or not based Figure 2 Schematic representation of planned study interventions. Each infant, after obtaining informed parental consent, will undergo two sequential early diagnostic assessments (EDAs) at predefined time points, followed by a standard diagnostic assessment (SDA) to categorise study cohort as chronic pulmonary hypertension (cPH) or no cPH, as per the standard currently used clinical definition. Blinded measurements will be performed for tricuspid annular plane systolic excursion (TAPSE, a marker of right ventricular function) and pulmonary artery acceleration time (PAAT, a marker of pulmonary vascular resistance) at both EDAs to calculate their early diagnostic characteristics (sensitivity, specificity and positive and negative likelihood ratios) to diagnose cPH by comparing to eventual diagnosis made at SDA. GA, gestational age; NICU, neonatal intensive care unit; PMA, postmenstrual age. o n A p ril 5 , 2 0 2 1 b y g u e st. P ro te cte d b y co p yrig h t. h ttp ://b m jo p e n .b m j.co m / B M J O p e n : first p u b lish e d a s 1 0 .1 1 3 6 /b m jo p e n -2 0 2 0 -0 4 4 9 2 4 o n 3 1 M a rch 2 0 2 1 . D o w n lo a d e d fro m http://bmjopen.bmj.com/ 5Thomas L, et al. BMJ Open 2021;11:e044924. doi:10.1136/bmjopen-2020-044924 Open access on the currently used definition, as noted above. If study participants have clinically indicated scans after 36 weeks PMA, the data regarding outcome of cPH are collected. Clinical data, including baseline demographics, respira- tory illness severity during first 3 weeks of age and expo- sure to relevant but unrelated pre- existing morbidities and treatments are also collected in addition to data regarding common neonatal outcomes at discharge and from the infants’ 18- month follow- up neurodevelop- mental clinical assessment. At the end of the recruitment, all deidentified data will be sent for analysis to a statisti- cian blinded to all other aspects of this study. Rationale for primary parameter selection PAAT and TAPSE are simple, reliable, bedside tests, which, in older patients, have been validated30 41–49 and correlate with prognosis in cPH.30 50–53 The normal data for neonates are well established and these markers are commonly used in NICUs, including study NICUs, for longitudinal monitoring; however, no specific diagnostic cut- offs currently exist. In previous work, we have estab- lished the reproducibility and normal values for various markers of ventricular function and PVR in neonates;54 55 intraclass correlation coefficient for PAAT and TAPSE was 0.90 and 0.97, respectively. We then confirmed the ability Table 1 Echocardiographic variables measured on study echocardiograms Index name Description Measurement Study specific Pulmonary artery acceleration time (PAAT) (Primary index) A marker of pulmonary vascular resistance (inversely related). Interval between the onset of ejection and peak flow velocity measured from a pulse wave Doppler tracing obtained by placing a 2 mm sample volume in the middle of the main pulmonary artery, at the tip of pulmonary valve leaflets. In addition, the ratio of total right ventricular (RV) ejection time to PAAT is also measured as a heart rate independent measure of PVR (directly related). Tricuspid annular plane systolic excursion (TAPSE) (primary index) A marker of RV longitudinal systolic function. Measure of the downward displacement of tricuspid annulus during contraction. From an apical four- chamber view by placing M- mode cursor through the tricuspid valve annulus. Two- dimensional peak global end- systolic longitudinal strain (pLS)—right ventricle (secondary index) A measure of myocardial deformation expressed as percentage change in length in systole from the baseline at end diastole. Performed offline using software, which uses frame to frame tracking of the unique ultrasound speckles within the myocardial wall. Longitudinal strain will be calculated for RV lateral and inferior wall from RV apical four chamber and three chamber views, respectively. Fractional area change (FAC- 4C) % (secondary index) A surrogate to ejection fraction and indicative of overall ‘pump function’. Manual tracing of the RV endocardial borders at respective phases of the cardiac cycle. From an apical four chamber view the end systolic area (ESA) and end diastolic area (EDA) are measured. FAC- 4C (%) =((EDA- ESA)/ EDA)x 100 Tissue Doppler imaging (TDI) (secondary index) Allows measurement of velocities directly from the myocardium to assess longitudinal systolic and diastolic RV function. Measure the peak systolic and diastolic velocities, and duration of various phases of a cardiac cycle. Measurements obtained at the RV basal segment. RV peak systolic pressure (RVsP) (secondary index) A direct measure of pulmonary artery systolic pressure. Calculated by measuring peak velocity (v) of tricuspid regurgitation using the modified Bernoulli equation. RVsP=4v2+right atrial pressure. Calculated whenever feasible. Right atrial pressure is predefined as 5 mm Hg in all cases. Assessment of shunts (secondary index) Patent ductus arteriosus and patent foramen ovale/ atrial septal defect. Visualised on colour Doppler assessment. Shunt diameter in millimetre and flow direction documented. Flow patterns in main and branch pulmonary arteries (secondary index) Presence of notching is considered a marker of high downstream resistance Visualised on pulse wave Doppler Presence or absence of midsystolic notching in the Doppler profile. PVR, pulmonary vascular resistance. o n A p ril 5 , 2 0 2 1 b y g u e st. P ro te cte d b y co p yrig h t. h ttp ://b m jo p e n .b m j.co m / B M J O p e n : first p u b lish e d a s 1 0 .1 1 3 6 /b m jo p e n -2 0 2 0 -0 4 4 9 2 4 o n 3 1 M a rch 2 0 2 1 . D o w n lo a d e d fro m http://bmjopen.bmj.com/ 6 Thomas L, et al. BMJ Open 2021;11:e044924. doi:10.1136/bmjopen-2020-044924 Open access of PAAT to detect reduction in PVR in healthy neonates during first day after birth.56 Among RV markers, TAPSE was found sensitive to these changes,57 while no change was seen on septal flattening. Study definitions CPH: infants will be classified as having significant cPH if they meet any of the following features at SDA:17 1. Right ventricular systolic pressure (RVSP) ≥0.5 systolic blood pressure. 2. Bidirectional (right- left in systole) or a right to left shunt across a PDA. 3. A flat interventricular septal motion in end- systole. Infants who meet the above criteria on EDAs will only be classified as cPH if the findings persist until SDA or if severity increases enough for clinicians to initiate cPH treatment. CNLD: infants are assessed at 36 weeks PMA to clas- sify the severity of CNLD based on standard well- defined objective clinical criteria:58 1. Mild CNLD: supplemental oxygen requirement for ≥28 days but off all respiratory support at 36 weeks PMA or discharge, whichever is earlier. 2. Moderate CNLD: supplemental oxygen requirement for ≥28 days and at 36 weeks PMA but fractional in- spired oxygen concentration (FiO 2 ) <0.30. 3. Severe CNLD: supplemental oxygen requirement for ≥28 days and at 36 weeks PMA with fractional inspired oxygen concentration (FiO 2 ) ≥0.30 or dependent on any positive pressure ventilatory support. Infants who are discharged from study NICU before 36 weeks PMA will be classified based on respiratory status at discharge. Outcome measures Primary clinical outcome The primary clinical outcome is final diagnosis of cPH, made based on above definition at SDA. Our intention is to develop a risk score using a combination of echocar- diographic markers, at each EDA, predicting the primary clinical outcome. A cut- off point of the risk score will then be determined using Youden’s method to define a new diagnostic test at each EDA. The sensitivity and specificity of the new diagnostic test for early prediction of cPH will be estimated. Secondary clinical outcomes Key prematurity- related clinical outcomes including composite of predischarge mortality or CNLD, CNLD, total duration of need for respiratory support, length of hospital stay, discharge on home oxygen and neurodevel- opmental status at 18–24 months corrected age. These secondary clinical outcomes are those which are well known to be worsen in relation to the diagnosis of cPH in preterm neonates. These will be used to corroborate and test the clinical validity of our newly identified early cPH diagnostic test, as we anticipate that the infants identified as cPH with the new diagnostic test developed by this study would demonstrate more adverse clinical outcomes than those without cPH. Statistical methods Sample size calculation: The incidence of cPH in the study population is esti- mated to at least 20%. The mean and SD or sensitivity and specificity of echocardiographic markers during the EDA time points are not known, making specific sample size calculations non- feasible. Presuming a disease prevalence of 20%, to estimate new diagnostic test with a sensitivity of 85% and an absolute precision of 0.07, a sample size of approximate 350 babies will be required.59 The actual incidence of cPH at SDA will be evaluated at the halfway stage in the recruited cohort, to confirm representative- ness of the estimated incidence. The sample size calcu- lation may be revised if the incidence in the recruited cohort is found to be significantly higher or lower than the estimate. Analysis ► The study population will be summarised descrip- tively. The baseline infant characteristics (including BW z- score), clinical measurements and echocardio- graphic indices (ie, PAAT and TAPSE), as well as the presence of shunts such as atrial septal defect, at each EDA will be compared between two groups: infants with/without cPH using the χ2 test for categorical variables and the student t- test or Wilcoxon Rank- Sum test for continuous variables, as appropriate. Changes over time in echocardiographic indices will also be compared using two- way repeat measures analysis of variance. ► We will conduct a multiple logistic regression at each EDA using backward variable selection procedure with variable stay criterion of p<0.1. All echocardio- graphic indices associated with cPH identified in the bivariate analysis (p<0.1) or based on clinical exper- tise, including the presence of shunts if relevant, will be included in the full model. Denote the final model as logitPr ( cPH = yes ) = a0 + a1x1 + a2x2 + . . . + akxk … (1) where x1, …, xk are the echocardiographic indexes that remain. A risk score (RScore) will then be defined as Rscore= a0 + a1x1 + a2x2 + . . . + akxk . A new diagnostic test can be defined as DTest=(Rscore